Polypeptides involved in the biosynthesis of streptogramins, nucleotide sequences coding for these polypeptides and their use

Information

  • Patent Grant
  • 6077699
  • Patent Number
    6,077,699
  • Date Filed
    Thursday, August 3, 1995
    29 years ago
  • Date Issued
    Tuesday, June 20, 2000
    24 years ago
Abstract
Disclosed is the nucleic acid sequences for polypeptides and enzymes involved in the biosynthesis of streptogramins from Streptomyces sp. and the corresponding amino acid sequences. Also disclosed are recombinant vectors and cells as well as methods of making the polypeptides, enzymes and streptogramins using the recombinant host cells.
Description

The present invention relates to novel polypeptides involved in the biosynthesis of streptogramins, and also comprises the isolation and identification of genes for the biosynthesis of the A and B components of streptogramins, the expression of these genes with the object of increasing the levels of production and their use for the construction of blocked mutants capable of leading to the synthesis of novel antibiotics or to derived forms of streptogramins
Streptogramins form a homogeneous group of antibiotics, consisting of a combination of two types of molecules which are chemically different; on the one hand polyunsaturated macrolactones (A-group components, two examples of structures of which are presented in FIG. 1), and on the other hand depsipeptides (B-group components, three examples of the structure of which are presented in FIG. 2). This group comprises many antibiotics (see Table 1), which are known by different names in accordance with their origin, including pristinamycins, mikamycins and virginiamycins (for a review, see Cocito 1979, 1983).
The A and B components have a synergistic antibacterial activity which can reach 100 times that of the separate components and which, in contrast to that of each component, is bactericidal (Cocito 1979). This activity is more especially effective against Gram-positive bacteria such as staphylococci and streptococci (Cocito 1979, Videau 1982). The A and B components inhibit protein synthesis by binding to the 50S subunit of the ribosome (Cocito 1979; for a review, see Di Giambattista et al. 1989).
Streptogramins are chiefly produced by actinomycetes, including many streptomycetes, presented in Table 1. In addition, streptogramins are also synthesized by eukaryotes such as Micromonospora which synthesizes vernamycins. Actinomycetes constitute a very important group of microorganisms on account of the large amount of secondary metabolites they produce, including many antibiotics (beta-lactams, tetracyclines, macrolides, aminoglycosides, polyacetates and the like), herbicides, anticancer agents, antifungal agents, immunomodulators and enzyme inhibitors. Many biosynthesis pathways relating to antibiotics belonging to miscellaneous classes as well as other secondary metabolites such as pigments (for a review, Chater 1990) have already been studied at the present time in actinomycetes. An important aspect of this group of bacteria is that the genes involved in the same biosynthesis pathway, structural genes and also resistance gene(s) and regulatory gene(s), are grouped together physically on the chromosome, constituting clusters which can reach more than 100 kb (Hopwood et al. 1986a, Hopwood et al. 1986b, Hallam et al. 1988, Anzai et al. 1987, Ohnuki et al. 1985). To date, no example has been found to contradict this observation. Such a structural organization is of great interest in the development of strategies for cloning biosynthesis genes. In effect, it is possible, starting from a single gene previously cloned by various techniques, a biosynthesis, resistance or regulatory gene, to walk along the chromosome and thus to isolate the set of genes of the biosynthesis cluster.
Our knowledge of the biosynthesis pathways of each of the components of streptogramins is still very incomplete, but the origin of the different parts of each molecule has been identified by radioactive labelling (Kingston et al. 1983). Thus, the A-type components are made up of two regions originating from the condensation of acetates and several amino acids such as serine and glycine, for example. As regards the B-type components, studies have shown that all the amino acids present in the peptide chain are derived from natural amino acids (Hook and Vining 1973). However, no polypeptide involved in these pathways has, to date, been purified in sufficient amounts to permit its molecular characterization, and no biosynthesis gene has been described. In the process of biosynthesis of the B-type components, two parts may be distinguished:
1) Synthesis of the precursors, or of their analogues, of the macrocycle: 3-hydroxypicolinic acid (3-hPic), L-2-aminobutyric acid, p-dimethylamino-L-phenylalanine (L-Abu), 4-oxo-L-pipecolic acid (4-oPip), L-phenylglycine (L-Phg).
2) Formation of the macrocycle from the precursors mentioned above, L-threonine and L-proline, or their analogues, with possible modification of these precursors or peptide N-methylation.
To date, only the probable metabolic origin of the precursors of the macrocycle of the B-type components has been determined by studies using labelled isotopes (Reed et al., 1986, Molinero et al., 1989, Reed et al., 1989).
The present invention results from the purification of polypeptides participating in the biosynthesis of streptogramins, as well as from the cloning of genes whose product participates in the biosynthesis of streptogramins. The term biosynthesis of streptogramins is understood to comprise the regulatory genes and the genes conferring resistance on the producing microorganisms. Thus, the present invention makes it possible to increase the levels of production of these metabolites by means of recombinant DNA techniques. Another benefit of the present invention lies in the possibility, by construction of mutants blocked in the different steps of this biosynthesis, of producing synthesis intermediates for each of the two components. These intermediates may serve as substrates for further modification for chemical, biochemical, enzymatic or microbiological means. Similarly, isolation of the biosynthesis genes makes it possible, by gene transfer between producing strains, to manufacture hybrid antibiotics having pharmacologically advantageous properties (Hopwood et al., 1985a, Hopwood et al., 1985b, Hutchinson et al. 1989). Another benefit of the present invention lies in the fact that it provides a better knowledge of the biosynthesis pathways of the metabolites classed as streptogramins. In effect, the invention enables bacterial or fungal strains to be constructed in which one or more proteins participating in the biosynthesis of streptogramins is/are expressed under the control of suitable expression signals. Such strains may then be used to carry out bioconversions. These bioconversions may be carried out either using whole cells, or using acellular extracts of the said cells. These bioconversions may enable a streptogramin to be converted to a derived form with an enzyme of a biosynthesis pathway. For example, pristinamycin IIB may be converted in this manner to pristinamycin IIA. The same reasoning may be applied to any biosynthesis intermediate.
A first subject of the invention hence relates to a nucleotide sequence coding for a polypeptide involved in the biosynthesis of streptogramins.
More especially, several genes whose product participates in the biosynthesis of streptogramins have been isolated from Streptomyces pristinaespiralis. Since the streptogramins produced by this strain are more commonly designated by the term pristinamycins (see Table 1), in what follows, reference will be made in some cases to genes for the biosynthesis of pristinamycins. However, it is clear that the results obtained apply to all the streptogramins. Pristinamycins I and II correspond, respectively, to the B and A components of streptogramins. Molecules of the pristinamycin II family and of the pristinamycin I family hence designate in what follows the A and B components of streptogramins, respectively.
The present invention describes in particular the isolation and characterization of the snaA, snaB, snaC, snaD, papA, papM, samS, snbA, snbC, snbD, snbE and snbR genes. These genes were isolated from a library of genomic DNA of S. pristinaespiralis. This library was obtained by partial digestion of genomic DNA S. pristinaespiralis with the restriction enzyme Sau3A. Large DNA fragments, from 40 to 50 kb on average, were cloned into cosmid pHC79 (Hohn, B., and Collins, J. F., 1980). After in vitro encapsidation, E. coli strains HB101 (Boyer et Roulland-Dussoix, 1969) and DH1 (Low, 1968) were transfected. The DNA library of S. pristinaespiralis thus occurs in two different strains of E. coli.
The snaA, snaB and samS (initially designated SnaC genes are present on cosmid pIBV1 (FIG. 4). The product of the snaA and snaB genes, corresponding to the polypeptides SnaA and SnaB, participates in the final step of biosynthesis of the II component of pristinamycins (conversion of pristinamycin IIB to pristinamycin IIA), corresponding to the oxidation of the 2,3 bond of D-proline. These two polypeptides constitute the two subunits of pristinamycin IIA synthase, the purification of which is described in the present invention. The product of the samS gene is considered to participate in the synthesis of SAM (methyl group donor) from ATP and methionine. The A component of most streptogramins is, in effect, methylated at C-4 (FIG. 1), and this methyl has been described (Kingston et al., 1983) as being derived from the methyl of methionine, very probably via a methylation reaction with SAM. The samS gene is hence considered to code for a SAM synthase (SamS; EC. 2.5.1.6) which is specific to the biosynthesis pathway of pristinamycins.
The snbA, snbR, papA and papM genes are present on cosmid pIBV2 (FIG. 5). The snbA gene corresponds, on the basis of the biochemical studies presented in Example 5, to the first step for synthesis of pristinamycins I. This comprises activation of the first acid of the chain, 3-hydroxypicolinic acid, by adenylation. The snbR gene might participate in the transport of molecules of the pristinamycin I (or possibly pristinamycin II) family out of the cell after synthesis, thereby conferring a resistance to this component on the producing strain. The papA gene corresponds, on the basis of sequence analyses (Example 8.8) and the study of a mutant disrupted in this gene (Example 9.3), to a gene for the biosynthesis of para-aminophenylalanine from chorismate. para-Aminophenylalanine is then dimethylated by the product of the papM gene, an N-methyltransferase described in the present invention, to form para-dimethylaminophenylalanine, which is then incorporated in pristinamycin IA. The papA and papM genes hence participate in the synthesis of one of the precursors of pristinamycin IA.
The snaA, snaD, snbC, snbD and snbE genes are present on cosmid pIBV3 (FIG. 6), which hence adjoins cosmid pIBV1 on which the snaA gene is already present. The snaD gene codes, on the basis of analysis of its sequence (Example 8.9) and the study of a mutant disrupted in this gene (Example 9.5), for a peptide synthase involved in the biosynthesis of pristinamycin II. The snbC gene, whose product is described in the present invention, participates in the incorporation of threonine and aminobutyric acid residues in the peptide chain of pristinamycin IA. The snbD gene, whose product is also described in the present invention, is involved in the incorporation of proline and para-dimethylaminophenylalanine residues in the peptide chain of pristinamycin IA. It also governs the N-methylation of the peptide bond between these 2 residues. Lastly, the snbE gene, whose product is also described in the present invention, participates in the incorporation of the last two residues of pristinamycin IA, namely phenylglycine and 4-oxopipecolic acid.
The snaC gene is present on cosmid pIBV4 (FIG. 7). It codes for an FMN:NADH oxidoreductase, also designated FMN reductase, described in the present invention and which supplies pristinamycin IIA synthase with FMNH.sub.2 from FMN and NADH. The snaC gene hence participates in the final step of the biosynthesis of pristinamycin IIA.
These different genes were subcloned from their cosmid of origin and their nucleic acid sequences were determined. The snaA, snaB and samS genes were subcloned on a 6-kb BamHI-BamHI fragment, a portion of which was sequenced (SEQ ID no. 1). The snbA gene was subcloned in a 5.5-kb EcoRI-BglII fragment, a portion of which was sequenced (SEQ ID no. 5). The snbR gene was subcloned in a 4.6-kb BglII-BglII fragment, a portion of which was sequenced (SEQ ID no. 6). A portion of the papA gene was subcloned in a 3.4-kb XhoI-XhoI fragment, a portion of which was sequenced (SEQ ID no. 9). The papM gene was subcloned in a 4.1-kb PstI-PstI fragment, a portion of which was sequenced (SEQ ID no. 10). A portion of the snaD gene was subcloned in a 1.5-kb BamHI-SstI fragment, a portion of which was sequenced (SEQ ID no. 8). A portion of the snbC gene was subcloned on a 6.2-kb SphI-SphI fragment, 2 regions of which were sequences (SEQ ID nos. 11 and 12). A portion of the snbD gene was subcloned on an 8.4-kb SphI-SphI fragment, 2 regions of which were sequenced (SEQ ID Nos. 13 and 14). A portion of the snbE gene was subcloned on a 6.6-kb SphI-SphI fragment, 2 regions of which were sequenced (SEQ ID Nos. 15 and 16). The snaC gene was subcloned in a 4-kb BamHI-BamHI fragment, a portion of which was sequenced (SEQ ID no. 7).
The proximity of the snaA, snaB, snaD, samS, snbC, snbD and snbE genes on the one hand, as well as the snbA, snbR, papA and papM genes, confirms the cluster localization of the genes for biosynthesis of the A and B components of streptogramins. Furthermore, the 4 cosmids described in the present invention are grouped together in a region of the chromosome whose size is estimated at 200 kb by pulsed-field electrophoresis, equivalent to 3% of the total genome (7500 kb) of Streptomyces pristinaespiralis (Example 13). It is hence obvious that the regions surrounding the genes identified in the present invention (snaA, snaB, snaD, samS, snbC, snbD and snbE; snbA, snbR, papA and papM; snaC) contain the other genes of the pristinamycin biosynthesis cluster, and that these genes may be used to localize the other genes for the biosynthesis of streptogramins.
Preferably, the subject of the invention is a nucleotide sequence chosen from:
(a) all or part of the snaA (SEQ ID no. 2), snaB (SEQ ID no. 3), snaC (SEQ ID no. 7), snaD (SEQ ID no. 8), papA (SEQ ID no. 9), papM (SEQ ID no. 10), samS (SEQ ID no. 4), snbA (SEQ ID no. 5), snbC (SEQ ID nos. 11 and 12), snbD (SEQ ID nos. 13 and 14), snbE (SEQ ID nos. 15 and 16) and snbR (SEQ ID no. 6) genes,
(b) the sequences adjacent to the genes (a) constituting the biosynthesis clusters and coding for the polypeptides involved in the biosynthesis of streptogramins,
(c) the sequences which hybridize with all or part of the genes (a) or (b) and which code for a polypeptide involved in the biosynthesis of streptogramins, and
(d) the sequences derived from the sequences (a), (b) and (c) owing to the degeneracy of the genetic code.
Still more preferably, the subject of the invention is the nucleotide sequences represented by the snaA (SEQ ID no. 2), snaB (SEQ ID no. 3), snaC (SEQ ID no. 7), snaD (SEQ ID no. 8), papA (SEQ ID no. 9), papM (SEQ ID no. 10), samS (SEQ ID no. 4), snbA (SEQ ID no. 5), snbC (SEQ ID nos. 11 and 12), snbD (SEQ ID nos. 13 and 14), snbE (SEQ ID nos. 15 and 16) and snbR (SEQ ID no. 6) genes.
Another subject of the invention relates to any recombinant DNA comprising a gene for the biosynthesis of streptogramins. More preferably, this is a recombinant DNA comprising all or part of cosmids pIBV1, pIBV2, pIBV3 or pIBV4 as shown in FIGS. 4 to 7, or all or part of sequences which hybridize with cosmids pIBV1 to pIBV4 or with fragments of these latter.
In a preferred embodiment of the invention, the nucleotide sequences defined above form part of an expression vector, which can be autonomously replicating or integrative.
As stated above, although the invention is more especially illustrated with the genes for the biosynthesis of pristinamycin, it is clear that the results obtained apply to all streptogramins.
More especially, the techniques developed in the present invention for purifying proteins or cloning genes for the biosynthesis of streptogramins from S. pristinaespiralis may be applied to other microorganisms producing streptogramins (see Table 1).
Thus, the purification of an enzymatic activity from S. pristinaespiralis makes it possible to purify the same activity from another strain producing streptogramin. The present invention may hence by applied to the cloning of genes for the biosynthesis of streptogramins from any producing microorganism, by purification of a protein participating in the biosynthesis and then, using the NH.sub.2 -terminal sequence thereof, synthesis of an oligonucleotide probe which enables the corresponding gene to be cloned. Chromosome walking then enables the whole biosynthesis cluster to be identified.
Furthermore, from the genes identified in the present application, it is possible, by hybridization, to clone the genes for the biosynthesis of streptogramins directly from the DNA of another producing microorganism. In effect, the genes for the biosynthesis of pristinamycins hybridize strongly with those for the other streptogramins. It is thus possible to clone, by hybridization, the genes for the biosynthesis of streptogramins using as a probe the sna, snb or pap genes, or fragments of the latter, or fragments adjacent to these containing, as is shown in the present invention, other sna and snb genes. This is due to the fact that: 1) the streptogramins produced by the different microorganisms have identical or similar structures (see FIG. 3), 2) the genes for the biosynthesis of streptogramins are organized in clusters, and 3) the enzyme systems responsible for this biosynthesis do not have an absolute specificity for their substrates.
Moreover, the cloning of genes involved in the biosynthesis of streptogramins may also be carried out using degenerate oligonucleotides, prepared from the sequences of the sna or snb genes mentioned above, or fragments of these genes, or fragments adjacent to these genes. It is thus possible to take one's pick of the genes for the biosynthesis of the A and B components of the different strains producing streptogramins. These strains can belong to the genus Streptomyces, and also to other genera (see Table 1). In addition, if the genomic DNA of the starting strains used has a G+C composition different from that observed in Streptomyces, the probes used may be synthesized with a codon bias specific to the genus or species from which it is desired to isolate the DNA.
Another subject of the present invention relates to the polypeptides resulting from the expression of the nucleotide sequences defined above. More especially, the present invention relates to polypeptides comprising all or part of the polypeptides SnaA (SEQ ID NO: 18), SnaB (SEQ ID NO: 19), SnaC (SEQ ID NO: 23), SnaD (SEQ ID NO: 24), PapA (SEQ ID NO: 25), PapM (SEQ ID NO: 26), SamS (SEQ ID NO: 20), SnbA (SEQ ID NO: 21), SnbC (SEQ ID NO: 27 and SEQ ID NO: 28), SnbD (SEQ ID NO: 29 and SEQ ID NO: 30), SnbE (SEQ ID NO: 31 and SEQ ID NO: 30) and SnbR (SEQ ID NO: 22) or of derivatives of these. Within the meaning used in the present invention, the term derivative denotes any molecule obtained by modification of a genetic and/or chemical nature of the peptide sequence. Modification of a genetic and/or chemical nature is understood to mean any mutation, substitution, deletion, addition and/or modification of one or more residues. Such derivatives may be generated for different purposes, such as, in particular, that of increasing the affinity of the peptide for its substrate(s), that of improving its levels of production, that of increasing its resistance to proteases, that of increasing and/or modifying its activity, or that of endowing it with novel biological properties. Among derivatives resulting from an addition, there may be mentioned, for example, chimeric polypeptides containing an additional heterologous portion attached to one end. The term derivative also comprises polypeptides homologous to the polypeptides described in the present invention and originating from other cell sources, and in particular from strains producing streptogramins.
The subject of the invention is also any recombinant cell containing a nucleotide sequence or a vector as defined above. The recombinant cells according to the invention can equally well be eukaryotic cells or prokaryotic cells. Among eukaryotic cells which are suitable, animal cells, yeasts or fungi may be mentioned. In particular, as regards yeasts, yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces or Hansenula may be mentioned. As regards animal cells, COS, CHO, C127 cells, Xenopus eggs, and the like, may be mentioned. Among fungi, special mention may be made of Micromonospora, Aspergillus ssp. or Trichoderma ssp. As prokaryotic cells, it is preferable to use the following bacteria: Actinomycetes, and Streptomyces in particular, E. coli (Example 11), Bacillus. Preferably, the recombinant cells of the invention are chosen from cells producing streptogramins (see Table 1). The recombinant cells of the invention may be obtained by any method which enables a foreign nucleotide sequence to be introduced into a cell. It can be, in particular, transformation, electroporation, conjugation, protoplast fusion or any other technique known to a person skilled in the art.
A further subject of the invention is a method for producing a polypeptide involved in the biosynthesis of streptogramins, according to which a recombinant cell as defined above is cultured and the polypeptide produced is recovered.
The subject of the invention is also the use of a recombinant cell as defined above, expressing at least one polypeptide involved in the biosynthesis of streptogramins, in a bioconversion reaction. In particular, these cells can enable a streptogramin to be converted into a derived form. For example, pristinamycin IIB can be converted in this manner to pristinamycin IIA. The same reasoning may be applied to any biosynthesis intermediate. These cells can also enable hybrid antibiotics having advantageous pharmacological properties to be manufactured (Hopwood et al. 1985a, Hopwood et al. 1985b, Hutchinson et al. 1989). These bioconversions may be carried out either using whole cells, or using acellular extracts of the said cells.
Another subject of the invention relates to the use of a nucleotide sequence as defined above for amplifying streptogramin production. The invention also relates to a method for producing streptogramins, according to which one or more nucleotide sequences according to the invention is/are introduced and/or amplified in a cell producing streptogramins or which is potentially a producer of streptogramins, the said cell is cultured under conditions of streptogramin production, and the streptogramins produced are recovered.
The overexpression of certain genes involved in the biosynthesis can enable the streptogramin A and/or B production of the producing strains to be increased. This overproduction may be carried out in several strains: either strains which produce only molecules of the streptogramin A family, or strains which produce only molecules of the streptogramin B family, or strains which produce both the A and B components. These overexpressions can result from an increase in the level of synthesis, and hence in the productivity, of the A and/or B components, either in an Erlenmeyer, or in small fermenters, or in large industrial fermenters. Moreover, the specific overexpression of a gene involved in the biosynthesis of an A or B component also makes it possible to vary the % of A and B components produced by the strain, and thus to obtain a better synergy between these molecules. In addition, the biosynthesis genes isolated from a microorganism producing streptogramins may be used to amplify production in another producing microorganism.
Another subject of the invention relates to a method for preparing cells blocked in a step of the pathway of biosynthesis of streptogramins, according to which a mutagenesis is performed on at least one gene of the biosynthesis pathway, on a cell producing streptogramins.
Preferably, the mutagenesis is performed in vitro or in situ, by suppression, substitution, deletion and/or addition of one or more bases in the gene in question, or by gene disruption.
Another aspect of the present invention lies, in effect, in the construction of mutants blocked in certain steps of biosynthesis of streptogramins. The value lies, on the one hand in the study of the functionality of the mutated proteins, and on the other hand in the production of strains producing biosynthesis intermediates. These intermediates may be modified, where appropriate after separation, either by adding particular components to the production media, or by introducing into the strains thus mutated other genes capable of modifying the intermediate by acting as a substrate for them. These intermediates may thus be modified by chemical, biochemical, enzymatic and/or microbiological means. In this context, the mutant SP92::pVRC505 of S. pristinaespiralis strain SP92 was constructed: S. pristinaespiralis SP92::pVRC505 was isolated by homologous integration in the snaA gene of a suicide plasmid pVRC505, constructed from the vector pDH5 and a fragment internal to the snaA gene. The following mutants were also constructed: SP92 samS::.OMEGA.amR; SP92::pVRC508; SP92::pVRC404 and SP92::pVRC1000 (Example 9).
The invention hence also relates to a method for preparing an intermediate of the biosynthesis of streptogramins, according to which:
a cell blocked in a step of the pathway of biosynthesis of streptogramins is prepared as described above,
the said cell is cultured, and
the accumulated intermediate is recovered.
The invention also relates to a method for preparing a molecule derived from streptogramins, according to which:
a cell blocked in a step of the pathway of biosynthesis of streptogramins is prepared as described above,
the said cell is cultured, and
the intermediate accumulated by this cell is modified, where appropriate after separation of the culture medium.
The present invention is illustrated by means of the examples which follow, which are to be considered as illustrative and non-limiting.





LIST OF FIGURES
FIG. 1: Example of structure of the A components of streptogramins.
FIG. 2: Example of structure of the B components of streptogramins.
FIG. 3: Other examples of structures of streptogramins.
FIG. 4: Diagram of cosmid pIBV1.
FIG. 5: Diagram of cosmid pIBV2.
FIG. 6: Diagram of cosmid pIBV3.
FIG. 7: Diagram of cosmid pIBV4.
FIG. 8: Reaction catalysed by pristinamycin IIA synthase.
FIG. 9: Reaction catalysed by 3-hydroxypicolinic acid:AMP ligase.
FIG. 10: Reaction catalysed by SnbC.
FIG. 11: Reaction catalysed by SnbD.
FIG. 12: Reaction catalysed by SnbE.
FIG. 13: Reaction catalysed by SnaC.
FIG. 14: Reaction catalysed by PapM.
FIGS. 15A and 15B: Diagram of plasmids pVRC402 (A) and pVRC501 (B).
FIG. 16: Diagram of plasmid pXL2045.
FIG. 17: Diagram of plasmid pVRC1105.
FIG. 18: Diagram of plasmid pVRC1106.
FIG. 19: Diagram of plasmid pVRC1104.
FIG. 20: Diagram of plasmid pVRC900.
FIG. 21: Diagram of plasmid pVRC1000.
FIG. 22: Diagram of plasmid pVRC509.
FIG. 23: Diagram of plasmid pVRC903.
FIG. 24: Diagram of plasmid pVRC409.
FIG. 25: Diagram of plasmid pVRC505.
FIG. 26: Diagram of plasmid pVRC701.
FIG. 27: Diagram of plasmid pVRC702.
FIG. 28: Diagram of plasmid pVRC508.
FIG. 29: Diagram of plasmid pVRC404.
FIG. 30: Diagram of plasmid pVRC507.
FIG. 31: Diagram of plasmid pVRC706.
FIG. 32: General map.
FIG. 33: Fragment of ADNg from S. virginae inserted in pIBV30
FIG. 34: Diagram of plasmid pVRC510.





MATERIALS
Bio-Sil SEC 125 and 250 columns (Bio-Rad)
MonoQ HR 5/5, 10/10 and 16/10 columns (Pharmacia)
PD-10 column (Pharmacia)
Superose 6 HR 10/30 column (Pharmacia)
Superdex 200 Hi-Load 16/60 and 75 HR 10/30 column (Pharmacia)
Superose 12 prep grade column (Pharmacia)
Vydac C4 and C18 columns (The Separations Group)
Nucleosil 5-C18 column (Macherey-Nagel)
Phenyl Superose HR 10/10 column (Pharmacia)
TSK G2000 SW column (Tosoh, Japan)
Phenyl Sepharose (Pharmacia)
FMN-agarose (Sigma)
Q Sepharose Fast Flow (Pharmacia)
Sephadex G-25 Fine (Pharmacia)
Centricon 10 or 30 (Amicon)
Centriprep 10 or 30 (Amicon)
Centrilutor (Amicon)
EXAMPLE 1
Isolation of Total DNA of Streptomyces pristinaespiralis Strain SP92
This example illustrates how S. pristinaespiralis SP92 DNA may be purified.
S. pristinaespiralis strain SP92 is derived from S. pristinaespiralis strain DS5647 (ATCC25486).
50 ml of YEME medium (34% sucrose, 5 mM MgCl.sub.2, 0.25% glycine (D. Hopwood et al. 1985)) are inoculated with 10.sup.8 S. pristinaespiralis SP92 spores, and the culture is incubated for 40 hours at 30.degree. C. with stirring at 280 rpm.
The mycelium is harvested and washed with 15 ml of 10.3% sucrose. Approximately 1 g of the mycelium pellet is taken up with 5 ml of TE supplemented with 34% of sucrose, to which are added 1 ml of lysozyme at a concentration of 50 mg/ml in 10 mM Tris-HCl solution pH 8.0 and 1 ml of 0.25 M EDTA pH 8.0. After incubation at 30.degree. C. for a period of 30 to 60 min, the mixture is clarified by adding 0.8 ml of 10% sarkosyl. 2 ml of 0.25 M EDTA pH 8.0, 10 ml of TE, 18 g of CsCl and 1.2 ml of ETB at a concentration 10 mg/ml are then added. The preparation is ultracentrifuged overnight at 55,000 rpm at 20.degree. C.
The chromosomal DNA, present in the CsCl gradient in the form of a band, is recovered using a Pasteur pipette. The ETB is removed by several washes with a solution of isopropanol saturated with TE buffer, 5 M NaCl. The DNA is precipitated by adding 3 volumes of TE and 4 volumes of isopropanol. After washing with 70% ethanol, the DNA is taken up in a suitable volume of TE. The total amount of DNA obtained varies between 250 and 500 .mu.g per g of mycelium.
EXAMPLE 2
Isolation of E. coli Plasmid DNA
This example illustrates how E. coli plasmid DNA is prepared from recombinant strains of E. coli.
2.1. Preparation of E.coli Plasmid DNA in Large Amounts
This example illustrates how maxi preparations of plasmid DNA are produced in E. coli.
This preparation is performed using a 500 ml culture in LB medium containing 150 .mu.g/ml of ampicillin. The extraction protocol is derived from the methods described by Birnboim and Doly (1979) and Ish-Horowicz and Burke (1981), and is described in Maniatis et al. (1989).
After this extraction, the plasmid DNA is purified using a CsCl gradient as described by Maniatis et al. (1989). The plasmid DNA is then precipitated by adding 3 volumes of TE and 4 volumes of isopropanol. After centrifugation, the pellet is taken up in 0.5 to 1 ml of TE.
2.2. Preparation of E. coli Plasmid DNA in Small Amounts
This example illustrates how minipreparations of plasmid DNA are produced in E. coli.
This preparation is carried out using 1.5 ml of culture in LB medium containing 150 .mu.g/ml of ampicillin. The procedure is that described by Birnboim and Doly (1979).
EXAMPLE 3
Construction of the Genomic DNA Library of S. pristinaespiralis SP92 in E. coli and Preparation of Hybridization Membranes
This example illustrates how a genomic DNA library of S. pristinaespiralis SP92 is produced in E. coli.
3.1. Preparation of Genomic DNA Fragments
This example illustrates how high molecular weight genomic DNA fragments may be prepared.
Total DNA of the strain SP92, prepared as described in Example 1, is partially digested with Sau3A (New England Biolabs, Beverly, Mass. 01915-5510 USA) in the buffer recommended by the supplier: 100 mM NaCl, 10 mM Tris-HCl (pH7.5), 10 mM MgCl.sub.2, 100 .mu.g/ml BSA. The amount of enzyme used to obtain high molecular weight DNA fragments was determined empirically. Approximately 0.025 enzyme units are used to digest 1 .mu.g of total DNA for 20 min at 37.degree. C. The reaction is then stopped by incubation for 15 min at 65.degree. C., and the enzyme is removed by adding an equal volume of phenol/chloroform. After centrifugation, the supernatant containing the partially digested total DNA is precipitated by adding 0.3 M final sodium acetate and 2.5 volumes of ethanol.
Approximately 100 .mu.g of total DNA are digested in this way, and DNA fragments between 30 and 50 kb in size are isolated with a 10-40% sucrose gradient. Their size is verified by electrophoresis on 0.4% agarose gel.
3.2. Preparation of Cosmid pHC79
This example illustrates how cosmid pHC79 is prepared from E. coli.
Cosmid pHC79 (Hohn, B. and Collins, 1980) comprises a portion of pBR322 (Bolivar, F. et al., 1977), the cro-cII region of .lambda. and the region containing the cos sequence of Charon 4A (Blattner, F. R. et al., 1977).
Extraction of the cosmid was carried out as described in Example 2.1., from an E. coli strain TGl (K12, .DELTA.(lac-pro) supE thi hsd DS F' traD36 proA.sup.+ B.sup.+ lacIq LacZ .DELTA.M15, Gibson, 1984).
500 ng of cosmid pHC79 are digested with BamHI (New England Biolabs, Beverly, Mass. 01915-5510 USA) in 20 .mu.l of buffer comprising 150 mM NaCl, 6 mM Tris-HCl pH 7.9, 6 mM MgCl.sub.2, 6 mM 2-mercaptoethanol, 100 .mu.g/ml BSA.
3.3. Ligation of the DNA Fragments and the Cosmid
This example illustrates how the fragments of the S. pristinaespiralis SP92 genome originating from an Sau3A digestion may be ligated with the BamHI-linearized vector pHC79.
Approximately 150 ng of cosmid linearized as described above were precipitated by means of ethanol with 350 ng of fragments of total DNA of S. pristinaespiralis SP92 prepared as described in Example 3.2. The pellet was taken up in 10 .mu.l of ligation buffer: 50 mM Tris-HCl pH 7.8, 10 mM MgCl.sub.2, 20 mM DTT, 1 Mm ATP, 50 .mu.g/ml of BSA, and 0.5 .mu.l of T4 DNA ligase at a concentration of 400,000 units per ml (New England Biolabs, Beverly, Mass. 01915-5510 USA) were added. Incubation was carried out overnight at 15.degree. C.
3.4. Carrying Out Encapsidation In Vitro
This example illustrates how the cosmids constructed in 3.3 are encapsidated in vitro.
Encapsidation of the hybrid cosmids after ligation was carried out using the Gigapack II Gold kit developed by Stratagene (Stratagene Cloning Systems, La Jolla, Calif. 92037, USA).
2.times.4 .mu.l of ligation mixture, equivalent to 2.times.70 ng of hybrid cosmids, were encapsidated in vitro according to the procedure described by the supplier.
3.5. Transfection of E. coli Strains DH1 and HB101
This example illustrates how the cosmids are introduced into E. coli.
Two transfections were carried out in parallel with E. coli strains DH1 (F.sup.- gyrA96 recA1 relA1 endA1 thi-1 hsdR17 supE44L-, Low 1968) and HB101 (F.sup.- supE44 hsdS20(rB.sup.- mB.sup.-) recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1, Boyer and Roulland-Dussoix 1969).
The cells were prepared according to the following protocol: a 100-ml preculture is produced in LB medium supplemented with 0.2% maltose and 10 mM MgSO.sub.4 for 4 to 5 hours until the OD.sub.600 reaches a value of 0.8. The culture is then centrifuged, and the pellet is taken up in 40 ml of 10 mM MgSO.sub.4 and diluted to OD.sub.600 =0.5 in the same solution. 200 .mu.l of the cell suspension thus prepared are mixed with 100 .mu.l of encapsidation mixture. After 20 min of contact at 37.degree. C., 1 ml of LB is added and the whole is incubated for 1 hour at 37.degree. C. The transfectants are then selected on solid LB medium containing 150 .mu.g/ml of ampicillin. The number of transfectants obtained is approximately 10.sup.4 per .mu.g of recombinant cosmid.
3.6. Storage of Genomic DNA Libraries of S. pristinaespiralis SP92
This example illustrates how the genomic DNA libraries of S. pristinaespiralis SP92 are stored.
After verification of the average size of the fragments inserted into cosmid pHC79, approximately 1500 colonies originating from each of the transfections carried out with the strains HB101 and DH1 are subcultured in 96-well microtitration plates containing 200 .mu.l of Hogness medium (LB medium supplemented with 8.8% glycerol, 3 mM sodium acetate, 55 mM K.sub.2 HPO.sub.4, 26 mM KH.sub.2 PO.sub.4, 1 mM MgSO.sub.4, 15 mM (NH.sub.4).sub.2 SO.sub.4, 150 .mu.g/ml ampicillin). These plates are incubated overnight at 37.degree. C. and then stored at -80.degree. C.
3.7. Preparation of Hybridization Membranes From Genomic Libraries of S. pristinaespiralis SP92
This example illustrates how the DNA of the colonies constituting the genomic libraries of S. pristinaespiralis SP92 is transferred onto a hybridization membrane.
These hybridization membranes were produced in duplicate for each of the 2 libraries according to the following protocol:
The 15 microtitration plates of each library are replicated using a replica plater on LB agar medium containing 150 .mu.g/ml of ampicillin. After growth overnight at 37.degree. C., colony transfer is performed onto a Biohylon Z.sup.+ membrane (Bioprope System) according to the following protocol: the membrane is cut to the appropriate size and left in contact with the colonies for 1 min. Denaturation is then performed by soaking the membrane with 0.5 M NaOH, 1.5 M NaCl solution for 5 min, followed by neutralization by soaking the membrane in 3 M sodium acetate solution for 5 min. The DNA is fixed to the membrane by exposure under a UV lamp for 5 min.
EXAMPLE 4
4.1. Preparation of Chromosomal DNA of S. pristinaespiralis Strain SP92 and Strains Derived From SP92 in the Form of Inserts For Pulsed-field Electrophoresis
This example illustrates how DNA of S. pristinaespiralis strain SP92 and strains derived from SP92 is prepared in the form of inserts for pulsed-field electrophoresis.
This preparation is made from a mycelium culture obtained in the following manner: 30 ml of YEME medium containing 0.25% of glycine are inoculated with 10.sup.8 spores of the strain under study, and the culture is incubated for 48 hours at 30.degree. C. and stirred at 280 rpm in 250-ml Erlenmeyers. The mycelium is then harvested by centrifugation for 10 min at 3800 rpm and washed twice with 10% sucrose. The mycelium pellet is then resuspended in 5 ml of solution I (250 mM EDTA pH 8.0, 20.6% sucrose). To 200 Ml of mycelium thereby obtained, 400 Ml of a lysozyme solution at a concentration of 50 mg/ml in solution I together with 800 Ml of 1% LMP agarose in 25 mM EDTA pH 8 and 10.3% sucrose, maintained at 42.degree. C., are added. The mixture maintained at 42.degree. C. is then poured into the wells of special combs, which are closed with adhesive tape and kept for 30 min at 4.degree. C. The mixture solidifies, and the 30 to 40 inserts thereby obtained and contained in the wells are carefully removed from the moulds.
The inserts are first rinsed for 30 min at 4.degree. C. in a solution containing 25 mM EDTA and 10.3% sucrose. They are then soaked in a solution of 500 mM EDTA, 1% lauryl sarcosyl and 1 mg/ml of proteinase K for twice 24 hours at 50.degree. C., stirring from time to time. The inserts are then washed for 3 times one hour in TE containing 1 mM PMSF, changing the solution after each wash. The inserts thereby obtained are stored at 4.degree. C. for not more than 4 months in 0.5 M EDTA pH 8.0.
4.2. Digestion of Inserts of DNA of S. pristinaespiralis Strain SP92 and Strains Derived From SP92 and Analysis by Pulsed-field Electrophoresis
This example illustrates how chromosomal DNA of S. pristinaespiralis strain SP92 and strains derived from SP92, prepared in the form of inserts as described in Example 4.1., is cut with different restriction enzymes for pulsed-field electrophoresis.
4.2.1. Digestion of Chromosomal DNA in the Form of Inserts
The inserts are first washed six times in TE, and then incubated twice for one hour in the buffer of the chosen restriction enzyme. Each insert is then placed in the lid of an Eppendorf tube containing 160 Ml of buffer of the restriction enzyme and 40 units of enzyme. The whole is covered with Parafilm, and the Eppendorf is closed to hold in place the Parafilm which enables any evaporation of the buffer to be avoided. The tubes are incubated at the desired temperature in an incubator overnight.
4.2.2. Analysis of Digested DNA by Pulsed-field Electrophoresis
The pulsed-field electrophoresis technique chosen for this study is that of the CHEF (Clamped Homogenous Electric Field) system developed by Chu et al. (1986), which makes it possible to obtain two homogeneous alternating fields oriented at 120.degree. with respect to one another and linear trajectories for the DNA molecules. The apparatus used is the "Pulsafor System" marketed by Pharmacia-LKB.
The electrophoretic migration paratmeters, such as the pulse time and the migration period, were varied so as to obtain an optimal separation of DNA fragments ranging in size between 10 and 2500 kb. The three migration conditions used are as follows: to separate large fragments from 200 to 1700 kb in size, the chosen migration is 40 hours with a pulse time of 90 seconds; to separate fragments from 50 to 400 kb in size, the chosen migration is 20 hours with a pulse time of 10 seconds followed by 20 hours with a pulse time of 30 seconds; lastly, to separate smaller fragments from 10 kb to 200 kb in size, the chosen migration is 24 hours with a pulse time of 10 seconds. For these three migration conditions, the voltage is set at a constant 150 volts, the temperature is maintained at 13.degree. C. and the electrophoresis gels contain 1.3% of agarose.
The inserts containing chromosomal DNA of S. pristinaespiralis strain SP92 and strains derived from SP92 are digested with the restriction enzymes as described above and are placed in the wells of the electrophoresis gel using two scalpel blades. The molecular weight markers used are "Yeast chromosome PFG marker" and "Lambda Ladder PFG marker" marketed by the company New England Biolabs. Migration is performed under one of the conditions described above and the gel is then stained in a bath of ETB (ethidium bromide) at a concentration of 4 Mg/ml for 20 min and thereafter decolorized in water for 20 min. After the gel is photographed, the DNA fragments are transferred onto a nylon membrane and then hybridized with [.alpha.-.sup.32 P]dCTP-labelled probes as described in Example 9.1.
EXAMPLE 5
Isolation of Cosmids Carrying the Genes Coding For Purified Proteins Involved in the Biosynthesis of Streptogramins
This example describes how, starting from a purified protein participating in biosynthesis of pristinamycins and whose NH.sub.2 -terminal sequence or an internal sequence has been established, it is possible to isolate a cosmid carrying the structural gene for this same protein from the genomic libraries produced above, or alternatively to identify the corresponding structural gene from among the genes carried by the cosmids and which have already been sequenced.
5.1. Isolation of Cosmids pIBV1 and pIBV3 Carrying One or Both Structural Genes For the Two Subunits of Pristinamycin IIA Synthase
5.1.1. Identification and Purification of One of the Proteins Involved in the Final Step of the Synthesis of Pristinamycins II: Pristinamycin IIA Synthase
As stated in the introduction, the final step of synthesis of pristinamycin IIA corresponds to an oxidation of the 2,3 bond of D-proline to dehydroproline. The protein responsible for this activity has been purified to homogeneity, as illustrated by this example.
5.1.1.A. Assay of Pristinamycin IIA Synthase Activity
This example illustrates the assay of an activity of the biosynthesis pathway of pristinamycin IIA which has never before been described and which possesses the noteworthy property of being expressed only during the period of production of pristinamycins. The enzyme in question is pristinamycin IIA synthase, which catalyses the conversion of pristinamycin IIB to pristinamycin IIA by oxidation of the D-proline residue of pristinamycin IIB to a 2,3-dehydroproline residue (FIG. 8) in the presence of molecular oxygen and FMNH.sub.2. The enzyme fractions to be assayed (0.002 to 0.005 units) are incubated for 1 h at 27.degree. C. in a total volume of 500 ml of 50 mM bis-tris propane buffer pH 6.8 containing NADH (500 .mu.M), FMN (5 .mu.M), pristinamycin IIB (20 .mu.M) and 0.02 units of FMN reductase (Boehringer Mannheim).
The pristinamycin IIA formed is assayed by HPLC after incubation is stopped by adding 500 .mu.l of 0.1 N hydrochloric acid and 500 .mu.of acetonitrile and centrifugation of the sample for 5 min at 5000 g. 150 .mu.l of the centrifugation supernatant are injected onto a 15-cm Nucleosil 5-C8 column eluted with a mixture of 34% of acetonitrile and 66% of 0.1 M phosphate buffer pH 2.9. Pristinamycins IIA and IIB are detected by means of their UV absorbance at 206 nm.
The unit of enzymatic activity is defined as the amount of enzyme needed to synthesize 1 .mu.mol of pristinamycin IIA per hour under the conditions described.
5.1.1.B. Purification of S. pristinaespiralis SP92 Pristinamycin IIA Synthase
This experiment illustrates how an enzyme of S. pristinaespiralis SP92 participating in the biosynthesis pathway of pristinamycin IIA may be purified.
Using the assay described above in Example 5.1.1.A, the purification of pristinamycin IIA synthase is carried out as described below taking care to freeze and store the active fractions at -30.degree. C. between successive steps if necessary.
150 g of a centrifugation pellet, washed with 0.1 M phosphate buffer pH 7.2 containing 10% v/v of glycerol, of an S. pristinaespiralis SP92 culture harvested at the beginning of the pristinamycin production phase are taken up with 450 ml of 50 mM bis-tris propane buffer pH 6.8 containing 5 mM DTT and 0.2 mg/ml of lysozyme. The suspension thereby obtained is incubated for 45 minutes at 27.degree. C. and then centrifuged at 50,000 g for 1 hour. The crude extract thereby collected is fractionated by ammonium sulphate precipitation. The protein fraction precipitating at between 40 and 55% saturation is desalted on a column of Sephadex G-25 Fine, and then injected (100 mg per injection) in pH 6.8 50 mM bis-tris propane buffer, 1 mM DTT onto a monoQ HR 10/10 column. The proteins are eluted with a linear KCl gradient (0 to 0.5 M). The fractions containing the enzymatic activity (detected by means of the test described in Example 5.1.1.A) are pooled and concentrated to 20 ml on Centriprep 10. After dilution with one volume of pH 6.8 50 mM bis-tris propane buffer, 1 mM DTT containing 2 M ammonium sulphate, the proteins are chromatographed (22.5 mg per injection) on a Phenyl Superose HR 10/10 column with a decreasing ammonium sulphate gradient (1.0 M to 0 M). The best fractions containing the desired activity are pooled, reconcentrated to 1 ml on Centriprep 10 and then applied (200 .mu.l per injection) to a Bio-Sil SEC 250 column. The activity peak is detected in this technique at a molecular weight centred at 77,000. The fraction containing the activity is injected onto a MonoQ ER 5/5 column in pH 6.8 50 mM bis-tris propane buffer, DTT 1 mM eluted with a linear KCl gradient (0 to 0.5 M).
After this step, the enzyme is pure and, in SDS-PAGE electrophoresis, two subunits of molecular weight estimated at 35,000 and 50,000 are detected. They are separated on a 25-cm Vydac C4 column eluted with a linear gradient of from 30 to 50% of acetonitrile in water containing 0.07% of trifluoroacetic acid.
TABLE 2______________________________________Purification of pristinamycin IIA synthasePurification Vol. Protein Sp. Act. Yield Purificationstep (ml) (mg) .mu.mol/h/mg (%) factor______________________________________Crude extract 490 1690 0.14 100 140-45% A.S. 60 1050 0.19 85 1.4MonoQ 10/10 95 45 3.0 58 21Phenyl Superose 8 2.8 12 14 86Bio-Sil SEC 5 1.3 18 14 130MonoQ 5/5 10 0.7 23 10 160______________________________________
The purification factor is calculated from the increase in specific activity of the fractions during the purification.
5.1.2. Production of Oligonucleotides From the Protein Sequences
This example describes how, starting from the NH.sub.2 -terminal sequences of the two subunits of pristinamycin IIA synthase purified as described in Example 5.1.1.B., it is possible to synthesize oligonucleotides. The two subunits of pristinamycin IIA synthase are referred to as SnaA and SnaB, and correspond to polypeptides of molecular weights 50,000 and 35,000, respectively, as descibed in Example 5.1.1.B.
The NH.sub.2 -terminal sequences of the proteins SnaA and SnaB, corresponding to the subunits of pristinamycin IIA synthase, were deduced by microsequencing. This is carried out by the Edman degradation technique, using an automated sequencer (Applied Biosystems model 407A) coupled to an HPLC apparatus for identification of the phenylthiohydantoin derivatives. About thirty residues were determined for each of them.
Protein SnaA: (see residues 2 to 29 on SEQ ID No. 18)
T A P(R) (R,W)R I T L A G I I D G P G G H V A A(W)R H P (A) T
Protein SnaB: (see residues 2 to 31 on SEQ ID No. 19)
T A P I L V A T L D T R G P A A T L G T I T(R)A V(R)A A E A
Moreover, sequences internal to these two polypeptides were determined after trypsin digestion of SnaA and SnaB and purification of the fragments obtained on a Vydac C18 HPLC column. The following internal sequences were found:
Protein SnaA: (see residues 365 to 384 on SEQ ID No. 18)
G A D G F N I D F P Y L P G S A D D F V
Protein SnaB: (see residues 122 to 136 on SEQ ID No. 19)
G L(-)D S F D D D A F V H D R
From the underlined regions in each of the sequences of the fragments internal to the proteins SnaA and SnaB, and in accordance with the degeneracy of the genetic code specific to Streptomyces (see Example 8), the following mixtures of oligonucleotides were synthesized with a Biosearch 8600 automated synthesizer. They were then purified by the technique already described (Sawadogo M. and Von Dyke M. W., 1991). The snaA and snaB genes denote the structural genes for the proteins SnaA and SnaB, respectively.
Mixture corresponding to the underlinedportion of the internal sequence of SnaA:ATC GAC TTC CCC TAC CTC CCC GG (SEQ ID NO:34) T T G T G G A TMixture corresponding to underlinedportion of the internal sequence of SnaB:TTC GAC GAT GAT GCA TTC GTC CAT GAC (SEQ ID NO:35) C C T G C C G
5.1.3. Labelling of the Mixtures of Synthetic Oligonucleotides and Hybridization With the Genomic DNA Libraries of the Strain SP92
This example describes how oligonucleotides specific for a gene for the biosynthesis of pristinamycins may be radioactively labelled and then hybridized with membranes onto which DNA of genomic libraries of S. pristinaespiralis SP92 has been transferred.
Labelling of the oligonucleotides is carried out by transfer at the 5'-terminal position of the [.gamma.-.sup.32 P]phosphate group of ATP with T4 polynucleotide kinase. This labelling is carried out as described in Maniatis et al. (1989). After labelling, the oligonucleotides are used without purification.
Approximately 2.times.500 ng of each mixture of oligonucleotides were labelled in this way with .sup.32 P and were used to hybridize each of the two libraries.
Hybridization of the membranes of each library is carried out according to a protocol derived from those developed by Meinkoth, J. and Wahl, G. (1984) and Hames, B. D. and Higgins, S. J. (1985): the 15 membranes are prehybridized for 3 hours at 50.degree. C. in 40 ml of a solution containing: Denhardt (.times.5) [Denhardt (.times.100): 2% (w/v) Ficoll, 2% (w/v) polyvinylpyrrolidone, 2% (w/v) BSA)], SSC (.times.5) [SSC (.times.20): 3 M NaCl, 0.3 M sodium citrate), 50 mM NaPO.sub.4 pH 6.5, 0.1% SDS, 250 .mu.g/ml salmon sperm DNA].
Hybridization is then carried out overnight at 50.degree. C. in 20 ml of the same solution to which the 500 ng of labelled oligonucleotides are added.
The filters are then washed in a solution of SSC (.times.6) and 0.5% SDS, twice for 30 min at room temperature and then empirically at gradually higher temperatures (50 to 65.degree. C.). The temperature of these latter washes is gradually increased after successive autoradiographic exposures in order to determine the specificity of the hybridizing clones with the mixtures of oligonucleotides.
5.1.4. Isolation of cosmids pIBV1 and pIBV3 and determination of the regions containing the snaA and snaB genes
This example illustrates how it is possible to isolate cosmids constructed as described in Example 3 containing genes for the biosynthesis of pristinamycins.
Cosmids pIBV1 and pIBV3 were isolated from two clones originating, respectively, from the library produced in the strain HB101 and from the library produced in the strain DH1 which hybridized with both mixtures of oligonucleotides simultaneously for pIBV1 and with the mixture of oligonucleotides originating from the internal sequence of the protein SnaA for pIBV3.
These cosmids were purified as described in Example 2. Cosmids pIBV1 and pIBV3 contain, respectively, a genomic DNA insert of S. pristinaespiralis SP92 whose sizes were estimated, respectively, at 30 kb and 34 kb. Maps (FIGS. 4 and 6) were established from digestions with different restriction enzymes, according to the protocols of the supplier (New England Biolabs, Beverly, Mass. 01915-5510 USA).
Southern hybridizations of pIBV1 and pIBV3 DNA, digested by means of different enzymes, with the mixtures of oligonucleotides enabled the region of this cosmid containing the snaA and/or snaB genes to be identified.
Southern hybridization was carried out as described in Maniatis et al. (1989). After separation of the restriction fragments by electrophoresis on 0.8% agarose gel, the DNA is transferred onto a Biohylon Z.sup.+ membrane (Bioprope System). Hybridization of the DNA thus transferred onto the membranes with the mixtures of oligonucleotides was carried out as described in Example 5.1.3.
These Southern hybridizations enabled it to be shown that cosmid pIBV1 possessed a 6-kb BamHI fragment containing the sequences homologous to the probes synthesized in Example 5.1.2 (originating from the proteins SnaA and SnaB), as well as a 2.5-kb EcoRI fragment internal to the BamHI fragment containing the sequences homologous to the probes originating exclusively from the protein SnaA. Furthermore, the hybridization signals obtained with cosmid pIBV3 showed that it possessed only the 2.5-kb EcoRI fragment containing the sequences homologous to probes originating exclusively from the protein SnaA.
5.2. Isolation of cosmid pIBV2 containing the structural gene for 3-hydroxypicolinic acid:AMP ligase (snbA)
This example illustrates how it is possible to obtain a cosmid as constructed in Example 3 containing at least one gene for the biosynthesis of pristinamycins I.
5.2.1 Identification and purification of the protein involved in the activation of 3-hydroxypicolinic acid
This example illustrates how the protein responsible for the activation of 3-hydroxypicolinic acid may be purified to homogeneity from S. pristinaespiralis SP92.
5.2.1.A. Assay of 3-hydroxypicolinic acid:AMP ligase
This example illustrates the assay of an activity of the biosynthesis pathway of pristinamycin IA which has never before been described and which possesses the noteworthy property of being expressed only during the period of production of pristinamycins. The enzyme in question is 3-hydroxypicolinic acid:AMP ligase, which catalyses the formation of the adenylate of 3-hydroxypicolinic acid (FIG. 9) from this free acid and ATP in the presence of MgCl.sub.2.
The enzyme fractions to be assayed (0.002 to 0.020 units) are incubated for 15 min at 27.degree. C. in a total volume of 250 .mu.l of pH 6.8 50 mM bis-tris propane buffer, 1 mM DTT, 10% v/v glycerol, in the presence of 3-hydroxypicolinic acid (1 mM), ATP (2 mM), MgCl.sub.2 (5 mM) and tetrasodium pyrophosphate labelled with the radioactive isotope 32 of the phosphorus atom (200 .mu.M).
The reaction is stopped by adding 1 ml of a suspension of activated charcoal at a concentration of 10 g/l in a mixture of 75% of 0.1 M tetrasodium pyrophosphate and 25% of 14% perchloric acid. After stirring, the charcoal is collected and washed with twice 1 ml of the pyrophosphate/perchloric acid mixture. The radioactive organic molecules are then eluted with three times 1 ml of a mixture of 50% of methanol and 50% of N ammonia solution into a counting vial containing 12 ml of water. The radioactivity is measured by the Cerenkov effect with a scintillation counter (PACKARD Minaxi TriCarb 4000).
The unit of enzymatic activity is defined as the amount of enzyme needed to incorporate 1 .mu.mol of pyrophosphate into ATP in the course of 1 hour under the conditions described above.
5.2.1.B. Purification of S. pristinaespiralis SP92 3-hydroxypicolinic acid:AMP ligase
This experiment illustrates how an enzyme of S. pristinaespiralis SP92 participating in the biosynthesis pathway of pristinamycin IA may be purified.
Using the assay described above in Example 5.2.1.A, the purification of 3-hydroxypicolinic acid:AMP ligase is carried out as described below, taking care to freeze the active fractions at -70.degree. C. and store them at -30.degree. C. between successive steps if necessary.
234 g of a centrifugation pellet, washed with 0.1 M phosphate buffer pH 7.2 containing 10% v/v of glycerol, of an S. pristinaespiralis SP92 culture harvested at the beginning of the pristinamycin production phase are taken up with 234 ml of pH 8.0 100 mM Tris-HCl buffer containing 4 mM DTE, 1 mM benzamidine, 1 mM PMSF, 15% v/v glycerol and 0.6 mg/ml of lysozyme. The suspension thereby obtained is incubated for 30 minutes at 27.degree. C. and then centrifuged at 50,000 g for 1 hour. The crude extract thereby collected is injected in pH 8.0 100 mM Tris-HCl buffer, 4 mM DTE, 1 mM benzamidine, 1 mM PMSF, 15% v/v glycerol onto a column (80 ml) of Q Sepharose Fast Flow. The proteins are eluted with a linear KCl gradient (0 to 0.4 M). The fractions containing the enzymatic activity (detected by means of the test described in Example 5.2.1.A) are pooled and diluted with one volume of pH 8.0 100 mM Tris-HCl buffer, 1 mM benzamidine, 1 mM PMSF, 15% v/v glycerol containing 2 M ammonium sulphate. The proteins are then chromatographed on a column (50 ml) of Phenyl Sepharose with a decreasing ammonium sulphate gradient (1.0 M to 0 M) in pH 8.0 100 mM Tris-HCl buffer, 1 mM benzamidine, 1 mM PMSF, 15% v/v glycerol. After the addition of 4 mM DTE, the active fractions are pooled, concentrated to 5 ml on Centriprep 10 and then applied to a column (100 ml) of Superose 12 prep grade. The fractions containing the desired activity are pooled and injected in pH 8.0 100 mM Tris-HCl buffer, 4 mM DTE, 1 mM benzamidine, 1 mM PMSF, 15% v/v glycerol (approximately 6 mg per injection) onto a column of MonoQ HR 5/5 eluted with a linear KCl gradient (0 to 0.4 M). The active fractions are pooled, concentrated to 1 ml on Centricon 10, diluted with 3 volume of pH 6.8 50 mM bis-tris propane buffer, 4 mM DTE, 1 mM benzamidine, 1 mM PMSF, 15% v/v glycerol, and then injected (2 mg per injection) in the latter buffer onto a column of MonoQ HR 5/5 eluted with a linear KCl gradient (0 to 0.3 M). The best fractions containing the desired ligase are pooled and then applied in pH 6.8 2.0 mM sodium phosphate buffer, 50 mM sodium sulphate to a Bio-Sil SEC 250 column. The activity peak is detected in this technique at a molecular weight centred at 60,000.
The protein possessing the activity of activation of 3-hydroxypicolinic acid is hereinafter designated SnbA.
After this step, the enzyme is pure and, in SDS-PAGE electrophoresis, its molecular weight is estimated at approximately 67,000.
TABLE 3______________________________________Purification of 3-hydroxypicolinic acid:AMP ligasePurification Vol. Protein Sp. Act. Yield Purificationstep (ml) (mg) .mu.mol/h/mg.sup.a (%) factor______________________________________Crude extract 246 2050 (0.06)Q Sepharose 40 188 0.47 100 1Phenyl Sepharose 70 35 2.21 88 4.7Superose 12 16 17 2.03 39 4.3MonoQ pH 8.0 4.5 9.0 2.09 21 4.5MonoQ pH 6.8 1.0 2.0 2.9 6.6 6.2Bio-Sil 250 2.5 0.23 12.4 3.2 26______________________________________ .sup.a The activity in the crude extract cannot be measured accurately owing to exchanges between pyrophosphate and ATP which are not specific t 3hydroxy-picolinic acid.
The purification factor is calculated from the increase in specific activity of the fractions during the purification.
5.2.2. Production of oligonucleotides from the protein sequence:
This example describes how, starting from the NH.sub.2 -terminal and internal sequences of the protein 3-hydroxpicolinic:AMP ligase, it is possible to synthesize oligonucleotides.
The NH.sub.2 -terminal sequence of the protein SnbA was deduced by microsequencing as described in Example 5.1.2. About twenty residues were identified in this way.
A sequence of approximately 20 amino acids internal to the protein SnbA was also identified after trypsin hydrolysis and purification of the fragments obtained on a Vydac C18 HPLC column.
NH.sub.2 -terminal sequence of the protein 3-hydroxypicolinic:AMP ligase:
(See residues 1 to 21 on SEQ ID NO:21)
M L D G S V P W P E D V A A K Y R A A G Y
Internal sequence of the protein 3-hydroxypicolinic:AMP ligase:
(See residues 448 to 467 on SEQ ID NO:21)
V S A (-) E V E G H L G A H P D V Q Q A A
From the underlined regions in each of the sequences, and in accordance with the degeneracy of the genetic code specific to Streptomyces (see Example 8), the following mixtures of oligonucleotides were synthesized:
Mixture corresponding to the underlinedportion of the NH.sub.2 -terminal sequence of the protein3-hydroxypicolinic:AMP ligase:5' 3' GTC CCC TGG CCC GAG GAC GTC GCC GCC AAG TAC (SEQ ID NO:36) G G G G G GMixture corresponding to the underlinedportion of the internal sequence of the protein3-hydroxypicolinic:AMP ligase:5' 3'GAG GTC GAG GGC CAC CTC GGC GCC CAC CCC GAC GTC CAG CAG GC (SEQ ID NO:37) G G G G G G G
5.2.3. Labelling of the mixtures of synthetic oligonucleotides and hybridization of the genomic DNA libraries of S. pristinaespiralis SP92.
This example describes how oligonucleotides specific for a gene for the biosynthesis of pristinamycins may be radioactively labelled and then hybridized with membranes onto which DNA of genomic libraries of S. pristinaespiralis has been transferred.
Labelling the oligonucleotides is carried out by transfer at the 5'-terminal position of the [.gamma.-.sup.32 P]phosphate group of ATP with T4 polynucleotide kinase, as described in Example 5.1.3.
Approximately 2.times.500 ng of each mixture of oligonucleotides were labelled in this way with .sup.32 P and were used to hybridize each of the two libraries.
Hybridization of the membranes of each library was carried out as described in Example 5.1.3.
5.2.4. Isolation of cosmid pIBV2 and determination of the region containing the structural gene for 3-hydroxypicolinic acid:AMP ligase
This example illustrates how it is possible to obtain a cosmid as constructed in Example 3 containing at least the structural gene for 3-hydroxypicolinic acid:AMP ligase.
Cosmid pIBV2 was isolated from a clone of the library produced in E. coli strain DH1 which hybridized with both mixtures of oligonucleotides simultaneously.
This cosmid was purified as described in Example 2. It contains a genomic DNA insert of S. pristinaespiralis SP92 whose size was estimated at 47 kb. A map (FIG. 5) was established from digestions with different restriction enzymes, as described in Example 5.1.4.
Southern hybridizations of pIBV2 DNA, digested by means of different enzymes, with the mixtures of oligonucleotides enabled the region containing the structural gene for 3-hydroxypicolinic acid:AMP ligase to be identified. Southern blotting and hybridizations were carried out as described in Example 5.1.4.
The hybridization results enabled it to be shown that cosmid pIBV2 possessed a 5.5-kb EcoRI-BglII fragment containing the sequence homologous to the probes synthesized in Example 5.2.2.
5.3. Demonstration of the presence of a portion of the structural gene for pristinamycin I synthase II (SnbC) on cosmid pIBV3
This example illustrates how it is possible to identify the presence of genes for the biosynthesis of pristinamycins I on a cosmid which has already been isolated (Example 5.1).
5.3.1. Identification of pristinamycin I synthase II involved in the incorporation of threonine and aminobutyric acid residues into the peptide chain pristinamycin IA
This example illustrates how the protein responsible for the incorporation of threonine and aminobutyric acid residues into the peptide chain of pristinamycin IA may be purified to homogeneity from S. pristinaespiralis SP92.
5.3.1.A. Assay of the partial activities of pristinamycin I synthase II
This example illustrates the assay of activities of the biosynthesis pathway of pristinamycin IA which have never before been described and which possess the noteworth property of being expressed only during the period of production of pristinamycins. The activities in question are the partial activities of the peptide synthase responsible for the incorporation of threonine and aminobutyric acid residues into the peptide chain of pristinamycin IA (FIG. 10) in the presence of ATP and MgCl.sub.2.
The threonine:AMP ligase and aminobutyric acid:AMP ligase activities are measured in an enzymatic test of ATP-pyrophosphate exchange similar to that described in 5.2.1.A for 3-hydroxypicolinic acid:AMP ligase.
The aminoacylation reactions of the enzyme with threonine or alanine (an analogue of aminobutyric acid which is found in pristinamycin IC) enable the peptide synthase to be differentiated from other enzymes which may effect an ATP-pyrophosphate exchange, and in particular aminoacyl-tRNA synthetases. The test of aminoacylation of the enzyme with tritium-labelled threonine described below is hence the one which was used in this example.
The enzymes fractions to be assayed (0.2 to 2 units) are incubated for 15 min at 27.degree. C. in a total volume of 250 .mu.l of pH 6.8 50 mM bis-tris propane buffer, 1 mM DTT, 10% v/v glycerol in the presence of 1 .mu.Ci of [3-.sup.3 H]-L-threonine (15 Ci/mmol), ATP (2 mM) and MgCl.sub.2 (5 mM).
The reaction is stopped by adding 150 .mu.l of 25% trichloroacetic acid solution. The precipitated proteins are collected on a microfilter and washed with 3 times 400 .mu.l of 7% trichloroacetic acid, before being eluted with twice 400 .mu.l of N sodium hydroxide into a counting vial containing 1 ml of N HCl and 12 ml of scintillation cocktail (Beckmann Readygel). The amount of radioactivity contained in this vial is measured with a scintillation counter (PACKARD Minaxi TriCarb 4000). It represents the amount of threonine bound covalently to the desired peptide synthase.
The unit of enzymatic activity is defined as the amount of enzyme needed to bind 1 picomole of threonine covalently in 15 min under the conditions described above.
5.3.1.B. Purification of pristinamycin I synthase II
This experiment illustrates how an enzyme of S. pristinaespiralis SP92 participating in the biosynthesis pathway of pristinamycin IA may be purified.
Using the acid described above in Example 5.3.1.A., purification of the peptide synthase responsible for the incorporation of threonine and aminobutyric acid residues into the peptide chain of pristinamycin IA is carried out as described below, taking care to work at 4.degree. C. and to store the active fractions at -70.degree. C.
150 g of a centrifugation pellet, washed with 0.1 M phosphate buffer pH 7.2 containing 10% v/v of glycerol, of an S. pristinaespiralis SP92 culture harvested at the beginning of the pristinamycin production phase are taken up with 450 ml of pH 8.0 100 mM Tris-HCl buffer containing 4 mM DTE, 1 mM benzamidine, 1 mM PMSF, 1 mM EDTA, 1 mM EGTA, 15% v/v glycerol. The suspension thereby obtained is ground using a French Press adjusted to a pressure of 5000 psi, and then centrifuged at 50,000 g for 1 hour. The crude extract thereby collected is injected in pH 8 100 mM Tris-HCl buffer, 4 mM DTE, 2 mM benzamidine, 2 mg/l leupeptin, 1 mg/l E-64, 15% v/v glycerol onto a column (200 ml) of Q Sepharose Fast Flow. The proteins are eluted with a linear KCl gradient (0 to 0.6 M). At outflow from the column, each fraction is treated with one-tenth of its volume of a solution of 1 mM PMSF, 5 mM EDTA, 5 mM EGTA. The fractions containing the enzymatic activity (detected by means of the test described in Example 5.3.1.A) are pooled and reconcentrated by ultrafiltration on Centriprep 30 to a final volume of 28 ml. This concentrate is injected in 4-ml aliquots onto a Superdex 200 Hi-Load 16/60 permeation column equilibrated in pH 6.8 50 mM bis-tris propane buffer, 1 mM benzamidine, 4 mM DTE, 0.2 mM Pefabloc, 1 mM EDTA, 0.1 M KCl, 20% v/v glycerol. After assaying, the active fractions are pooled and reconcentrated to 15 ml on Centriprep 30, then desalted on PD-10 in pH 8.0 100 mM Tris-HCl buffer, 4 mM DTE, 2 mM benzamidine, 2 mg/l leupeptin, 1 mg/l E-64, 20% v/v glycerol and applied in two portions to a MonoQ HR 10/10 column equilibrated and eluted with a linear gradient of from 0.4 M KCl in this same buffer. The fractions containing the desired activity are pooled, reconcentrated on Centriprep 30 and then Centricon 30 to a final volume of 1 ml and injected in five portions onto a column of Superose 6 HR 10/30 in pH 6.8 50 mM bis-tris propane buffer, 1 mM benzamidine, 4 mM DTE, 0.2 mM Pefabloc, 1 mM EDTA, 0.1 M KCl, 20% v/v glycerol. The activity peak is detected in this technique at a molecular weight centred at 450.000.
After this step the enzyme is pure and, in SDS-PAGE electophoresis, its molecular weight is estimated at approximately 240,000. This band also contains all radioactivity of the protein labelled by aminoacylation with tritiated threonine.
At this stage, the maximal activity of the enzyme using a concentration of 100 .mu.Ci/ml of threonine (15 ci/mmol) amounts to 3670 units/mg; the enzyme is also capable of forming adenylates with L-aminobutyric acid or L-alanine; an aminoacylation reaction of the enzyme with tritiated alanine is detected, and the maximal activity in the presence of 200 .mu.Ci/ml of [2,3-.sup.3 H]-L-alanine (15 Ci/mmol) is 2290 pmol/mg in 15 min.
TABLE 4______________________________________Purification of pristinamycin I synthase IIPurification Vol. Protein Sp. Act..sup.a Yield Purificationstep (ml) (mg) (units/mg) (%) factor______________________________________Crude extract 445 4700 (1) -- --Q Sepharose 308 834 7 100 1Superdex 200 120 105 22 40 3.1MonoQ HR 15 11.5 96 19 14Superose 6 7.5 2.8 122 6 17______________________________________ .sup.a The activity in the crude extract cannot be measured accurately.
The purification factor is calculated from the increase in specific activity of the fractions during purification.
5.3.2. Production of oligonucleotides from the protein sequence
This example describes how, starting from internal sequences of pristinamycin I synthase II, it is possible to synthesize oligonucleotides.
The internal sequences of the peptide synthase which is responsible for the incorporation of threonine and aminobutyric acid residues into the peptide chain of pristinamycin IA were deduced by microsequencing as described in Example 5.1.2. after trypsin hydrolysis and purification of the fragments obtained on a Vydac C18 column.
pristinamycin I synthase II(See residues 49 to 61 on SEQ ID NO:28) (SEQ ID NO:38)1 5 10L A A F N D T A R P V P R1 5 10 15 20V P A A F V P L D A L P L T G N G V L D
From the underlined regions in these sequences, and in accordance with the degeneracy of the genetic code specific to Streptomyces (see Example 8), the following mixtures of oligonucleotides were synthesized:
Mixture corresponding to the underlinedportion of the sequence 1 internal to the proteinpristinamycin I synthase II: (SEQ ID NO:39)5' 3' GCC GCC TTC AAC GAC ACC GCC CGC CC G G G G GMixture corresponding to the underlinedportion of sequence 2 internal to the proteinpristinamycin I synthase II: (SEQ ID NO:4)5' 3' TTC GTC CCC CTC GAC GCC CTC CCC CT G G G G G G
5.3.3. Labelling of the mixtures of synthetic oligonucleotides and Southern hybridization of cosmid PIBV3 DNA
This example describes how oligonucleotides specific for a gene for the biosynthesis of pristinamycins I may be radioactively labelled and then hybridized with a membrane onto which cosmid pIBV3 DNA has been transferred.
Labelling of the oligonucleotides is carried out by transfer at the 5'-terminal position of the [.gamma.-.sup.32 P]phosphate group of ATP with T4 polynucleotide kinase, as described in 5.1.3.
Approximately 500 ng of the mixture of oligonucleotides were labelled in this way with .sup.32 P, and were used for Southern hybridization of pIBV3 DNA digested with different enzymes. These hybridizations enabled it to be shown that a portion of the structural gene for pristinamycin I synthase II was carried by cosmid pIBV3, and enabled the region containing this gene to be identified. Southern blotting and hybridization were carried out as described in Example 5.1.4.
The hybridization results enabled it to be shown that cosmid pIBV3 possessed a 6.2-kb SphI fragment containing the sequence homologous to the probes synthesized in Example 5.3.2.
5.4. Demonstration of the presence of a portion of the structural gene for pristinamycin I synthase III (SnbD) on cosmid pIBV3
This example illustrates how it is possible to identify the presence of genes for the biosynthesis of pristinamycins I on a cosmid which has already been isolated (Example 5.1).
5.4.1. Identification of pristinamycin I synthase III involved in the incorporation of proline and p-dimethylaminophenylalanine residues into the peptide chain of pristinamycin IA
This example illustrates how the protein responsible for the incorporation of proline and p-dimethylaminophenylalanine residues into the peptide chain of pristinamycin IA may be purified to homogeneity from S. pristinaespiralis SP92.
5.4.1.A. Assay of partial activities of pristinamycin I synthase III
This example illustrates the assay of activities of the biosynthesis pathway of pristinamycin IA which have never before been described and which possess the noteworthy property of being expressed only during the period of production of pristinamycins. The activities in question are partial activities of the peptide synthase responsible for the incorporation of proline and para-dimethylaminophenylalanine residues into the peptide chain of pristinamycin IA (FIG. 11) in the presence of SAM, ATP and MgCl.sub.2.
The proline:AMP ligase and p-dimethylaminophenylalanine:AMP ligase activities are measured in an enzymatic test of ATP-pyrophosphate exchange similar to that described in 5.2.1.A. for 3-hydroxypicolinic acid:AMP ligase.
The aminoacylation reactions of the enzyme with proline and p-dimethylaminophenylalanine make it possible to differentiate the peptide synthase from other enzymes which may perform a ATP-pyrophosphate exchange, and in particular aminoacyl-tRNA synthases. The same applies to the N-methylation of the .alpha.-amino function of p-dimethylaminophenylalanine acylated on the enzyme. The latter test characteristic of N-methylation is hence the one which was used in this example.
The enzyme fractions to be assayed (0.2 to 2 units) are incubated for 15 min at 27.degree. C. in a total volume of 250 .mu.l of pH 6.8 50 mM bis-tris propane buffer, 1 mM DTT, 10% v/v glycerol in the presence of 1 .mu.Ci of [methyl-.sup.3 H]-SAM (15 Ci/mmol), para-dimethylamino-L-phenylalanine (1 mM), ATP (2 mM) and MgCl.sub.2 (5 mM).
The reaction is stopped by adding 150 .mu.l of 25% trichloroacetic acid solution. The precipitated proteins are collected on a microfilter and washed with 3 times 400 .mu.l of 7% trichloroacetic acid, before being eluted with twice 400 .mu.l of N sodium hydroxide into a counting vial containing 1 ml N HCl and 12 ml of scintillation cocktail (Beckmann Readygel). The amount of radioactivity contained in this vial is measured with a scintillation counter (PACKARD Minaxi TriCarb 4000). It represents the amount of N-methylated para-dimethylaminophenylalanine bound covalently to the desired peptide synthase.
The unit of enzymatic activity is defined as the amount of enzyme needed to bind 1 picomole of N-methylated p-dimethylaminophenylalanine covalently in 15 min under the conditions described above.
5.4.1.B. Purification of pristinamycin I synthase III
This experiment illustrates how an enzyme of S. pristinaespiralis SP92 participating in the biosynthesis pathway of pristinamycin IA may be purified.
Using the assay described above in Example 5.4.1.A, purification of the peptide synthase responsible for the incorporation of proline and para-dimethylaminophenylalanine residues into the peptide chain of pristinamycin IA is carried out as described below, taking care to work at 4.degree. C. and to store the active fractions at -70.degree. C.
250 g of a centrifugation pellet, washed with 0.1 M phosphate buffer pH 7.2, 1 mM PMSF, 5 mM EDTA, 5 mM EGTA, 0.5 M KCl, 10% v/v glycerol, of an S. pristinaespiralis SP92 culture harvested at the beginning of the pristinamycin production phase are taken up with 750 ml of pH 8.0 100 mM Tris-HCl buffer containing 4 mM DTE, 5 mM benzamidine, 0.2 mM Pefabloc, 1 mM EDTA, 1 mM EGTA, 2 mg/l leupeptin, 2 mg/l STI, 2 mg/l aprotinin, 1 mg/l E-64, 20% v/v glycerol. The suspension thereby obtained is ground using a French Press adjusted to a pressure of 5000 psi, and then centrifuged at 50,000 g for 1 h. The crude extract thereby collected is fractionated by ammonium sulphate precipitation. The protein fraction coming out at between 0 and 35% ammonium sulphate saturation is redissolved in the disruption buffer and desalted on a column of Sephadex G 25 Fine equilibrated and eluted in this same buffer. The proteins thus prepared are injected in pH 8.0 100 mM Tris-HCl buffer, 4 mM DTE, 2 mM benzamidine, 2 mg/l leupeptin, 1 mg/l E-64, 20% v/v glycerol onto a column (200 ml) of Q Sepharose Fast Flow, and are then eluted with a linear KCl gradient (0 to 0.6 M). At outflow from the column, each fraction is treated with one-tenth of its volume of a solution of 2 mM Pefabloc, 5 mM EDTA, 5 mM EGTA, 5 mM benzamidine. The fractions containing the enzymatic activity (detected by means of the test described in Example 5.4.1.A) are pooled and precipitated with ammonium sulphate at 80% saturation. The proteins which have come out are redissolved in pH 6.8 50 mM bis-tris propane buffer, 1 mM benzamidine, 1 mM DTE, 0.2 mM Pefabloc, 1 mM EDTA, 1 mM EGTA, 2 mg/l leupeptin, 0.15 M NaCl, 20% v/v glycerol, and injected in 5 4-ml aliquot portions onto a Superdex 200 Hi-Load 16/60 permeation column equilibrated and eluted in this same buffer. After assay, the active fractions are pooled and reconcentrated to 3 ml on Centriprep 30, then rediluted to 20 ml with pH 8.0 100 mM Tris-HCl buffer, 4 mM DTE, 1 mM benzamidine, 1 mM PMSF, 20% v/v glycerol and applied in two portions to a MonoQ HR 10/10 column equilibrated and eluted with a linear gradient from 0.4 M KCl in this same buffer. The best fractions containing the desired activity are pooled and used as material for characterization of the activities of the enzyme and for its microsequencing.
After this step, the enzyme is pure and, in SDS-PAGE electrophoresis, its molecular weight is estimated at approximately 250,000. This band also contains all the radioactivity of the protein labelled by aminoacylation with tritiated SAM and para-dimethylaminophenylalanine. In permeation on Superose 6 HR 10/30, the native molecular weight of the enzyme is estimated at 700,000.
At this stage, the enzyme is also capable of forming adenylates with proline; an aminoacylation reaction of the enzyme with tritiated proline is detected, and the maximal activity in the presence of 200 .mu.Ci/ml of [5-.sup.3 H]-L-proline (34 Ci/mmol) is 2490 pmol/mg in 15 min.
TABLE 5______________________________________Purification of pristinamycin I synthase IIIPurification Vol. Protein Sp. Act..sup.a Yield Purificationstep (ml) (mg) (units/mg) (%) factor______________________________________Crude extract 800 8100 (4) -- --35% A.S. 200 4000 (6) -- --Q Sepharose 132 498 46 100 1Superdex 200 45 39.5 417 71 9MonoQ HR 9 5.3 1070 25 23______________________________________ .sup.a The activity in the crude extract and after ammonium sulphate precipitation cannot be measured accurately.
The purification factor is calculated from the increase in specific activity of the fractions during the purification.
5.4.2. Production of oligonucleotides from the protein sequence
This example describes how, starting from internal sequences of pristinamycin I synthase III, it is possible to synthesize oligonucleotides.
An internal sequence of the peptide synthase responsible for the incorporation of proline and para-dimethylaminophenylalanine residues into the peptide chain of pristinamycin IA was deduced by microsequencing as described in Example 5.1.2. after cyanogen bromide treatment and purification of the fragments obtained on a Vydac C18 HPLC column.
Sequence internal to the proteinpristinamycin I synthase III1 (see residues 2 to 20 on SEQ ID NO:29)1 5 10 15 20 D D D P Y R A Y A L A H L A G
From the underlined region in this sequence, and in accordance with the degeneracy of the genetic code specific to Streptomyces (see Example 8), the following mixture of oligonucleotides was synthesized:
Mixture corresponding to the underlinedportion of the sequence internal to the proteinpristinamycin I synthase III:5' 3' GTC ACC CCG TAC CGC GCC TAC (SEQ ID NO:41) G G C G G
5.4.3. Labelling of the mixtures of synthetic oligonucleotides and Southern hybridization of cosmid pIBV3 DNA
This example describes how oligonucleotides specific for a gene for the biosynthesis of pristinamycins I may be radioactively labelled and then hybridized with a membrane onto which cosmid pIBV3 DNA has been transferred.
Labelling of the oligonucleotides is carried out by transfer at the 5'-terminal position of the [.gamma.-.sup.32 P]phosphate group of ATP with T4 polynucleotide kinase, as described in 5.1.3.
Approximately 500 ng of the mixture of oligonucleotides were labelled in this way with .sup.32 P, and were used for Southern hybridization of pIBV3 DNA digested with different enzymes. These hybridizations enabled it to be shown that a portion of the structural gene for pristinamycin I synthase III was carried by cosmid pIBV3, and enabled the region containing this gene to be identified. Southern blotting and hybridization were carried out as described in Example 5.1.4.
The hybridization results enabled it to be shown that cosmid pIBV3 possessed an 8.4-kb SphI fragment containing the sequence homologous to the probes synthesized in Example 5.4.2.
5.5. Demonstration of the presence of a portion of the structural gene for pristinamycin I synthase IV (SnbE) on cosmid pIBV3
This example illustrates how it is possible to identify the presence of genes for the biosynthesis of pristinamycins I on a cosmid which has already been isolated (Example 5.1).
5.5.1. Identification of the peptide synthase (referred to as pristinamycin I synthase IV) responsible for the incorporation of the phenylglycine residue into the peptide chain of pristinamycin IA
5.5.1.A. Assay of enzymatic activities carried by the peptide synthase (pristinamycin I synthase IV) responsible for the incorporation of the phenylglycine residue into the peptide chain of pristinamycin IA
This example illustrates the assay of an enzymatic activity of the biosynthesis pathway of pristinamycin IA which has not been described hitherto and which possesses the noteworthy property of being expressed only during the period of production of pristinamycins in the wild-type microorganism. The activity in question is that of the peptide synthase (pristinamycin I synthase IV) responsible for the incorporation of the L-phenylglycine residue into the peptide chain (FIG. 12) in the presence of ATP and MgCl.sub.2. The phenylglycine:AMP ligase activity of pristinamycin I synthase IV is measured in an enzymatic test of ATP-pyrophosphate exchange similar to that described in 5.2.1.A. for 3-hydroxypicolinic acid:AMP ligase activity, in the presence of L-phenylglycine (1 mM) and KCl (50 mM) in the incubation buffer.
5.5.1.B. Purification of the peptide synthase responsible for the incorporation of the phenylglycine residue (pristinamycin I synthase IV) into the peptide chain of pristinamycin IA
This example illustrates how an enzyme of S. pristinaespiralis SP92 participating in the biosynthesis pathway of pristinamycin IA may be purified. Using the assay described above in Example 5.5.1.A. The purification of pristinamycin I synthase IV is carried out as described below. All the operations are performed at 4.degree. C. The fractions containing the activity are frozen immediately and stored at -70.degree. C.
70 g of wet cells, harvested as described in Example 5.2.1.B., are resuspended in 250 ml of cell lysis buffer (100 mM Tris-HCl pH 8.0 containing 25% of glycerol, 4 mM DTE, 1 mM EGTA, 1 mM EDTA, 1 mM PMSF, 1 mg/l E-64, 2 mg/l STI, 2 mg/l .alpha..sub.2 -macroglobulin, 1 mg/l leupeptin, 2 mg/l aprotinin, 5 mM benzamidine, 0.6 mg/ml lysozyme. The solution thereby obtained is kept stirring at 4.degree. C. for 1 h and then centrifuged at 50,000 g for 1 h. The supernatant is then injected in the cell lysis buffer onto a column of Sephadex G-25, and the excluded fraction (approximately 250 mg of protein injected in each chromatographic run) is injected onto a column of Mono Q HR 16/10 (Pharmacia) equilibrated with 100 mM Tris-HCl buffer pH 8.0, 4 mM DTE, 1 mM EGTA, 1 mM EDTA, 1 mg/l E-64, 2 mg/l STI, 20% glycerol. The proteins are eluted with a linear gradient of from 0 to 0.6 M KCl and, at outflow from the column, each fraction is treated with one-tenth of its volume of a solution of 2 mM Pefabloc, 5 mM EGTA, 5 mM EDTA. The fractions containing the activity are pooled and then mixed with 1 volume of 100 mM Tris-HCl pH 8.0 15% glycerol, 1 mM PMSF, 1 mM benzamidine, 4 mM DTT, 3.4 M ammonium sulphate per 3 volumes of fraction. The solution is injected onto a column of Phenyl Superose HR 10/10 (one-fifth of the solution is injected at each chromatographic run), and the proteins are eluted with a decreasing linear gradient of from 0.9 to 0 M ammonium sulphate. The fractions containing the activity are pooled. The solution is concentrated to 3500 .mu.l in a Centriprep 30 and injected in two portions onto a Superdex 200 Hi-Load 16/60 column equilibrated and eluted with 50 mM bis-tris propane buffer pH 6.8 containing 20% of glycerol, 0.15 M NaCl, 4 mM DTT, 1 mM PMSF, 1 mM benzamidine, 1 mM EDTA. The active fraction is diluted with 9 volumes of 50 mM bis-tris propane buffer pH 6.8 containing 25% of glycerol, 4 mM DTT, 1 mM PMSF, 1 mM benzamidine, and then injected onto a column of Mono Q HR 5/5 equilibrated in the same buffer. The desired activity is eluted with a linear gradient of from 0 to 0.4 M KCl and concentrated to 630 .mu.l in a Centricon-30. The desired protein is then purified by electrophoresis on 6% polyacrylamide gel after denaturation of the sample by heating for 10 min at 80.degree. C. with an SDS/mercaptoethanol mixture. After electrophoresis and staining of the gel with Coomassie blue, the gel band containing the protein is cut out and the protein is electroeluted from the gel in a Centrilutor.
Note: the band corresponding to pristinamycin I synthase IV is identified by comparison with a tritiated (by covalent binding to tritiated phenylglycine; see description in Example 5.2.2.) pristinamycin I synthase IV standard.
After this step, the enzyme is pure in electrophoresis (SDS-PAGE). Its molecular weight is estimated at approximately 170,000.
5.5.2. Labelling of pristinamycin I synthase IV by thioesterification of radioactive phenylglycine on the enzyme
After activation in the form of an adenylate through phenylglycine:AMP ligase activity, phenylglycine is transferred to a thiol group of the active site of the enzyme before being incorporated into the peptide chain during elongation (general process of biosynthesis of peptide antibiotics known by the name of "thiotemplate mechanism"). Generally speaking, radioactive labelling of the protein effecting the activation of amino acid may hence be performed by preparing the thioester derivative with a radioactive form of the amino acid.
As an example, the radioactive labelling of pristinamycin I synthase IV is accomplished by incubating 50 .mu.g of the protein (active fraction emerging from the Mono Q HR 5/5 chromatography column; see above in Example 5.5.1.B.) for 1 hour at 27.degree. C. with 100 .mu.Ci of (RS)-2-phenyl[2-.sup.3 H]glycine (18 Ci/mmol; Amersham) in 70 .mu.l of 50 mM bis-tris propane buffer pH 6.8 containing 20% of glycerol, 25 mM MgCl.sub.2, 5 mM ATP, 0.15 M NaCl, 4 mM DTT, 1 mM PMSF, 1 mM benzamidine, 1 mM EDTA. After denaturation (SDS alone without mercaptoethanol), the proteins are separated by electrophoresis (SDS-PAGE, 6% gel) and visualized with Coomassie blue. Analysis of the radioactivity profile by counting the protein bands as well as by autoradiography (Hyperfilm MP; fluorography after impregnation of the gel with Amersham Amplify) discloses a single radioactive band with a molecular weight of 170,000.
TABLE 6______________________________________Purification of pristinamycin I synthase IVPurification Protein Sp. Act. Protein PurificationStep (mg) (cpm/mg).sup.a (mg) factor______________________________________Crude extract 2200 3.6 -- --MonoQ 16/10 136 58 100 16Phenyl Superose 32.6 175 72 49Superdex 200 3.1 870 34 240MonoQ 5/5 2.0 1000 25 280Electroelution 0.1 -- -- --SDS-PAGE______________________________________ .sup.a The specific activity cannot be measured accurately in the crude extract owing to the high level of nonphenylglycine-dependent ATPpyrophosphate exchange. The specific activity value was calculated fro the number of units present at emergence from the first chromatographic step expressed with reference to the amount of protein in the crude extract.
5.5.3. Other activities carried by pristinamycin I synthase IV
Purification of the peptide synthase responsible for the incorporation of phenylglycine, described in Example 5.5.2., led to a pure protein of molecular weight 170,000. This protein does not activate the other amino acids tested, especially pipecolic acid or 4-oxopipecolic acid. A second preparation of this protein, performed under the conditions described in 5.5.1.B. eliminating, however, the Phenyl Superose step, starting from another culture of S. pristinaespiralis SP92, the crude extract of which was prepared in a French Press as described in 5.4.1B, led to a protein which, at emergence from the Mono Q HR 5/5 step, was equivalent in purity to that obtained at the same step in the example described in 5.5.1.B., but possessed a molecular weight of approximately 250,000 in SDS-PAGE. This new preparation was competent for the activation and thioesterification of phenylglycine, but possessed, in addition, an ATP-pyrophosphate exchange activity with L-pipecolic acid (1 mM) in the exchange test similar to that described in 5.2.1.A. for 3-hydroxypicolinic acid. Moreover, it could be shown that the 170,000 protein does not possess ATP-pyrophosphate exchange activity with L-pipecolic acid even in preparations of the protein that are still very impure. It should be noted that S. pristinaespiralis SP92 naturally produces small amounts of a pristinamycin IA analogue having a pipecolic acid residue in place of 4-oxopipecolic acid. Hence this demonstrates that the peptide synthase responsible for the incorporation of phenylglycine (pristinamycin I synthase IV) also catalyses the incorporation of the preceding residue (probably pipecolic acid). The difference in molecular weight obtained for pristinamycin I synthase IV in the two preparations (170,000 and 250,000) is attributed to a phenomenon of partial proteolytic cleavage in the first case, leading to loss of the activity of activation of L-pipecolic acid.
5.5.4. Synthesis of oligonucleotides from the protein sequence
This example describes how, starting from an internal sequence of pristinamycin I synthase IV, it is possible to set about testing for the corresponding gene using suitably chosen oligonucleotides.
An internal sequence of pristinamycin I synthase IV of 15 amino acids was identified after cyanogen bromide cleavage of the purified protein and purification of the fragments obtained on a Vydac C18 HPLC column.
Sequence internal to the proteinpristinamycin I synthase IV(See residues 82 to 98 on SEQ ID NO:32)1 5 10 15 GD V F L N N T R L I Q N F R P R
From the underlined region in this sequence, and in accordance with the degeneracy of the genetic code specific to Streptomyces (see Example 8), the following mixture of oligonucleotides was synthesized:
Mixture corresponding to the underlined portion of the internal sequence of the protein pristinamycin I synthase IV:
(SEQ ID NO 42)5' 3' ACG CGC CTC ATC CAG AAC TTC CGC CC C G G G T T
5.5.5. Labelling of the mixtures of synthetic oligonucleotides and Southern hybridization of cosmid pIBV3 DNA
This example describes how oligonucleotides specific for a gene for the biosynthesis of pristinamycins I may be radioactively labelled and then hybridized with a membrane onto which cosmid pIBV3 DNA has been transferred.
Labelling of the oligonucleotides is carried out by transfer at the 5'-terminal position of the [.gamma.-.sup.32 P]phosphate group of ATP with T4 polynucleotide kinase, as described in 5.1.3.
Approximately 500 ng of the mixture of oligonucleotides were labelled in this way with .sup.32 P, and were used for Southern hybridization of pIBV3 DNA digested with different enzymes. These hybridizations enabled it to be shown that a portion of the structural gene for pristinamycin I synthase II was carried by cosmid pIBV3, and enabled the region containing this gene to be identified. Southern blotting and hybridization were carried out as described in Example 5.1.4.
The hybridization results enabled it to be shown that cosmid pIBV3 possessed a 6.6-kb SphI fragment containing the sequence homologous to the probes synthesized in Example 5.5.4.
5.6. Isolation of cosmid pIBV4 containing the structural gene for FMN reductase (snaC)
This example illustrates how it is possible to obtain a cosmid as constructed in Example 3 containing at least one gene for the biosynthesis of PII.
5.6.1. Identification of FMN reductase associated with pristinamycin IIA synthase
This example illustrates how the protein responsible for reduction of FMN by NADH to form the FMNH.sub.2 needed for the reaction catalysed by pristinamycin IIA synthase may be purified to homogeneity from S. pristinaespiralis SP92.
5.6.1.A. Assay of FMN reductase activity
This example illustrates the assay of an activity of the biosynthesis pathway of pristinamycin IIA which has never before been described and which possesses the noteworthy property of being expressed only during the period of production of pristinamycins. The enzyme in question is FMN reductase, also referred to as NADH:FMN oxidoreductase, which catalyses the reduction of FMN to FMNH.sub.2 (FIG. 13) in the presence of NADH. FMN reductases catalysing the same reaction which are specific or otherwise for NADH or NADPH and associated with other biosynthesis pathways have been described elsewhere (Duane et al., 1975, Jablonski et al., 1977, Watanabe et al., 1982).
Two assays are used to detect this activity:
The first is based on a coupling with the pristinamycin IIA synthase described in Example 5.1.1., and is used for the first steps of the purification. The enzyme fractions to be assayed (0.002 to 0.005 units) are incubated for 1 hour at 27.degree. C. in a total volume of 500 .mu.l of 50 mM bis-tris propane buffer pH 6.8 containing NADH (500 .mu.M), FMN (2 .mu.M), pristinamycin IIB (20 .mu.M) and 0.01 units of pristinamycin IIA synthase described in Example 5.1.1. The pristinamycin IIA formed is assayed by HPLC as described in Example 5.1.1.A.
The unit of enzymatic activity is defined as the amount of enzyme needed to synthesize 1 .mu.mol of pristinamycin IIA per minute under the conditions described above.
The second assay is a spectrophotometric assay, and can be employed only with at least partially purified fractions. The enzyme fractions to be assayed (0.006 to 0.030 units) are incubated for 13 min at 27.degree. C. in a total volume of 3 ml of 50 mM bis-tris propane buffer pH 6.8 containing NADH (500 .mu.M) and FMN (2 .mu.M). After 7 min of incubation, 6 readings of the optical density of 340 nm taken at 1-min intervals are performed against a reference curve without enzyme. The activity in .mu.mol/min is calculated by dividing the slope of decrease per min in the optical density by a factor of 6.2 (optical density of 1 mol of NADH at 340 nm).
The unit of enzymatic activity is defined as the amount of enzyme needed to consume 1 .mu.mol of NADH per minute under the conditions described above.
5.6.1.B. Purification of S. pristinaespiralis SP92 FMN reductase
This experiment illustrates how an enzyme of S. pristinaespiralis SP92 participating in the biosynthesis pathway of pristinamycin IIA may be purified.
Using the assays described above in Example 5.6.1.A., the purification of FMN reductase is carried out as described below, taking care to freeze and store the active fractions at -30.degree. C. between successive steps if necessary.
500 g of a centrifugation pellet, washed with 0.1 M phosphate buffer pH 7.2 containing 10% v/v of glycerol, of an S. pristinaespiralis SP92 culture harvested at the beginning of the pristinamycin production phase are taken up with 1500 ml of 50 mM bis-tris propane buffer pH 6.8 containing 5 mM DTT, 10% v/v of glycerol and 0.2 mg/ml of lysozyme. The suspension thereby obtained is incubated for 45 min at 27.degree. C. and then centrifuged at 50,000 g for 1 hour. The crude extract thereby collected is fractionated by ammonium sulphate precipitation. The protein fraction precipitating at between 40 and 75% saturation is desalted on a column of Sephadex G-25 Fine and then injected in pH 6.8 50 mM bis-tris propane buffer, 5 mM DTT, 10% v/v glycerol onto a column (300 ml) of Q Sepharose Fast Flow. The active proteins are not retained on the column, and they are desalted on a column of Sephadex G-25 Fine and then reinjected in pH 8.2 50 mM Tris-HCl buffer, 5 mM DTT, 10% v/v glycerol onto a column (35 ml) of Q Sepharose Fast Flow and eluted with a linear KCl gradient (0 to 0.5 M). The fractions containing the enzymatic activity (detected by means of the first test described in Example 5.6.1.A) are pooled, desalted on a column of Sephadex G-25 Fine and then injected in pH 8.2 50 mM Tris-HCl buffer, 5 mM DTT, 10% v/v glycerol onto a MonoQ HR 10/10 column. The proteins retained are eluted directly by the same buffer to which 0.2 M KCl has been added. They are collected in a volume of 1 ml, which is immediately reinjected onto a column of Superdex 75 HR 10/30 eluted with pH 6.8 50 mM bis-tris propane buffer, 1 mM DTT, 10% v/v glycerol. The fractions containing the desired activity (detected from this step onwards by means of the spectrophotometric test as described in Example 5.6.1.A) are pooled and the total volume of the pool is made to 7 ml; these 7 ml are injected onto a column packed with 8 ml of FMN-agarose; the column is washed with pH 6.8 50 mM bis-tris propane buffer, 1 mM DTT, 10% v/v glycerol, and then eluted with the same buffer containing 10 .mu.M FMN. The active fractions are pooled, desalted on PD-10 columns, injected in pH 8.2 50 mM Tris-HCl buffer, 5 mM DTT, 10% v/v glycerol onto a MonoQ HR 5/5 column and eluted with a linear KCl gradient (0 to 0.25 M).
After this step, the enzyme is pure. In SDS-PAGE electrophoresis, a single fairly broad and is seen, centred at a molecular weight estimated at 28,000, while, in Bio-Sil SEC 125 gel permeation chromatography, this protein forms a symmetrical peak centred around a molecular weight of approximately 30,000.
For sequencing, the protein is desalted on a 25-cm Vydac C4 column eluted with a linear gradient of from 30 to 70% of acetonitrile in water containing 0.07% of trifluoroacetic acid.
TABLE 7______________________________________Purification of FMN reductasePurification Vol. Protein Sp. Act..sup.a,b Yield PurificationSteps (ml) (mg) (units/mg) (%) factor______________________________________Crude extract 1620 5100 0.004.sup.a 100 140-75% A.S. 155 2930 0.005.sup.a 68 1.2Q seph. pH 6.8 357 180 0.058.sup.a 49 14Q Seph. pH 8.2 153 15 0.36.sup.a 25 85MonoQ HR 10/10 1.0 8.8 0.50.sup.a 19 120 4.4.sup.bSuperdex 75 1.5 3.1 7.4.sup.b 12 200FMN-agarose 7.5 0.28 96.sup.b 14 2600MonoQ HR 5/5 3.0 0.29 68.sup.b 11 1900Bio-Sil 125 7.5 0.18 106.sup.b 10 2900______________________________________ .sup.a assay coupled to pristinamycin IIA synthase .sup.b spectrophotometric assay
The purification factor is calculated from the increase in specific activity of the fractions during the purification.
5.6.2. Production of oligonucleotides from the protein sequence
This example describes how, starting from NH.sub.2 -terminal and internal sequences of the protein FMN reductase, it is possible to synthesize oligonucleotides.
The NH.sub.2 -terminal sequence of FMN reductase was deduced by microsequencing as described in Example 5.1.2. About 30 residues were identified in this way.
(NH.sub.2 -Terminal sequences beginning at the 4th and at the 11th residue were also found in the sample sequenced.)
Two sequences internal to FMN reductase, of 13 and 21 amino acids, were also identified after trypsin hydrolysis and purification of fragments obtained on a Vydac C18 column.
NH.sub.2 -Terminal sequence of the protein FMN reductase(See residues 2 to 25 on SEQ ID NO:23)1 5 10 15 20 25T G A D D P A R P A V G P Q S F R D A M A Q L A S P VInternal sequence of the protein FMN reductase:(See residues 102 to 122 on SEQ ID NO:23)1 5 10 15 20F A G G E F A A W D G T G V P Y L P D A K(See residues 149 to 161 on SEQ ID NO:23)1 5 10T G D P A K P P L L W Y R
From the underlined regions in each of the sequences, and in accordance with the degeneracy of the genetic code specific to Streptomyces (see Example 8), the following mixtures of oligonucleotides were synthesized:
Mixture corresponding to the NH.sub.2 -terminal sequence of theprotein FMN reductase:5' 3'TTC CGC GAC GCC ATG GCC CAG CTC GC (SEQ ID NO:43) G G G GMixtures corresponding to the internal sequences of theprotein FMN reductase:5' 3'TTC GCC GGC GGC GAG TTC GCC GCC TGG GAC GGC ACC GG (SEQ ID NO:44) G G G G G G5' 3'GAC CCC GCC AAG CCC CCC CTG CTG TGG TAC CG (SEQ ID NO:45) G G G G C C
5.63. Labelling of the mixtures of synthetic oligonucleotides and hybridization of the genomic DNA libraries of S. pristinaespiralis SP92.
This example describes how oligonucleotides specific for a gene for the biosynthesis of pristinamycins may be radioactively labelled and then hybridized with membranes onto which DNA of genomic libraries of S. pristinaespiralis has been transferred.
Labelling the oligonucleotides is carried out by transfer at the 5'-terminal position of the [.gamma.-.sup.32 P]phosphate group of ATP with T4 polynucleotide kinase, as described in Example 5.1.3.
Approximately 2.times.500 ng of each mixture of oligonucleotides were labelled in this way with .sup.32 P and were used to hybridize each of the two libraries.
Hybridization of the membranes of each library was carried out as described in Example 5.1.3.
5.6.4. Isolation of cosmid pIBV4 and determination of the region containing the structural gene for FMN reductase (snaC)
Cosmid pIBV4 was isolated from a clone of the library produced in E. coli strain HB101 which hybridized with all three mixtures of oligonucleotides simultaneously.
This cosmid was purified as described in Example 2. It contains a genomic DNA insert of S. pristinaespiralis SP92 whose size was estimated at 48 kb. A map (FIG. 7) was established from digestions with different restriction enzymes, as described in 5.1.4.
Southern hybridizations of pIBV4 DNA, digested by means of different enzymes, with the mixtures of oligonucleotides enabled the region containing snaC, the structural gene for FMN reductase, to be identified. Southern blotting and hybridizations were carried out as described in Example 5.1.4.
The hybridization results enabled it to be shown that cosmid pIBV4 possessed a 4-kb BamHI-BamHI fragment containing the sequences homologous to the probes synthesized in Example 5.6.3.
5.7 Demonstration of the presence of the structural gene for p-aminophenylalanine (phenyl-N)-methyltransferase on cosmid pIBV2
This example illustrates how it is possible, starting from a purified protein, to identify the corresponding structural gene from among the genes which have already been analyzed and sequenced as described in Examples 6.7 and 7.8 and which have also been expressed in E. coli as described in Example 11.
5.7.1. Identification and purification of the protein involved in the methylation of p-aminophenylalanine to p-dimethylaminophenylalanine
This examples illustrates how the protein responsible for the methylation of p-aminophenylalanine to p-dimethylaminophenylalanine [p-aminophenylalanine (phenyl-N)-methyltransferase] may be purified to homogeneity from S. pristinaespiralis strain SP92, and how it may also be obtained pure from a recombinant strain of E. coli.
5.7.1. A. Assay of the activity of methylation of p-aminophenylalanine to p-methylaminophenylalanine and of the activity of methylation of p-methylaminophenylalanine to p-dimethylaminophenylalanine
This examples illustrates the assay of two terminal activities of the biosynthesis of p-dimethylaminophenylalanine, a component of pristinamycin IA. These activities have never before been described, and possess the noteworthy property of being expressed only during the period of production of pristinamycins. They are the methylation of p-aminophenylalanine to p-methylaminophenylalanine (methylation 1) on the one hand, and the methylation of p-methylaminophenylalanine to p-dimethylaminophenylalanine (methylation 2), both of these activities utilizing SAM as a methyl group donor (FIG. 14).
The enzyme fractions to be assayed (1 to 20 units) are incubated for 30 min at 27.degree. C. in a total volume of 200 .mu.l of pH 6.8 50 mM bis-tris propane buffer containing SAM (200 .mu.M) in which the methyl group is radioactively labelled with isotope 14 of the carbon atom (2Ci/mol), in the presence of p-amino-L-phenylalanine (1mM) for the assay of methylation 1 or of p-methylamino-L-phenylalanine (2.5 mM) for the assay of methylation 2.
The reaction is stopped by adding 16 .mu.l of 37% hydrochloric acid and then 20 .mu.l of sodium heptane sulphonate as a concentration of 240 g/l. After centrifugation, 150 .mu.l of supernatant are injected into the HPLC system in the following gradient mode:
______________________________________mobile phase: eluent A = 1.2 g of sodium heptanesulphonate + 2.5 ml of glacial acetic acid + water (qs 1000 ml) eluent B = 1.2 g of sodium heptanesulphonate + 2.5 ml of glacial acetic acid + 300 ml of acetonitrile + water (qs 1000 ml) gradient: t(min) %B 0 30 16 30 17 100 20 100 21 30 25 30stationary phase: 150 .times. 4.6 mm Nucleosil 5 .mu.m C18 column (Macherey-Nagel)______________________________________
At outflow from the column, the substrates and products of the enzymatic reaction are quantified by absorption at 254 nm. This detection is coupled to an in-line radiochemical detection by means of a Berthold LB506 detector equipped with a type GT400-U4 solid scintillation cell. This enables the incorporation of radioactive methyl groups into the reaction products to be monitored specifically.
The unit of enzymatic activity for methylation 1 (for methylation 2) is defined as the amount of enzyme needed to incorporate 1 nmol of methyl groups into p-aminophenylalanine (into p-methylaminophenylalanine).
5.7.1. B. Purification from S. pristinaespiralis SP92 of the SAM-dependent N-methyltransferase catalyzing the methylation of p-aminophenylalanine to p-dimethylaminophenylalanine [p-aminophenylalanine (phenyl-N)-methyltransferase]
This experiment illustrates how an enzyme of S. pristinaespiralis SP92 participating in the biosynthesis pathway of pristinamycin IA may be purified.
Using the assay described above in Example 5.7.1.A, the purification of the SAM-dependent N-methyltransferase is carried out as described below, taking care to freeze and store the active fractions at -70.degree. C. between successive steps if necessary.
240 g of a centrifugation pellet, washed with pH 7.2 100 mM phosphate buffer, 1 mM PMSF, 5 mM EDTA, 5 mM EGTA, 0.5 M KCl, 10% v/v glycerol, of an S. pristinaespiralis SP92 culture harvested at the beginning of the pristinamycin production phase are taken up in 480 ml of pH 8.0 0.1 M Tris-HCl buffer containing 4 mM DTE, 5 mM benzamidine, 0.2 mM Pefabloc, 100 .mu.g/l E-64, 2 mg/l leupeptin, 1 mM EDTA, 1 mM EGTA, 2 mg/l STI, 2 mg/l aprotinin, 20% v/v glycerol and 2 mg/ml lysozyme, this buffer being maintained at +4.degree. C. The suspension thereby obtained is stirred vigorously at 4.degree. C. After 30 min of stirring, 0.2 mg/ml deoxyribonuclease I and 5 mM MgCl.sub.2 are added. After 90 min of stirring, the extract is centrifuged for 1 hour at 50,000 g. The supernatant is divided into 3 fractions of approximately 180 ml. Each one is desalted by gel permeation on a 500 ml column of Sephadex G-25 Fine equilibrated at the natural flow rate in pH 6.8 20 mM bis-tris buffer containing 4 mM DTE, 5 mM benzamidine, 0.2 mM Pefabloc, 100 .mu.g/l E-64, 2 mg/l leupeptin, 1 mM EDTA, 1 mM EGTA, 2 mg/l STI, 2 mg/l aprotinin, 20% v/v glycerol. The protein eluate is then chromatographed (400 mg of protein at each cycle) on a MonoQ HR 16/10 column at a flow rate of 6 ml/min with an increasing linear gradient of sodium chloride (0 to 0.3 M) in pH 6.8 20 mM bis-tris buffer containing 4 mM DTE, 2 mM benzamidine, 100 .mu.g/l E-64, 2 mg/l leupeptin, 20% v/v glycerol. At outflow from the column, the fractions are supplemented with 10% v/v of pH 6.8 20 mM bis-tris buffer containing 4 mM DTE, 30 mM benzamidine, 2 mM Pefabloc, 100 .mu.g/l E-64, 2 mg/l leupeptin, 5 mM EDTA, 5 mM EGTA, 10 mg/l STI, 10 ml/l aprotinin, 20% v/v glycerol. Under these conditions, both methylation activities (1 and 2) are detected identically in the exclusion fractions and the first elution fractions. These fractions are pooled and concentrated by ultrafiltration on CentriPrep 10. This concentrate is made to 0.85 M ammonium sulphate and then chromatographed (20 to 80 mg at each cycle) on a Phenyl Superose HR 10/10 column at a flow rate of 1 ml/min with a decreasing linear gradient of ammonium sulphate (0.85 to 0M) in pH 6.8 50 mM bis-tris buffer containing 4 mM DTE, 2 mM benzamidine, 100 .mu.g/l E-64, 2 mg/l leupeptin, 1 mM EGTA, 10% v/v glycerol. At outflow from the column, the fractions are supplemented with 10% v/v of pH 6.8 50 mM bis-tris buffer containing 4 mM DTE, 30 mM benzamidine, 2 mM Pefabloc, 100 .mu.g/l E-64, 2 mg/l leupeptin, 1 mM EDTA, 1 mM EGTA, 10 mg/l STI, 10 mg/l aprotinin, 10% v/v glycerol. Under these conditions, both methylation activities (1 and 2) are detected identically in the elution fractions corresponding to approximately 0.15 M ammonium sulphate. These fractions are pooled, concentrated by ultrafiltration on Centricon 10, desalted on PD-10 columns equilabrated in pH 8.2 (at 5.degree. C.) 50 mM Tris buffer containing 4 mM DTE, 2 mM benzamidine, 100 .mu.g/l E-64, 2 mg/l leupeptin, 20% v/v glycerol, and the chromatographed (approximately 10 mg at each cycle) on a MonoQ HR 5/5 column equilibrated in the same buffer at a flow rate of 1 ml/min. Under these conditions, the two activities are not retained on the column. At outflow from the column, the exclusion fractions hence containing these two activities are supplemented with 10% v/v of pH 8.2 50 mM Tris buffer containing 4 mM DTE, 30 mM benzamidine, 2 mM Pefabloc, 100 .mu.g/l E-64, 2 mg/l leupeptin, 1 mM EDTA, 1 mM EGTA, 20% v/v glycerol. These fractions are then concentrated by ultrafiltration on Centricon 10 and thereafter chromatographed on a 300.times.7.5 mm 10 .mu.m TSK G2000 SW column equilibrated at a flow rate of 0.5 ml/min in pH 7.0 50 mM Hepes buffer containing 4 mM DTE, 0.2 mM Pefabloc, 1 mM EDTA, 1 mM EGTA, 10% v/v glycerol, 0.15 M sodium chloride. The two activities co-elute in this technique at a retention time corresponding to a molecular weight close to 30,000. After this step, a preponderant protein is visible in SDS-PAGE. It is located at around 32,000.
TABLE 8______________________________________Purification of the enzyme methylatingp-aminophenylalanine to p-dimethylaminophenylalaninePurification Vol. Protein SP.Act. Yield PurificationSteps (ml) (mg) (units.sup.a /mg) (%) factor______________________________________Crude extract 510 1800 29 -- --G-25 Fine 560 1560 34 102 1.17MonoQ HR 16/10 670 82 430 67 14.8Phenyl Superose 10 3.48 6300 42 217MonoQ HR 5/5 7 0.88 17200 29 593TSK G2000 0.8 0.113 40300 8.7 1390______________________________________
This refers to units of enzymatic activity for methylation 1. At each step, the value of the units of enzymatic activity for methylation 2 was equal to 120% of that of the units for methylation 1.
The purification factor is calculated from the increase in specific activity of the fractions during the purification.
5.7.1.C. Purification from E. coli pVRC706 of the recombinant protein of S. pristinaespiralis SP92 displaying the SAM-dependent N-methyltransferase activity catalysing the methylation of p-aminophenylalanine to p-dimethylaminophenylalanine.
This experiment illustrates how an enzyme of S. prisinaespiralis SP 92 participating in the biosynthesis pathway of pristinamycin IA and expressed in E. coli by cloning of the papM gene may be purified.
Using the assay described above in Example 5.7.1.A., we showed that crude extracts of the recombinant strain E. coli::pVRC706 display a strong activity for methylation 1 and for methylation 2, whereas in the control E. coli strain (pMTL23) neither of these two activities was detected. The purification of the SAM-dependent p-aminophenylalanine (phenyl-N)-methyltransferase catalyzing the methylation of p-aminophenylalanine to p-dimethylaminophenylalanine was then carried out.
Under the same conditions as those described in Example 5.7.1.B., except for a chromatography step which was eliminated (step of purification on MonoQ HR 5/5), we purified to homogeneity a protein which possesses a molecular weight in chromatography on a TSK G2000 column and in SDS-PAGE identical to those possessed by the protein purified in Example 5.7.1.B.
TABLE 9______________________________________Purification of the enzyme methylatingp-aminophenylalanine to p-dimethylaminophenylalaninefrom E. coli strain pVRC706Purification Vol. Protein SP.Act. Yield PurificationSteps (ml) (mg) (units.sup.a /mg) (%) factor______________________________________Crude extract 15 190 235 -- --G-25 Fine 22 175 231 91 1MonoQ HR 16/10 24 13.4 2100 63 8.9Phenyl Superose 3.0 0.39 35500 31 145TSK G2000 0.8 0.092 45200 9.3 192______________________________________
This refers to units of enzymatic activity for methylation 1. At each step, the value of the units of enzymatic activity for methylation 2 was equal to 120% of that of the units for methylation 1.
The purification factor is calculated from the increase in specific activity of the fractions during the purification.
5.7.2. Identification of the structural gene for p-aminophenylalanine (phenyl-N)-methyltransferase
The NH.sub.2 -terminal sequence of the 32,000 protein purified in Example 5.7.1. B was determined by microsequencing as described in Example 5.1.2. Ten residues were determined:
TAAAPTLAQA
The NH.sub.2 -terminal sequence of the 32,000 protein purified in Example 5.7.1.C was determined by microsequencing as described in Example 5.1.2. Ten residues were determined:
TAAAPTLAQA
In both cases the same residues are found, and this sequence corresponds exactly to the beginning of the protein sequence which is deduced from the sequence of the papM gene (see residues 2 to 11 on SEQ ID NO: 26). The purified p-aminophenylalanine (phenyl-N)-methyltransferase is hence the protein PapM.
EXAMPLE 6
Subcloning of DNA fragments cloned into cosmids as prepared in Example 3 and containing the genes of interest
This example illustrates how, starting from cosmids constructed as described in Example 3 and containing genes for the biosynthesis of pristinamycins II or pristinamycins I, it is possible to subclone DNA fragments containing these genes.
These subclonings were performed in order to be able to deduce subsequently the nucleic acid sequence of the genes identified, as well as to carry out the different construction presented in the examples which follow.
6.1. Isolation of the 5.5-kb EcoRI-BglII fragment containing the structural gene for 3-hydroxypicolinic acid:AMP ligase
This example describes how, starting from cosmid pIBV2 containing the structural gene for 3-hydroxypicolinic acid:AMP ligase, it is possible to subclone a DNA fragment of smaller size containing this gene.
Approximately 10 .mu.g of cosmid pIBV2 were cut successively with the restriction enzymes BglII and EcoRI (New England Biolabs) under the conditions recommended by the supplier. The restriction fragments thereby obtained were separated by electrophoresis on 0.8% agarose gel, and the 5.5-kb BglII-EcoRI fragment was isolated by electroelution as described in Maniatis et al. (1989).
Approximately 100 ng of pUC19 (Viera and Messing 1982) cut with Bam HI and EcoRI were ligated with 200 ng of the 5.5-kb BglII-EcoRI fragment under the conditions described in Example 3.3.
After transformation of the strain TG1 and selection of the transformants on solid LB medium containing 150 .mu.g/ml of ampicillin and 20 .mu.g/ml of X-gal according to the technique described by Maniatis et al. (1989), a clone carrying the desired fragment was isolated. The recombinant plasmid was designated pVRC402. Its restriction map is presented in FIG. 15(A). It was shown by hybridization, in Example 5.2., that the 5.5-kb EcoRI-BglII fragment contains the structural gene for S. pristinaespiralis SP92 3-hydroxypicolinic acid:AMP ligase. Plasmid pVRC402 hence contains the structural gene for S. pristinaespiralis SP92 3-hydroxypicolinic acid:AMP ligase.
6.2. Isolation of 4.6-kb BglII-BglII fragment from cosmid pIBV2
This example describes how, starting from cosmid pIBV2, it is possible to subclone a DNA fragment of smaller size for the purpose of identifying, in the regions adjacent to the structural gene for 3-hydroxypicolinic acid:AMP ligase, the presence of other genes involved in the biosynthesis of pristinamycins I.
The different cloning steps were carried out as described above.
Approximately 10 .mu.g of cosmid pIBV2 were cut with BglII. The restriction fragments thereby obtained were separated by electrophoresis on 0.8% agarose gel, and the 4.6-kb BglII-BglII fragment was isolated by electroelution.
Approximately 100 ng of pUC19 cut with BamHI were ligated with 200 ng of the BglII-BglII fragment.
A clone carrying the desired fragment was isolated after transformation of the strain TGl as described in Example 6.1. The recombinant plasmid was designated pVRC501. Its restriction map is presented in FIG. 15(B).
6.3. Isolation of the 6-kB BamHI-BamHI fragment containing the structural genes for the two subunits of pristinamycin IIA synthase
This example describes how, starting from cosmid pIBVl, it is possible to subclone a DNA fragment of smaller size containing the structural gene for the two subunits of pristinamycin IIA synthase.
The different cloning steps were carried out as described above.
Approximately 10 .mu.g of cosmid pIBV1 were cut with BamHI. The restriction fragments thereby obtained were separated by electrophoresis on 0.8% agarose gel, and the 6-kb BamHI fragment was isolated by electroelution.
Approximately 100 ng of pBKS.sup.- (Stratagene Cloning Systems, La Jolla Calif.) cut with Bam HI were ligated with 200 ng of the 6-kb BamHI fragment.
A clone carrying the desired fragment was isolated after transformation of the strain TG1. The recombinant plasmid was designated pXL2045. Its restriction map is presented in FIG. 16. It was shown by hybridization, in Example 5.1, that the 6-kb BamHI fragment contains the snaA and snaB genes coding for the two subunits of S. pristinaespiralis SP92 pristinamycin IIA synthase. Plasmid pXL2045 hence contains the snaA and snaB genes coding for the two subunits of S. pristinaespiralis SP92 pristinamycin IIA synthase.
6.4. Isolation of the 6.2-kb SphI fragment containing a portion of the structural gene for pristinamycin I synthase II
This example describes how, starting from cosmid pIBV3, it is possible to subclone a DNA fragment of smaller size containing a portion of the structural gene for pristinamycin I synthase II.
The different cloning steps were carried out as described above.
Approximately 10 .mu.g of cosmid pIBV3 were cut with SphI. The restriction fragments thereby obtained were separated on 0.8% agarose gel, and the 6.2-kb SphI fragment was isolated by the technique of the "Geneclean" kit marketed by the company Bio101-Ozyme.
Approximately 100 ng of pUC19 cut with SphI were ligated with 200 ng of the 6.2-kb SphI fragment.
A clone carrying the desired fragment was isolated after transformation of the strain TG1. The recombinant plasmid was designated pVRC1105. Its restriction map is presented in FIG. 17.
6.5. Isolation of the 8.4-kb SphI fragment containing a portion of the structural gene for pristinamycin I synthase III
This example describes how, starting from cosmid pIBV3, it is possible to subclone a DNA fragment of smaller size containing a portion of the structural gene for pristinamycin I synthase III.
The different cloning steps were carried out as described above.
Approximately 10 .mu.g of cosmid pIBV3 were cut with SphI. The restriction fragments thereby obtained were separated on 0.8% agarose gel, and the 8.4-kb SphI fragment was isolated by the technique of the "Geneclean" kit marketed by the company Bio101-Ozyme.
Approximately 100 ng of pUC19 cut with SphI were ligated with 200 ng of the 8.4-kb SphI fragment.
A clone carrying the desired fragment was isolated after transformation of the strain TG1. The recombinant plasmid was designated pVRC1106. Its restriction map is represented in FIG. 18.
6.6. Isolation of a 6.6-kb SphI fragment containing a portion of the structural gene for pristinamycin I synthase IV
This example described how, starting from cosmid pIBV3, it is possible to subclone a DNA fragment of smaller size containing a portion of the structural gene for pristinamycin I synthase IV.
The different cloning steps were carried out as described above.
Approximately 10 .mu.g of cosmid pIBV3 were cut with SphI. The restriction fragments thereby obtained were separated on 0.8% agarose gel, and the 6.6-kb SphI fragment was isolated by the technique of the "Geneclean" kit marketed by the company Bio101-Ozyme.
Approximately 100 ng of pUC19 cut with SphI were ligated with 200 ng of the 6.6-kb SphI fragment.
A clone carrying the desired fragment was isolated after transformation of the strain TG1. The recombinant plasmid was designated pVRC1104. Its restriction map is presented in FIG. 19.
6.7 Isolation of the 17-kb HindIII-HindIII fragment containing cosmid pHC79 and carrying the genes located upstream of 3-hydroxypicolinic acid:AMP ligase (pristinamycin I synthase I)
This example describes how, starting from cosmid pIBV2 containing the structural gene for 3-hydroxypicolinic acid:AMP ligase, it is possible to delete a large portion of this cosmid and retain only the portion located upstream of 3-hydroxypicolinic acid:AMP ligase.
Approximately 200 ng of cosmid pIBV2 were cut with the restricted enzyme HindIII. The enzyme was denatured for 30 min at 85.degree. C. as recommended by the supplier. Cosmid pIBV2 digested in this way was precipiated with ethanol as described in Maniatis et al. (1989) and religated with itself in a volume of 50 .mu.l.
After transformation of the strain TG1 and selection of the transformants on solid LB+150 .mu.g/ml of amplicillin according to the technique described by Maniatis et al. (1989), a clone containing cosmid pHC79 and the portion located upstream of 3-hydroxypicolinic acid:AMP ligase (the whole corresponding to a size of approximately 17 kb) was isolated. The recombinant plasmid was designated pVRC900. Its restriction map is presented in FIG. 20.
6.8. Isolation of the 1.4-kb BamHI-SstI fragment originating from cosmid pIBV3
This example describes how, starting from cosmid pIBV3 containing the snaA gene coding for the large subunit of PIIA synthase, it is possible to subclone a DNA fragment located upstream in order to study and sequence it.
Approximately 10 .mu.g of cosmid pIBV3 were cut successively with the restriction enzymes SstI and BamHI. The restriction fragments thereby obtained were separated on 0.8% agarose gel, and the 1.4-kb BamHI-SstI fragment was isolated by the technique of the "Geneclean" kit marketed by the company Bio101-Ozyme.
Approximately 100 ng of pDH5 (Hilleman et al. 1991) cut with BamHI and SstI were ligated with 200 ng of the BamHI-SstI fragment under the conditions described in Example 3.3.
After transformation of the strain TG1 and selection of the transformants of solid LB+150 .mu.g/ml of ampicillin+X-gal according to the technique described by Maniatis et al. (1989), a clone carrying the desired fragment was isolated. The recombinant plasmid was designated pVRC1000. Its restriction map is represented in FIG. 21.
6.9. Isolation of the 4-kb BamHI-BamHI fragment containing the structural gene for FMN reductase
This example describes how, starting from cosmid pIBV4 containing the structural gene for FMN reductase (snaC), it is possible to subclone a DNA fragment of smaller size containing this gene.
The different cloning steps were carried out as described above.
Approximately 10 .mu.g of cosmid pIBV4 were cut with the restriction enzyme BamHI. The restriction fragments thereby obtained were separated on 0.8% agarose gel, and the 4-kb BamHI-BamHI fragment was isolated by electroelution.
Approximately 100 ng of pUC19 cut with BamHI were ligated with 200 ng of the 4-kb BamHI-BamHI fragment.
After transformation of E. coli strain DH5.alpha. (supE44 DlacU169 (f801acZDM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1) (Hanahan, 1983) and selection of the transformants on solid LB+150 .mu.g/ml of ampicillin+X-gal according to the technique described by Maniatis et al. (1989), a clone carrying the desired fragment was isolated. The recombinant plasmid was designated pVRC509. Its restriction map is presented in FIG. 22.
EXAMPLE 7
Sequence of the isolated DNA fragments containing the genes for the biosynthesis of pristinamycins of S. pristinaespiralis sp92
This example illustrates the sequencing of DNA fragments carrying, on the one hand genes involved in the biosynthesis of pristinamycins of the pristinamycin I family, and on the other hand genes involved in the biosynthesis of pristinamycins of the pristinamycin II family, of the S. pristinaespiralis strain.
7.1. Sequencing of a 5-kb BamHI-XhoI fragment
This example illustrates how the nucleotide sequence of a fragment containing the snaA and snaB genes of S. pristinaespiralis SP92 may be obtained.
The BamHI-KhoI fragment is part of the 6-kb BamHI-BamHI fragment which was cloned into phasmid pBKS.sup.- to give plasmid pXL2045 described in Example 6.3. Subfragments of this 5-kb BamHI-XhoI insert were then obtained by enzymatic digestion and thereafter subcloned into phages M13mp18 or M13mp19 (Messing et al, 1981) in both orientations. The subcloning sites used are the following: EcoRI, PstI, PstI, NruI, EcoRI, NruI, NotI, SalI, SstI, XhoI, SalI and XhoI, and are shown in FIG. 16.
These different inserts were sequenced by the chain-termination reaction method, using as a synthetic primer the universal primer (Maniatis et al, 1989) or oligonucleotides which are synthesized (as is described in Example 5) and are complementary to a sequence of 20 nucleotides of the insert to be sequenced.
The overlap between these different inserts enabled the total nucleotide sequence to be established on both strands of the BamHI-XhoI fragment which comprises 5392 bp (SEE ID no. 1).
7.2. Sequencing of a region of 1870 bp of the 5.5-kb EcoRI-BglII fragment
This example illustrates how the nucleotide sequence of a fragment containing the snbA gene of S. pristinaespiralis SP92 may be obtained.
The region of 1870 bp sequenced is part of the 5.5-kb EcoRI-BglII fragment which was cloned into plasmid pUC19 to give plasmid pVRC402 described in Example 6 (FIG. 15(A). Subfragments of the 5.5-kb EcoRI-BglII insert were obtained by enzymatic cleavage and then subcloned into phages M13mp18 or M13mp19 in both orientations. The subcloning sites are HindIII, PstI and HindIII, and are shown in FIG. 15(A).
The overlap between these fragments enabled the total sequence of the Sau3A-Sau3A region, which comprises 1870 bp (SEQ ID no. 5), to be established.
7.3. Sequence of a region of 1830 bp in the 4.6-kb BglII-BglII fragment
This example illustrates how the nucleotide sequence of a fragment adjacent to that which contains the snbA gene of S. pristinaespiralis SP92 may be obtained.
This sequence was deduced by subcloning the 1-kb BamHI-PstI and 2.1-kb PstI-EcoRI fragments (FIG. 15(B)) from pVRC501 (Example 6) into the vectors M13mp18 and M13mp19. The PstI site was traversed by subcloning a 423-bp Sau3A-Sau3A fragment overlapping this site, followed by sequencing. The sequence of 1830 bp thereby obtained is shown in (SEQ ID no. 6).
7.4 Sequencing of two regions of 227 bp and 247 bp of the 6.2-kb SphI fragment
This example illustrates how the nucleotide sequence of fragments containing a portion of the structural gene for pristinamycin I synthase II (snbC) of S. pristinaespiralis may be obtained.
The regions of 227 and 247 bp sequenced are parts of the 6.2-kb SphI fragment which was cloned into plasmid puC19 to give plasmid pVRC1105 described in Example 6.4 (FIG. 17). Subfragments of the 6.2-kb SphI insert were obtained by enzymatic cleavage and then subcloned into phages M18mp18 or M13mp19 in both orientations. The subcloning sites are XhoI, PstI and BglII, and are shown in FIG. 17. The 227-bp PstI-BglII fragment was sequenced completely, and 247 bp were sequenced from the 900-bp XhoI fragment: these sequences are presented in SEQ ID nos. 11 and 12.
7.5 Sequencing of two regions of 192 bp and 474 bp of the 8.4-kb SphI fragment
This example illustrates how the nucleotide sequence of fragments containing portions of the structural gene for pristinamycin I synthase III (snbD) of S. pristinaespiralis may be obtained.
The regions of 192 and 474 bp sequenced are parts of the 8.4-kb SphI fragment which was cloned into plasmid pUC19 to give plasmid pVRC1106 described in Example 6.5 (FIG. 18). Subfragments of the 8.4-kb SphI insert were obtained by enzymatic cleavage and then subcloned into phages M13mp18 or M13mp19 in both orientations. The subcloning sites are XhoI, PstI, SphI and BglII, and are shown in FIG. 18.
The 192-bp BglII-SphI and 474-bp PstI-XhoI fragments were sequenced completely: these sequences are presented in SEQ ID nos. 13 and 14.
7.6 Sequencing of two regions of 485 bp and 291 bp of the 6.6-kb SphI fragment
This examples illustrates how the nucleotide sequence of fragments containing portions of the structural gene for pristinamycin I synthase IV (snbE) of S. pristinaespiralis may be obtained.
The regions of 291 and 485 bp sequenced are parts of the 6.6-kb SphI fragment which was cloned into plasmid pUC19 to give plasmid pVRC1104 described in Example 6.6 (FIG. 19). Subfragments of the 6.6-kb SphI insert were obtained by enzymatic cleavage and then subcloned into phages M13mp18 or M13mp19 in both orientations. The subcloning sites are XhoI, PstI and SphI, and are shown in FIG. 19. The 485-bp XhoI-SphI fragment was sequenced completely, and 291 bp were sequenced from the 1500-bp PstI fragment: these sequences are presented in SEQ ID nos. 15 and 16.
7.7. Sequence of a region of 645 bp in a 3.4-kb XhoI-XhoI fragment isolated from pVRC900
This example illustrates how the nucleotide sequence of a fragment located upstream of that which contains the snbA gene of S. pristinaespiralis may be obtained.
To deduce this sequence, the 3.4-kb XhoI-XhoI fragment was subcloned beforehand into the vector pUC18 from the vector pVRC900 described in 6.7. The different cloning steps were carried out as described in 6.1:plasmid pVRC900 was digested with the restriction enzyme XhoI, and the fragments thereby obtained were separated on 0.8% agarose gel. The 3.4-kb XhoI-XhoI fragment was purified by electroelution and was ligated with pUC18 cut with the restriction enzyme SalI. After transformation into TG1, a clone carrying the 3.4-kb XhoI-XhoI fragment was isolated. The recombinant plasmid was referred to as pVRC903. Its restriction map is presented in FIG. 23.
The 645-bp sequence was then deduced by subcloning the 1.4-kb PvuII-EcoRI and 0.9-kb PvuII-EcoRI fragments (FIG. 23) from pVRC903 described above into the vectors M13mp18 and M13mp19. To carry out these clonings, the vectors M13mp18 and M13mp19 were first digested with the restriction enzyme BamHI; the cohesive ends thereby liberated were filled in with the large fragment of DNA polymerase I (Klenow: New England Biolabs) according to the technique described by Maniatis et al. (1989) so as to generate blunt ends compatible with the ends liberated by PuvII digestion; the vectors were then digested with the restriction enzyme EcoRI. The PvuII site was traversed by subcloning a 2.2-kb PstI-PstI fragment, isolated from pVRC903, overlapping this site. The sequence of 645 bp thereby obtained is shown in SEQ ID no. 9.
7.8. Sequence of a region of 1050 bp in a 4.1-kb PstI-PstI fragment isolated from pVRC900
This example illustrates how the nucleotide sequence of a fragment located upstream of that which contains the snbA gene of S. pristinaespiralis may be obtained.
To deduce this sequence, a 4.1-kb PstI-PstI fragment was subcloned beforehand into the vector pUC19 from the vector pVRC900 described in 6.7. The different cloning steps were carried out as described in 6.1. Plasmid pVRC900 was digested with restriction enzyme PstI, and the fragments thereby obtained were separated on 0.8% agarose gel. The 4.1-kb PstI-PstI fragment was purified by electroelution and was ligated with pUC19 cut with the restriction enzyme PstI. After transformation into TG1, a clone carrying the 4.1-kb PstI-PstI fragment was isolated. The recombinant plasmid was referred to as pVRC409. Its restriction map is presented in FIG. 24.
This sequence was then deduced by subcloning the 0.7-kb XhoI-XhoI and 1-kb XhoI-StuI fragments (FIG. 24) from pVCR409 described above into the vectors M13mp18 and M13mp19. The XhoI site internal to the sequence was traversed by double-strand sequencing from plasmid pVRC409. The sequence of 1050 bp thereby obtained is shown in SEQ ID no. 10.
7.9. Sequence of a region of 640 bp in the 1.4-kb BamHI-SstI fragment
This example illustrates how the nucleotide sequence of a fragment adjacent to that which contains the snA and snaB genes of S. pristinaespiralis coding for the two subunits of PIIA synthase may be obtained.
This sequence was deduced by subcloning the 1.4-kb BqmHI-SstI fragment (FIG. 21) from pVRC1000 (Example 6.8) into the vectors M13mp18 and M13mp19 (see Example 7.1). The sequence of 640 bp obtained is shown in SEQ ID no. 8.
7.10. Sequencing of the XhoI-KpnI region of 694 bp present in the 4-kb BamHI-BamHI fragment
This example illustrates how the nucleotide sequence of a fragment containing the snaC gene of S. pristinaespiralis may be obtained.
The region of 694 bp sequenced is part of the 4-kb BamHI-BamHI fragment which was cloned into plasmid pUC19 to give plasmid pVRC509 described in Example 6.9. A 694-bp XhoI-KpnI fragment, obtained by double digestion of plasmid pVRC509 with the restriction enzymes XhoI and KpnI and which hybridizes with the 3 oligonucleotide probes described in 5.6, was cloned into phages M13mp18 and m13mp19. The XhoI and KpnI subcloning sites are shown in FIG. 22.
The sequence of the 694-bp fragment thereby obtained is presented in SEQ ID no. 7.
EXAMPLE 8
Analysis of the nucleotide sequences by determination of the open reading frames
This example illustrates how it is possible to determine the open reading frames present in the nucleotide sequences defined in Example 7, and to identify the genes involved in the biosynthesis of pristinamycins I and pristinamycin II of S. pristinaespiralis SP92 as well as the polypeptides encoded by these genes.
8.1. 5-kb BamHI-XhoI fragment (pXL2045)
This example illustrates how it is possible to determine the open reading frames present within the 5-kb BamHI-XhoI fragment isolated above and sequenced as described in Examples 6 and 7.
We looked for the presence of open reading frames within the 5-kb BamHI-KhoI fragment utilizing the fact that Streptomyces DNA has a high percentage of G and C bases as well as a strong bias in the use of the codons of which the coding frames are composed (Bibb et al. 1984). The Staden and McLachlan (1982) method enables the probability of the coding frames to be calculated on the basis of the use of the codons of Streptomyces genes which are already sequenced and collated in a file containing 19673 codons obtained from the BISANCE data-processing server (Dessen et al. 1900).
This method enabled four highly probable open reading frames, which are shown in Table 9, to be characterized within the 5-kb BamHI-XhoI fragment. They are designated frames 1 to 4 according to their position starting from the BamHI site. For each one, their length in bases, their position within the fragment (the BamHI site being located at position 1) and also the molecular weight in kDa of the corresponding protein are given. Frames 1, 3 and 4 are coded by the same strand and frame 2 by the complementary strand (FIG. 16).
TABLE 10______________________________________Frame number MW in kDa ofnumber and number of of amino the proteingene name Position nucleotides acids encoded______________________________________1 (snaA) 48-1313 1266 422 46.52 2530- 1203 401 -- 13283 (snaB) (inv) 831 277 29 2692-4 (samS) 3522 1206 402 43 3558- 4763______________________________________
Frames 1 and 3 correspond respectively to the proteins SnaA (SEQ ID NO: 18) and SnaB (SEQ ID NO: 19) isolated above as described in Example 5 and for which the cloning of the genes is detailed in Example 6. In effect, the NH.sub.2 -terminal sequences of the products of ORFs 1 and 3 are identical to the NH.sub.2 -terminal sequences found for the proteins SnaA and SnaB, respectively, in Example 5.1.2, apart from the amino-terminal methionine which has been excized. Moreover, the molecular masses calculated from the sequences are comparable to the apparent molecular masses of the proteins SnaA and SnaB, estimated, respectively, in SDS-PAGE as described in Example 5.
Comparison of the product of open reading frame no. 4 with the protein sequences contained in the NBRF bank reveals a homology with various S-adenosylmethioine (or SAM) synthases, in particular of E. coli (Markham et al., (1984), of rat (Horikawa et al., 1989) and of S. cerevisiae (Thomas et al., 1988). The percentage homology values calculated over the whole of the sequence using Kanehisa's (1984) algorithm vary from 51.8 to 55.4%.
These sequence comparisons hence enable it to be demonstrated that the product of open reading frame no. 4 is an SAM synthase involved in the biosynthesis of pristinamycins I or II. This gene was designated SamS (SEQ ID no. 4).
The demonstration of the involvement of the SamS gene in the biosynthesis of pristinamycins is confirmed by the construction of the SP92 mutant disrupted in this gene, as described in Example 9.2.
Comparison of the sequence of the product of open reading frame no. 2 with the protein sequences contained in the Genpro bank reveals that an internal portion of this protein is 36% homologous with an internal portion of the first open reading frame of the insertion sequence (IS891) of Anabena (Bancroft and Wolk, 1989). This result suggests that open reading frame no. 2, designated ORF 401, belongs to an insertion sequence, and that there is hence an insertion sequence located between the snaA and snaB genes.
8.2. 1870-bp Sau3A-Sau3A fragment (pVRC4021)
This example illustrates how it is possible to determine open reading frames present within the 1870-bp Sau3A-Sau3A fragments isolated above and sequenced as described in Examples 6 and 7.
The search for open reading frames for the Sau3A-Sau3A fragment was performed as above. A single complete open reading frame could be demonstrated in this way. Its characteristics are as follows: this frame extends from position 109 to position 1858 of the Sau3A-Sau3A fragment, which corresponds to a frame of 1749 bases coding for a protein of 582 amino acids having a molecular mass of 61400 Da. This protein corresponds to the protein SnbA purified above as described in Example 5 and for which the cloning of the gene is detailed in Example 6. In effect, the NH.sub.2 -terminal sequence of the product of the ORF present on the Sau3A-Sau3A fragment is identical to the NH.sub.2 -terminal sequence found for the protein SnbA in Example 5.2. The molecular mass of 61400 Da calculated from the sequence is comparable to the apparent molecular mass of the protein SnbA, estimated at 67000 Da in SDS-PAGE and at 6000 Da by gel permeation as described in Example 5.2.1.B.
The snbA gene hence codes for the enzyme which catalyses the formation of the acyladenylate 3-hydroxypicolinyl-AMP from one molecule of 3-hydroxypicolinic acid and one molecule of ATP: 3-hydroxypicolinic acid:AMP ligase (SEQ ID no. 5).
8.3. 1830-bp fragment (pVRC501)
This example illustrates how it is possible to determine the open reading frames present within the 1830-bp fragment sequenced from the 3.1-kb BamHI-EcoRI fragment isolated above.
The search for open reading frames for the 1830-bp fragment was performed as above. A single complete open reading frame could be demonstrated in this way. Its characteristics are as follows: the probable beginning of this frame is located at position 103 and the end at position 1686 of the region of 1830 bp sequenced from the BamHI-EcoRI fragment, which corresponds to a protein of 528 amino acids having an approximate molecular weight of 54000.
Comparison of the sequence of this protein with the sequences contained in the Genepro bank reveals that it is homologous to proteins having a transport function for various metabolites, in particular for tetracycline in various microorganisms (Khan and Novick, 1983; Hoshino et al., 1985), actinorhodine (Fernandez-Moreno et al., 1991) and methylenomycin (Neal and Chater, 1987b) in S. coelicolor.
These data indicate that the product of the open reading frame contained in the 3.1-kb BamHI-EcoRI fragment is a transport protein enabling pristinamycins I (and possibly pristinamycins II) to be exported out of the cell. This protein was designated SnbR (SEQ ID NO: 22) and the corresponding gene snbR (SEQ ID no. 6).
Analysis of the hydrophobicity profile of the protein SnbR by the method of Kyte and Doolittle (1982) corroborates its membrane localization and hence its transport function.
8.4. 1050-bp fragment (pVRC409)
This example illustrates how it is possible to determine the open reading frames present within the 1050-bp fragment sequenced above from pVRC409 as described in Example 7.8.
The search for open reading frames for the 1050-bp fragment was performed as above. A single complete open reading frame could be demonstrated in this way. Its characteristics are as follows: this phase extends from position 84 to position 962 of the sequenced portion, which corresponds to a frame of 878 bases coding for a protein of 292 amino acids having a molecular mass of 32000 Da. This protein was referred to as protein PapM. It was, moreover, purified from S. pristinaespiralis strain SP92 as described in Example 5. The molecular mass of 32000 Da calculated from the sequence is identical to the apparent molecular mass of 32000 Da estimated on SDS-PAGE as described in Example 5. Moreover, the NH.sub.2 -terminal sequence of this protein, deduced as described in Example 5, corresponds well to the NH.sub.2 -terminal sequence of the protein PapM (SEQ ID NO: 26) identified by analysis of the open reading frames of the sequence of 1050 bp (SEQ ID no. 10).
8.5. 220-bp and 247-bp fragments (pVRC1105)
This example illustrates how it is possible to determine the open reading frames present within the 227-bp and 247-bp fragments sequenced from pVRC1105 as described in Examples 6 and 7.
The search for open reading frames for these two fragments was performed as above. An incomplete reading frame could be demonstrated in both cases over the whole length of the fragment.
The sequence obtained from the open reading frame identified on the 247-bp fragment isolated from the 900-bp XhoI fragment contains one of the internal sequences of the protein SnbC purified as described in Example 5.
Comparison of the product of the open reading frames identified on the 227-bp and 247-bp fragments isolated from pVRC1105 with sequences of the Genpro bank reveals that they are homologous to peptide synthases. The one deduced from the 227-bp fragment displays 24.5% homology with Acremonium chrysogenum (.alpha.-aminoadipyl)cysteinylvaline synthetase (Gutierrez et al. 1991). The one deduced from the 247-bp fragment displays 34.9% homology with Bacillus gramicidin S synthase II (Hori et al. 1991) and 28% homology with Acremonium chrysogenum (.alpha.-aminoadipyl)cysteinylvaline synthetase (Gutierrez et al. 1991).
This confirms that cosmid pIBV3 isolated in Example 5.1 does indeed contain a portion of the structural gene for pristinamycin I synthase II described in Example 5.3, designated SnbC (SEQ ID NO: 27 and SEQ ID NO: 28).
8.6. 192-bp and 474-bp fragments (pVRC1106)
This example illustrates how it is possible to determine the open reading frames present within the 192-bp and 474-bp fragments sequenced from pVRC1106 as described in Examples 6 and 7.
To search for open reading frames for these two fragments was performed as above. An incomplete reading frame could also be demonstrated on the 192-bp fragment isolated from pVRC1106. Its characteristics are as follows: this frame begins at position 29 of the portion sequenced in the direction of BGlII. No stop codon was identified, indicating that this open frame is not terminated.
The sequence obtained from the open reading frame identified on the 192-bp BglII-SphI fragment contains the internal sequence of the protein SnbD purified as described in Example 5, which proves, in fact, to be the NH.sub.2 -terminal sequence of the protein.
An incomplete reading frame could be demonstrated over the whole length of the 474-bp XhoI-PstI fragment.
Comparison of the product of the open reading frame identified on the 474-bp fragment isolated from pVRC1106 with the sequences of the Genpro bank reveals that this protein fragment displays from 30 to 40% homology with peptide synthases, for example 39.4% with Bacillus gramicidin S synthase II (Hori et al. 1991) and and 34% with Acremonium chrysegenum (.alpha.-aminoadipyl)cysteinylvaline synthetase (Gutierrez et al. 1991).
This confirms that cosmid pIBV3 isolated in Example 5.1 does indeed contain a portion of the structural gene for pristinamycin I synthase III described in Example 5.4, designated SnbD (SEQ ID nos. 13 and 14).
8.7. 291-pb and 485-bp fragments (pVRC1104)
This example illustrates how it is possible to determine the open reading frames present within the 291-bp and 485-bp fragments sequenced from pVRC1104 as described in Examples 6 and 7.
The search for open frames for these two fragments was performed as above. An incomplete reading frame could be demonstrated in both cases over the whole length of the fragment.
The sequence obtained from the open frame identified on the 291 fragment isolated from the 1450-bp PstI fragment contains the internal sequence of the protein SnbE purified as described in Example 5.
Comparison of the product of the open frame identified on the 485-bp XhoI-SphI fragment isolated from pVRC1104 with the sequences of the Genpro bank reveals that it is homologous to peptide synthases, for example 34.7% homologous with Bacillus gramicidin S synthase II (Hori et al. 1991) and 36.2% with Acremonium chrysogenum (.alpha.-aminoadipyl)cysteinylvaline synthetase (Gutierrez et al. 1991).
This confirms that cosmid pIBV3 isolated in Example 5.1 does indeed contain a portion of the structural gene for pristinamycin I synthase IV described in Example 5.5, designated SnbE (SEQ ID 15 and 16).
8.8 645-bp fragment (pVRC903)
This example illustrates how it is possible to determine the open reading frames present within the 645-bp fragment sequenced above from plasmid pVRC903 as described in Example 6.7.
The search for open reading frames for the 645-bp fragment was performed as above. An incomplete open reading frame could be demonstrated in this way. Its characteristics are as follows: this frame affords two possibilities for initiation of translation, a GTG at position 61 and a GTG at position 70 of the sequenced portion (the ATG located at position 124 was not taken into account owing to the sequence homologies described later). Analysis of the probabilities of the presence of Shine-Dalgarno regions does not make it possible to distinguish which of these codons corresponds to the initiation. No stop codon was identified, which indicates that this open reading frame is not terminated. The gene identified in this way was referred to as papA, and the corresponding protein was referred to as protein PapA (SEQ ID no. 9).
comparison of the product of the open reading frame identified in the 3.4-kb KhoI--KhoI fragment isolated from pVRC900 with sequences contained in the Genpro bank reveals that it is homologous to the II components of proteins of the p-aminobenzoate synthase and anthranilate synthase type, involved, respectively, in the synthesis of p-aminobenzoic acid (folic acid precursor) and in the synthesis of anthranilic acid (tryptophan precursor) of various microorganisms. It displays, in particular, a 48% homology with the protein TrpG of Azospirillum (Zimmer, W., Aparicio, C., and Elmerich, C. Mol. Gen. Genet. (1991) 229:41-51) and a 47% homology with the protein PabA of Klebsiella pneumoniae (Kaplan, J. B., Merkel, W. K. and Nichols, B. P. J. Mol. Biol. (1985) 183:327-340). The proteins TrpG and PabA carry the glutaminase activity involved in the transamination of chorismic acid. The homologies demonstrated tend to show that the protein PapA might be involved as well in the activity of transamination of chorismic acid. Chorismic acid is proposed as a precursor of p-dimethylaminophenylalanine, a component of pristinamycins I, by analogy with the synthesis of chloramphenicol, an antibiotic deduced by Streptomyces (Sharka, B., Westlake, D. W. S. and Vining, L. C. (1970Chem. Zvesti 24, 66-72).
the role of the protein PapA will be shown subsequently (Example 9.3.) by analysis of mutants of the strain SP92 in the papA gene.
8.9. 1.5-kb BamHI-SstI Fragment (PVRC1000)
This example illustrates how it is possible to determine the open reading frames present within the 1.5-kb BamHI-SstI fragment isolated above and sequenced as described in Examples 6.8 and 7.9.
The search for open reading frames for the sequenced region of 640 bp present in the 1.5-kb BamHI-SstI fragment was performed as described in Example 8.1. A single complete open reading frame could be demonstrated in this way. No initiation and no termination of translation could be demonstrated, which indicates that the sequenced region of 640 bp is probably internal to a much larger reading frame, designated snaD (SEQ ID no 8).
Comparison of the protein sequence encoded by the region of 640 bp with the protein sequences contained in the Genpro and NBRF banks reveals that this protein is 20-25% homologous to an internal portion of peptide synthases such as B. brevis gramicidin synthase I (Hori et al. 1989), B. brevis tyrocidin synthase I (Weckermann et al. 1988) and Acremonium chrysogenum ACV synthase (Gutierrez et al. (1991).
These data indicate that the protein partially encoded by the region of 640 bp is probably a peptide synthase involved in the biosynthesis of the peptide portion of pristinamycins II: in effect, all the peptide synthases involved in the biosynthesis of pristinamycins I have already been identified in other regions of the S. pristinaespiralis chromosome, as is described in Examples 5.2, 5.3, 5.4 and 5.5.
8.10. 694-bp Fragment (pVRC509)
This example illustrates how it is possible to determine the open reading frames present within the 694-bp fragment sequenced above from pVRC509 as described in Examples 6 and 7.
The search for open reading frames for the 694-bp fragment was performed as above. An incomplete open reading frame could be demonstrated in this way. Its characteristics are as follows: this frame begins at position 210 of the sequenced portion. No stop codon was identified, which indicates that this open frame is not terminated. Hence the molecular mass of the corresponding protein cannot e calculated and compared to the apparent molecular mass of 28,000 Da of the FMN reductase, estimated on SDS-PAGE as described in Example 5.6. On the other hand, the NH.sub.2 -terminal sequence of the protein identified in this way by analysis of the open reading frames of the sequence of 694-bp is identical to that NH.sub.2 -terminal sequence of the proteins SnaC purified as described in Example 5. Similarly, the two internal protein sequences of the FMN reductase described in 5.6 occur in the protein deduced from the sequenced portion. This confirms that the gene isolated from cosmid pIBV4 does indeed correspond to the protein FMN reductase described in Example 5.6, designated SnaC (SEQ ID no 7).
A study of the DNA fragments of S. pristinaespiralis strain SP92 carried by cosmids pIBV1 to pIBV2 demonstrated the presence of several genes involved in the biosynthesis of pristinamycins II and pristinamycins I. The snaA, snaB and samS genes code for enzymes participating in the biosynthesis of pristinamycins II, pristinamycin IIA synthase and probable SAM synthase, and are grouped together physically on a large DNA fragment cloned into plasmid pIBV1. Similarly, the snbA, snbR, papA and papM genes--which code for proteins participating in the biosynthesis of pristinamycins I, 3-hydroxypicolinic acid:AMP ligase (SnbA), the protein SnbR probably responsible for the transport of pristinamycins I, the protein Papa involved in the biosynthesis of p-aminophenylalanine from chorisimic acid, and p-aminophenylalanine (phenyl-N)-methyltransferase (PapM)--are grouped together on a large DNA fragment cloned into cosmid pIBV2. Similarly, the snaA and snaD genes on the one hand--which code for proteins participating in the biosynthesis of pristinamycins II, the protein SnaD probably being a peptide synthase--and the snbC, snbD and snbE genes on the other hand--which code for the 3 peptide synthases SnbC, SnbD and SnbE involved in the formation of the peptide chain of pristinamycin I from its 6 separate amino acids--are grouped together on a large DNA fragment cloned into cosmid pIVB3. These results confirm the hypothesis of the grouping together of the genes for the biosynthesis of pristinamycins II, and also of the genes for the biosynthesis of pristinamycins I, and afford the possibility of cloning the other genes involved in these biosynthesis, by chromosome walking, upstream and downstream of the regions studied.
Furthermore, it is possible by hybridization of the total DNA of the different strains producing streptogramins (see Table 1) with the snaA, snaB, snaC, snaD, samS, snbA, snbR, snbC, snbD, snbE, papA and papM genes, or with the genes identified by chromosome walking, or with fragments of these genes, to isolate the genes corresponding to the same functions in the other microorganisms producing streptogramins. This makes it possible, by the same approach as that envisaged for the pristinamycins, to isolate all the genes involved in the biosynthesis of the different streptogramins.
EXAMPLE 9
Genetic Study of DNA Fragments by Gene Disruption
This example illustrates how it is possible to demonstrate the involvement of genes in the biosynthesis of streptogramins by constructing strains derived from a producing strain and mutated in these genes by disruption, and by analysing the phenotype of such mutants. This example shows, furthermore, how to obtain strains that are left producing only one or other of the A and B components of streptogramins, or producing A and B components with ratios different from those observed with the strain SP92.
9.1. Construction of a Mutant of S. pristinaespiralis SP92 Disrupted in the snaA Gene
This example illustrates how it is possible, by disruption of the snaA gene, to construct a strain of S. pristinaespiralis SP92 which no longer produces pristinamycin IIA and which produces, in contrast, pristinamycin IIB.
This mutant was constructed for the purpose of confirming the functionality of the protein SnaA and of providing an intermediate of pristinamycin II production capable of being modified subsequently.
Its construction was carried out using a suicide vector capable of replicating in E. coli only but carrying a resistance marker which is expressed in Streptomyces. This vector, pDH5, was developed by Hillemann et al. (1991).
9.1.1. Construction of Plasmid pVRC505
This example illustrates how it is possible to construct a plasmid which does not replicate in S. pristinaespiralis SP92 and which may be used to disrupt the snaA gene by single homologous recombination.
Plasmid pVRC505 was constructed to produce the SP92 chromosomal mutant disrupted in the snaA gene from plasmid pXL2045 described in Example 6.3.
The 6-kb BamHI fragment, cloned into pXL2045 (FIG. 16), was cut with the restriction enzymes EcoRI and PstI. After separation of the fragments thereby generated by electrophoresis on 1% agarose gel, a 0.7-kb fragment containing the 5' end of the snaA gene was isolated and purified by Geneclean (Bio101, La Jolla, CA).
100 ng of vector pDH5 linearized by an EcoRI/PstI double digestion were ligated with 100 ng of the 0.7-kb fragment, as described in Example 3.3. A clone carrying the desired fragment was isolated after transformation of the strain TG1. The recombinant plasmid was designated pVRC505. Plasmid pVRC505 was prepared as described in Example 2.1. Its restriction map is presented in FIG. 25.
9.1.2. Isolation of the SP92 Mutant Disrupted in the snaA Gene by Homologous Recombination
This example illustrates how the mutant of S. pristinaespiralis SP92 disrupted in the snaA gene was constructed.
This mutant was isolated by transformation of the strain SP92 with the suicide plasmid pVRC505.
The preparation of the protoplasts and their transformation were carried out as described in D. Hopwood et al. (1985).
The strain SP92 was cultured in YEME medium, 34% sucrose, 5 mm MgCl.sub.2, 0.25% glycine for 40 hours at 30.degree. C. The mycelium was converted to protoplasts in the presence of lysozyme, and 5.times.1 .mu.g of pVRC505 were used for the transformation (by the method employing PEG) of the protoplasts. After overnight regeneration of the protoplasts on R2YE medium (D. Hopwood et al. 1985), the recombinants were selected by overlaying 3 ml of SNA medium (D. Hopwood et al. 1985) containing 2.6 .mu.g/ml of thiostrepton.
Of the 5 transformations carried out, 3 thiostrepton-resistant clones were isolated. This gives a recombinant efficiency of less than 1 per .mu.g of DNA. These recombinants result from integration by single homologous recombination between the snaA gene carried by the chromosome of the strain SP92 and the 0.7-kb fragment of the suicide plasmid pVRC505. The small size of the fragment inserted into pVRC505, 0.7-kb, explains the low recombination efficiency.
The spores of the recombinants were isolated by plating out and growth on R2YE medium supplemented with 400 .mu.g/ml of thiostrepton, and plated out again on the same medium to obtain isolated colonies.
In order to verify the position of integration of plasmid pVRC505, various Southern blots of the total DNA of several recombinant clones, which was digested with the appropriate restriction enzymes, were produced and hybridized with the vector pDH5 and the 0.7-kb fragment, used successively as probes after labelling by random priming (Random Primed DNA labeling kit, Boehringer Mannheim, France) with [.alpha.-.sup.32 P]-dCTP, as described in Maniatis et al. (1989). The hybridization results show the appearance in the genome of the recombinant clones of an additional EcoRI-PstI band, of the size of the vector pDH5 and which hybridizes with the latter, as well as of an additional EcoRI--EcoRI band which hybridizes with both the 2 probes. One of these mutants were designated SP92::pVRC505. This mutant corresponds well to the integration of plasmid pVRC505 in the snaA gene by single homologous recombination.
9.1.3. Production of Pristinamycins by the Mutant SP92::pVRC505
This example illustrates how it is determined that the mutant of S. pristinaespiralis SP92 disrupted in the snaA gene by integration of plasmid pVRC505 no longer produces pristinamycin IIA while continuing to produce pristinamycin IIB.
The mutant SP92::pVRC505, as well as the strain SP92 as control strain, were culture in liquid production medium. Fermentation was carried out as follows: 0.5 ml of a suspension of spores of the strains mentioned are added under sterile conditions to 40 ml of inoculum medium in a 300-ml Erlenmeyer. The inoculum medium consists of 10 g/l of corn steep, 15 g/l of sucrose, 10 g/l of (NH.sub.4).sub.2 SO.sub.4, 1 g/l of K.sub.2 HPO.sub.4, 3 g/l of NaCl, 0.2 g/l of MgSO.sub.4.7H.sub.2 O, and 1.25 g/l of CaCO.sub.3. The pH is adjusted to 6.9 with sodium hydroxide before the introduction of calcium carbonate. The Erlenmeyers are stirred for 44 hours at 27.degree. C. on a rotary stirrer at a speed of 325 rpm. 2.5 ml of the above culture when 44 hours old are added under sterile conditions to 30 ml of production medium in a 300-ml Erlenmeyer. The production medium consists of 25 g/l of soya flour, 7.5 g/l of starch, 22.5 g/l of glucose, 3.5 g/l of feeding yeast, 0.5 g/l of zinc sulphate and 6 g/l of calcium carbonate. The pH is adjusted to 6.0 with hydrochloric acid before the introduction of calcium carbonate. The Erlenmeyers are stirred for 24, 28 and 32 hours at 27.degree. C. At each time, 10 g of must are weighed into a smooth Erlenmeyer, and 20 ml of mobile phase composed of 62.5% of acetonitrile and 37.5% of 0.1 M KH.sub.2 PO.sub.4 solution (adjusted to pH 3.0 with H.sub.3 PO.sub.4), and which enables the pristinamycins to be extracted, are added to this. After stirring on a stirrer (325 rpm) for 20 min at room temperature, the whole is filtered through filter paper and the pristinamycins are assayed by HPLC as described in Example 5.1.1.A.
The results showed that, under the fermentation conditions implemented, the mutant SP92::pVRC505 produced an amount of pristinamycin I equivalent to that of the SP92 control, this being the case for all 3 times tested. In contrast, whereas the control produced approximately 70% of pristinamycin IIA and 30% of pristinamycin IIB at 24, 28 and 32 hours of fermentation, the mutant SP92::pVRC505 produced 100% of pristinamycin IIB for these same times, in amounts equivalent to the sum of pristinamycin IIA+pristinamycin IIB produced by the strain SP92. Hence the mutant is indeed blocked in a step of biosynthesis of pristinamycin II which corresponds to the oxidation of the 2,3 bond of the D-proline of the intermediate pristinamycin IIB. This mutant hence accumulates pristinamycin IIB. This shows well the functional involvement of SnaA in the conversion of pristinamycin IIB to pristinamycin IIA.
This example shows that it is possible, starting from cloned genes for biosynthesis, to construct strains that are mutated in the steps of biosynthesis of pristinamycin. This was shown for pristinamycin II, but the same results may be obtained for pristinamycins I and, by extension, for the different components of streptogramins. Strains producing different intermediates may thus be obtained. These strains may be used to produce novel molecules by chemical, biochemical, enzymatic, and the like, modification(s) of the said intermediates. A block in an early step of the biosynthesis pathway of one or other of the components of streptogramins may also lead to mutated strains that are left producing only one or other of the components.
9.2. Construction of a Mutant of S. pristinaespiralis SP92 Disrupted in the samS Gene
This example illustrates how it is possible, by disruption of the samS gene, to construct a strain of S. pristinaespiralis SP92 which produces 35% less PIA and 10 times as much PIB (the chemical structures are shown in FIG. 2) relative to the wild-type SP92 strain. This mutant was constructed for the purpose of confirming the presumed SAM synthase function for the protein encoded by the samS gene, and for obtaining a strain that synthesizes more PIB than the wild-type SP92 strain.
9.2.1. Construction of Plasmid pVRC702
From plasmid pXL2045 (described in Example 6.3), the 3.2-kb BamH1-EcoR1 fragment was isolated by enzymatic cleavage and purified after electrophoresis on 1% agarose gel by the Geneclean kit method (see Example 6.8). This fragment carries snaB gene as well as the samS gene (FIG. 16). This fragment is then cloned into a plasmid pUC18 in the following manner: 50 ng of pUC18 were linearized by double digestion using the enzymes EcoR1 and BamH1, and then ligated in the presence of T$ DNA ligase (Biolabs) with 300 ng of the 3.2-kb BamH1-EcoR1 fragment. After transformation of competent cells of E. coli strain TG1 with this ligation mixture, a recombinant clone possessing plasmid pUC18 with the 3.2-kb insert could be isolated, and this was designated pVRC701 (FIG. 26).
Plasmid pVRC702 is derived from plasmid pVRC701 by the introduction between the two Sst1 sites located in the middle of the samS gene (FIG. 27) of a cassette carrying the amR gene coding for resistance to amramycin and geniticin. To this end, a 2.2-kb BamH1--BamH1 fragment carrying the .OMEGA.amR cassette was first isolated by BamH1 digestion of plasmid pHP45.OMEGA.amR (given by J. L. Pernodet, Laboratoire de Genetique d'Orsay) using the same technique as above. 200 ng of this fragment were then ligated with 50 ng of plasmid pUC1318 (Kay and McPherson, 1987) linearized with the enzyme BamH1, and this ligation mixture was introduced into competent E. coli TG1 cells. From the recombinant zone possessing plasmid pUC1318 containing the .OMEGA.amR cassette, 50 ng of a 2.2-kb Sst1--Sst1 fragment containing the .OMEGA.amR cassette could be reisolated by partial cleavage using the enzyme Sst1, and this fragment was ligated with 30 ng of plasmid PVRC701 cut with Sst1 (FIG. 26) to give, after transformation of competent E. coli TG1 cells, plasmid PVRC702, the structure odf which is detailed in FIG. 27.
Plasmid pVRC702 thereby obtained was prepared in large amounts according to the method described above in Example 2.1.
9.2.2. Construction of the Strain Having the samS::.OMEGA.amR Chromosomal Gene
This strain was obtained by transformation of S. pristinaespiralis protoplasts with 1 .mu.g of the suicide plasmid pVRC702 which is incapable of replicating in a Streptomyces cell. The protocols for preparation of the protoplasts and for transformation are the same as above (Example 9.1). The only modifications made with respect to Example 10.1 relate to the selection antibiotic. In the present case, the recombinant protoplasts after regeneration for 18 hours at 30.degree. C. on R2YE medium are selected in the presence of 50 .mu.g/ml final of geniticin (Sigma Chemical Co.). Thus, an overlayer composed of 3 ml of SNA containing 383 .mu.g/ml of geniticin is added to each dish of R2YE.
In this way, it was possible to isolate 500 geniticin-resistant recombinant clones, which may result either from an integration of plasmid pVRC702 into the chromosome following a single homologous recombination between chromosomal and plasmid samS genes (in the case of single crossing-over), or from an exchange between the chromosomal samS gene and the plasmid samS::.OMEGA.amR plasmid gene following a double homologous recombination event (in the case of double crossing-over). In these two cases in point, the .OMEGA.amR cassette becomes transferred onto the chromosome of the strain, and endows it with an amR resistance which is stable over generations.
The recombinant clones were isolated by plating out and growth on HT7 medium containing 50 .mu.g/ml final of geneticin, and then analyzed by the colony hybridization technique. Hybridization of the clones with a first probe obtained as described in Example 9.1 from the 2.7-kb BamH1-EcoRI fragment originating from pVRC702 and corresponding to pUC18, as well as with a second probe corresponding to the 2.2-kb BamH1 fragment carrying the .OMEGA.amR cassette, enable the cases of single crossing-over (hybridizing with both probes) to be distinguished from the cases of double crossing-over (hybridizing only with the second probe). The 3 clones resulting from a double crossing-over thereby selected were purified by plating out and growth on YVD medium containing 50 mg/ml final of geneticin, and stocks of spores were obtained.
In order to verify the genomic structure of the 3 double recombinants, various Southern blots of the chromosomal DNA of these clones digested with the enzymes EcoRI and BamHI were produced and hybridized with the following three probes: the probe corresponding to the .OMEGA.amR cassette obtained from the 2.2-kb BamHI fragment of pVRC702, the probe corresponding to pUC18 obtained from the 2.7-kb BamHI-EcoRI fragment of pVRC701, and lastly a probe obtained from the 1.3-kb EcoRI-SstI fragment of pVRC701 carrying the snaB gene and the beginning of samS. These hybridizations enabled it to be verified that the three clones tested did indeed result from a double homologous recombination event permitting replacement of the intact chromosomal samS gene by the mutated samS gene interrupted by the .OMEGA.amR cassette.
One of these three mutant clones was designated SP92 samS::.OMEGA.amR.
9.2.3. Production of Pristinamycins by the Mutant Strain samS::.OMEGA.amR
This example illustrates how it is determined that the mutant SP92 samS::.OMEGA.amR having the disrupted samS gene produces 35% less pristinamycin IA and 10-fold more pristinamycin IB than the wild-type SP92 strain.
The mutant SP92 samS::.OMEGA.amR as well as the control SP92 strain were cultured in liquid production medium, and their productions of pristinamycin II and pristinamycin I were assayed as described in Example 9.1.
The results showed that, under the fermentation conditions implemented, the mutant SP92 samS::.OMEGA.amR produces an amount of pristinamycins II equivalent to that of the SP92 control for all three times tested. In contrast, the mutant SP92 samS::.OMEGA.amR produces approximately 35% less pristinamycin IA and 10-fold more pristinamycin IB than the control strain at all three times tested. The IB form of pristinamycins thus represents 20% of the collective total type I pristinamycins produced by the mutant SP92 samS::.OMEGA.amR, whereas the control strain synthesizes only of the order of 1% of PIB. The IB form of pristinamycins differs from the IA form in that the fifth residue is p-methylaminophenylalanine, instead of p-dimethylaminophenylalanine for pristinamycin IA. The fact that the mutant SP92 samS::.OMEGA.amR produces more pristinamycin IB and less pristinamycin IA shows that disruption of the samS gene causes a decrease in the degree of methylation of the fifth residue of pristinamycins I, and hence that the samS gene is probably involved in the biosynthesis of the methyl donor, SAM, that is to say that it codes for a SAM synthase.
9.3 Construction of a Mutant of S. pristinaespiralis SP92 Disrupted in the papA Gene
This example illustrates how it is possible, by disruption of the papA gene, to construct a strain of S. pristinaespiralis SP92 which no longer produces PI. This mutant is constructed for the purpose of confirming the functionality of the PapA protein. Its construction was carried out using the suicide vector pDH5 described in Example 9.1.
9.3.1. Construction of Plasmid pVRC508
This example illustrates how it is possible to construct a plasmid which does not replicate in S. pristinaespiralis SP92 and which may be used to disrupt the papA gene by single homologous recombination.
Plasmid pVRC508 was constructed to produce the SP92 chromosomal mutant disrupted in the papA gene from plasmid pVRC903 described in Example 7.7.
In Example 7.7, the cloning of the 1.4-kb PvuII-EcoRI fragment into M13mp18 from plasmid pVRC903 for the purpose of sequencing the papA gene was described (this fragment corresponds to the 1.4-kb PvuII-XhoI fragment present in the vector pVRC900, FIG. 23).
This construction in M13mp18 was digested with the restriction enzyme HindIII and EcoRI. After separation of the vector M13mp18 and the 1.4-kb fragment containing a portion of the papA gene on 0.8% agarose gel, the latter fragment was isolated and purified by Geneclean (Bio101, La Jolla, CA). The localization of the fragment in the papA gene is presented in FIG. 23.
100 ng of vector pDH5 linearized by a double digestion with the restriction enzymes HindIII and EcoRI were ligated with 200 ng of the 1.4-kb fragment as described in Example 3.3. A clone carrying the desired fragment was isolated after transformation of the strain TG1. The recombinant plasmid was designated pVRC508. Plasmid pVRC508 was prepared as described in Example 2.1. Its restriction map is presented in FIG. 28.
9.3.2. Isolation of the SP92 Mutant Disrupted in the papA Gene by Homologous Recombination
This example illustrates how the mutant of S. pristinaespiralis SP92 disrupted in the papA gene was constructed. This mutant was isolated by transformation of the strain SP92 with the suicide plasmid pVRC508. The preparation of the protoplasts and their transformation were carried out as described in Example 9.1. After transformation of protoplasts of the strain SP92, the recombinants were selected by overlaying 3 ml of SNA medium containing 2.6 mg/ml of thiostrepton. Of the 5 transformations carried out with 5 times 1 .mu.g of plasmid pVRC508, ten thiostrepton-resistant clones were isolated. This gives a recombinant efficiency of approximately 2 per .mu.g of DNA. These recombinants result from integration by single homologous recombination between the papA gene carried by the chromosome of the strain SP92 and the 1.4-kb fragment of the suicide plasmid pVRC508.
The spores of the recombinants were isolated by plating out and growth on R2YE medium containing 400 .mu.g/ml of thiostrepton, and plated out again on the same medium to obtain isolated colonies.
In order to verify the position of integration of plasmid pVRC508, various Southern blots of the total DNA of several recombinant clones, purified as described above, were produced and hybridized with the vector pDH5 and the 1.4-kb fragment, used successively as probes after labelling by random priming with [.alpha.-.sup.32 P]dCTP as described in Maniatis et al. (1989). The hybridization results show the disappearance from the genome of the recombinant clones digested with the restriction enzyme EcoRI (site flanking the 1.4-kb fragment) of the 6.8-kb EcoRI band, and the appearance of two additional bands relative to the control SP92 strain, of 2.4 hybridizing with the 1.4-kb fragment, and the other of 10.5 kb hybridizing both with pDH5 and with the 1.4-kb fragment. Digestion of the recombinant clones with the restriction enzyme PstI shows the appearance of two additional bands relative to the control SP92 strain, one of 1.0 kb hybridizing with the 1.4-kb fragment, and the other of 5.1 kb hybridizing both with pDH5 and with the 1.4-kb fragment. One of these mutants were designated SP92::pVRC508.
9.3.3. Production of Pristinamycins by the Mutant SP92::pVRC508
This example illustrates how it is determined that the mutant of S. pristinaespiralis SP92 disrupted in the papA gene by integration of plasmid pVRC508 no longer produces PI.
The mutant SP92::pVRC508, as well as the strain SP92 as control strain, were cultured in liquid production medium. The fermentation and also the assay of pristinamycins I and II were carried out as described in Example 9.1.
The results showed that, under the fermentation conditions implemented, whereas the control SP92 strain produced a standard amount of pristinamycins I, no trace of type I pristinamycins was detected in the fermentation must be of the mutant SP92::pVRC508. Moreover, the production of pristinamycins II by the mutant SP92::pVRC508 is equivalent to that of the SP92 control. The mutant SP92::pVRC508 is left producing only pristinamycins II. These results show clearly that the papA gene codes for a protein involved in the biosynthesis of pristinamycins I.
To check the absence of polarity of the disruption carried out in the mutant SP92::pVRC508, the latter was fermented in the presence p-dimethylaminophenylalanine. The mutant SP92::pVRC508 was fermented as described above, with the addition, at 17 hours of fermentation, of 100 mg/l of p-dimethylaminophenylalanine. Under these conditions of complementation, the mutant SP92::pVRC508 produces an amount of pristinamycins I equivalent to that produced by the strain SP92. The production of pristinamycins II is equivalent in both strains. This enables us to conclude that the mutant SP92::pVRC508 does not produce pristinamycins I because it is indeed disrupted in a gene that participates in the biosynthesis of p-dimethylaminophenylalanine (the papA gene). Complementation of this mutant with p-dimethylaminophenylalanine restores its capacity to produce pristinamycins I, proving that the mutation has no polar effect on the synthesis of other pristinamycin I precursors or on the condensation of these precursors.
This example shows that it is possible, starting from cloned genes for biosynthesis, to construct strains that are mutated in the steps of biosynthesis of pristinamycins, and especially pristinamycins I. This example also shows that it is possible, by this approach, to construct strains of S. pristinaespiralis specifically producing pristinamycins II and, by extension, strains specifically producing pristinamycins I. This same approach could also be used for other strains of actinomycetes producing streptogramins.
9.4. Construction of Mutant of S. pristinaespiralis SP92 Disrupted in the snbA Gene
This example illustrates how it is possible, by disruption of the snbA gene, to construct a strain of S. pristinaespiralis SP92 which no longer produces pristinamycins I. This mutant was constructed for the purpose of confirming the functionality of the SnbA protein. Its construction was carried out using the suicide vector pDH5 described in Example 9.1.
9.4.1. Construction of Plasmid pVRC404
This example illustrates how it is possible to construct a plasmid which no longer replicates in S. pristinaespiralis SP92 and which may be used to disrupt the snbA gene by single homologous recombination.
Plasmid pVRC404 was constructed from plasmid pVRC402 described in Example 6.2, to produce the SP92 chromosomal mutant disrupted in the snbA gene. Plasmid pVRC402 was digested with the restriction enzyme XhoI and HindIII. After separation of the fragments thereby generated by electrophoresis on 0.8% agarose gel, a 1170-bp fragment containing an internal portion of the snbA gene was isolated and purified by Geneclean (Bio101, La Jolla, CA). The localization of the fragment in the snbA gene is presented in FIG. 15A.
100 ng of vector pDH5 linearized by an SmaI digestion were ligated with 200 ng of the 1173-bp fragment as described in Example 3.3. A clone carrying the desired fragment was isolated after transformation of the strain TG1. The recombinant plasmid was designated pVRC404. Plasmid pVRC404 was prepared as described in Example 2.1. Its restriction map is presented in FIG. 29.
9.4.2. Isolation of the SP92 Mutant Disrupted in the snbA Gene by Homologous Recombination
This example illustrates how the mutant of S. pristinaespiralis SP92 disrupted in the snbA gene was constructed.
This mutant was isolated by transformation of the strain SP92 with the suicide plasmid pVRC404. The preparation of the protoplasts and their transformation were carried out as described in Example 9.1. After transformation of protoplasts of the strain SP92, the recombinants were selected by overlaying 3 ml of SNA medium containing 2.6 mg/ml of thiostrepton. Of the 5 transformations carried out with 5 times 1 .mu.g of plasmid pVRC404, about thirty thiostrepton-resistant clones were isolated. This gives a recombinant efficiency of approximately 5 per .mu.g DNA. These recombinants result from integration by single homologous recombination between the snbA gene carried by the chromosome of strain SP92 and the 1170-bp fragment of the suicide plasmid pVRC404. The spores of the recombinants were isolated by plating out and growth on R2YE medium+400 mg/ml of thiostrepton, and plated out again on the same medium to obtain isolated colonies. In order to verify the position of integration of plasmid pVRC404, various Southern blots of the total DNA of several recombinant clones, purified as described above, were produced and hybridized with the vector pDH5 and the 1170-kb fragment, used successively as probes after labelling by random priming with [.alpha.-.sup.= P]dCTP as described in Maniatis et al. (1989). The hybridization results show the appearance in the genome of the recombinant clones digested with the restriction enzymes XhoI and HindIII of an additional 4.7-kb XhoI-HindIII band relative to the control SP92 strain (vector pDH5+1.17 kb), hybridizing both with pDH5 and with the 1170-bp fragment. Digestion of the recombinant clones with the restriction enzyme PflMI (sites flanking tbhe 1170-bp XhoI-HindIII fragment) shows the disappearance of the 3.1-kb PflMI--PflMI band and the appearance of a band at 8.8 kb hybridizing with both probes. These results indicate that the genomic structure of the clones analysed is indeed that expected after a homologous recombination event between pVRC404 and the chromosomal snbA gene. One of these mutants was designated SP92:pVRC404.
9.4.3. Production of Pristinamycins by the Mutant SP92::VRC404
This example illustrates how it is determined that the mutant of S. pristinaespiralis SP92 disrupted in the snbA gene by integration of plasmid pVRC404 no longer produces PI.
The mutant SP92::pVRC404, as well as the strains SP92 as control strain, were cultured in liquid production medium. The fermentation and also the assay of pristinamycins I and II were carried out as described in Example 9.1. The results showed that, under the fermentation conditions implemented, whereas the control SP92 strain produces a standard amount of pristinamycins I, no trace of pristinamycins I was detected in the fermentation must of the mutant SP92::pVRC404. Moreover, the production of pristinamycins II by the mutant SP92::pVRC404 is equivalent to that of the SP92 control. The mutant SP92::pVRC404 is left producing only pristinamycins II. This shows clearly that the snbA gene codes for a protein SnbA involved in the biosynthesis of pristinamycins I, as had been shown during the purification in Example 5.2.
This example shows, as in the preceding example, that it is possible, starting from cloned genes for biosynthesis, to construct strains that are mutated in the steps of biosynthesis of pristinamycins, and especially pristinamycins I. This example also shows that is possible, by this approach, to produce strains of S. pristinaespiralis specifically producing pristinamycins II and, by extension, strains specifically producing pristinamycins I, as described in the following example: 9.5. This same approach could also be used for other strains of actinomycetes producing streptogramins.
9.5. Construction of a Mutant of S. pristinaespiralis SP92 Disrupted in the snaD Gene Probably Coding for a Peptide Synthase Involved in the Biosynthesis of Pristinamycins II
This example illustrates how it is possible, by disruption of the snaD gene probably coding for a peptide synthase involved in the biosynthesis of pristinamycins II, to construct a strain of S. pristinaespiralis SP92 which no longer produces pristinamycins II.
This mutant was constructed for the purpose of confirming the functionality of the snaD gene, and of obtaining a strain derived from SP92 left synthesizing only pristinamycins I.
Its construction was carried out using plasmid PVRC1000 described in Example 6.8, derived from the suicide vector pDH5, capable of replicating in E. coli only and carrying a resistance marker which is expressed in Streptomyces (see Example 9.1).
9.5.1. Construction of Plasmid pVRC1000
This example illustrates how it is possible to construct a plasmid which does not replicate in S. pristinaespiralis SP92 and which may be used to disrupt the snaD gene by single homologous recombination. The construction of plasmid pVRC1000 carrying a portion of the snaD gene is described in Example 6.8.
9.5.2. Isolation of the SP92 Mutant Disrupted in the snaD Gene by Homologous Recombination
This example illustrates how the mutant of S. pristinaespiralis SP92 disrupted in the snaD gene was constructed. This mutant was isolated by transformation of the strain SP92 with the suicide plasmid pVRC1000. The preparation of the protoplasts and their transformation were carried out as described in Example 9.1. Of the 5 transformations carried out with 1 mg of pVRC1000, approximately 1500 thiostrepton-resistant clones were isolated. This gives a recombinant efficiency of approximately 375 per .mu.g of DNA. These recombinants result from integration by single homologous recombination between then snaD gene carried by the chromosome of the strain SP92 and the 1.5-kb BamHI-SstI fragment of the suicide plasmid pVRC1000. About twenty recombinants were subcultured on R2YE medium containing 400 .mu.g/ml of thiostrepton, and the spores of these recombinants were isolated by plating out again and growth on R2YE medium containing 400 .mu.g/ml of thiostrepton.
In order to verify the position of integration of plasmid pVRC1000, various Southern blots of the total DNA of 7 recombinant clones, purified as described above, were produced and hybridized with the vector pDH5 and the 1.5-kb BamHI-SstI fragment contained in PVRC1000, used successively as probes after labelling with [.alpha.-.sup.32 P]dCTP as described in Example 9.1. The hybridization results show the appearance in the genome of the 7 recombinant clones of a 13.8-kb EcoRI band and an approximately 17-kb BglII band hybridizing with both probes, as well as a 3.7-kb EcoRI band hybridizing with the 1.2-kb BamHI-StsI probe. One of these mutants was designated SP92::pVRC1000 and corresponds well to the integration of plasmid pVRC1000 in the snaD gene by single homologous recombination.
9.5.3. Production of Pristinamycins by the Mutant SP92::pVRC1000
This example illustrates how it is determined that the mutant of S. pristinaespiralis SP922 disrupted in the snaD gene by integration of plasmid pVRC1000 no longer produces pristinamycins II, but only pristinamycins I. The mutant SP92::pVRC1000, as well as the control SP92 strain, were cultured in liquid production medium, and their productions of pristinamycins II and I were assayed as described in Example 9.1.
The results showed that, under the fermentation conditions implemented and for all three times tested, the mutant SP92::pVRC1000 produces 0 mg/l of pristinamycins Ii and an amount of pristinamycins I equivalent to that of the SP92 control. Hence this mutant is indeed blocked in a step of biosynthesis of pristinamycins II, which shows that the snaD gene codes for an enzyme involved in the biosynthesis of pristinamycins II, and very probably for a peptide synthase.
This example shows, as in the preceding example, that it is possible, starting from cloned genes for biosynthesis, to construct strains that are mutated in the steps of biosynthesis of pristinamycins, and especially pristinamycins II. This example also shows that it is possible, by this approach, to produce strains of S. pristinaespiralis specifically producing pristinamycins I and, by extension, strains specifically producing pristinamycins II. This same approach could also be used for other strains of actinomycetes producing streptogramins.
EXAMPLE 10
Complementation of a Non-Producing Mutant of the Strain SP92
This example shows how it is possible to express genes for the biosynthesis of pristinamycins. This expression was implemented more especially for the snaA and snaB genes carried by cosmid pIBV1 in a mutant strain derived from SP92: SP120. This mutant does not produce pristinamycin IIA. It accumulates the last intermediate of the biosynthesis pathway of pristinamycin II: pristinamycin IIB.
10.1. Cloning of the snaA and snaB Genes into the Shuttle Vector pIJ903
This example illustrates how a subfragment of cosmid pIVB1 containing the snaA and snaB genes was cloned into a vector capable of replicating both in E. coli and in Streptomyces.
The vector pIJ903 (Lydiate, D. J. et al., 1985) is a low copy number (1 to 3 per cell) shuttle vector capable of replicating both in E. coli as a result of its origin of replication of pBR322, and in Streptomyces as a result of its origin of replication of SCP2. The ampicillin resistance gene permits selection in E. coli, and the thiostrepton resistance gene permits selection in Streptomyces.
Cosmid pIBV1 was digested with the restriction enzyme SstI. A large 7.6-kb DNA fragment carrying the snaA and snaB genes was isolated by electrophoresis on 0.8% agarose gel and electroeluted. 500 ng of this fragment were ligated with 100 ng of the vector pUC1813 (Kay and McPherson, 1987) linearized with SstI. After transformation of E. coli strain DH5.alpha. (supE44 .DELTA.lacU169 (f80lacZ.DELTA.M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1), and selection of the transformants on solid LB containing 150 .mu.g/ml of ampicillin and 20 .mu.g/ml of X-gal, a clone carrying the 7.6-kb fragment was isolated. The plasmid was designated pVRC506. A preparation of this recombinant plasmid was carried out as described in Example 2.1.
Cloning into the vector pIJ903 was carried out at the HindIII site. Plasmid pVRC506 was cut with HindIII, and 7.6-kb fragment carrying the snaA and snaB genes was isolated by electrophoresis on 0.8% agarose gel and electroeluted. 500 ng of this fragment were ligated with 500 ng of the vector pIJ903 linearized with HindIII. After transformation of E. coli strain DH5.alpha. and selection of the transformants on solid LB containing 150 .mu.g/ml of ampicillin, a clone carrying the 7.6-kb fragment was isolated. The plasmid was designated pVRC507. A preparation of this recombinant plasmid was carried out as described in Example 2.1. Its map is presented in FIG. 30.
10.2. Expression of the snaA and snaB Genes in the Mutant SP120
This example illustrates how it is possible to produce the proteins SnaA and SnaB in S. pristinaespiralis SP92 by introducing a plasmid carrying the corresponding structural genes into this strain. Expression of the snaA and snaB genes was carried out after transformation of the mutant strain SP120 with 500 ng of plasmid pVRC507. Transformation of the protoplasts of SP120 and selection of the transformants with thiostrepton were carried out as described in Example 9.1.2.
Many transformants were obtained in this way, and 3 of them were chosen for the tests of production in a liquid medium. The strain SP120 carrying plasmid pIJ903 was chosen as control. The fermentations and also the extraction of the biosynthesis products were carried out as described in Example 9.1.3.
The results showed that, under the fermentation conditions implemented, whereas the control (SP120 carrying plasmid pIJ903) produced 100% of P IIB and 0% of P IIA at 24, 28 and 32 hours of fermentation, the 3 clones of the strain SP120 transformed with plasmid pVRC507 produced, for these same times, approximately 85 to 80% of pristinamycin IIB and 15 to 20% of pristinamycin IIA, the sum of which is equivalent in amount to the pristinamycin IIB production of the control strain (SP120 carrying plasmid pIJ903). The clones carrying pVRC507 were indeed partially complemented for the step of biosynthesis of pristinamycins II corresponding to the oxidation of the 2,3 bond of the D-proline of the intermediate pristinamycin IIB. This was confirmed by enzymatic assay of pristinamycin IIA synthase activity, as described in Example 5.1.1.A, for the strains SP120 carrying pVRC507 and SP120 carrying pIJ903. Whereas the control strain SP120 carrying pIJ903 displays no pristinamycin IIA synthase activity, the strain SP120 carrying pVRC507 displays PIIA synthase activity.
This example shows that it is possible to express genes for the biosynthesis of streptogramins. This expression was studied more especially for the genes coding for pristinamycin IIA synthase, but the other genes for the biosynthesis of pristinamycins II and pristinamycins I, as well as those involved in the biosynthesis of the components of the different streptogramins, may be expressed in this way. This expression may be carried out in mutant strains as is the case in Example 10, but also in producing strains in order to increase the levels of streptogramin production. The expression may be modified by cloning the genes into a vector having a different copy number (low or high) or into an integrative vector, by deregulation of these genes, by cloning these genes under a homologous or heterologous promoter (strong or specifically regulated promoter). Expression of the different genes for the biosynthesis of streptogramins may also be carried out in heterologous strains using appropriate expression vectors in order to produce hybrid antibiotics.
EXAMPLE 11
Expression of the papM Gene of S. pristinaespiralis in E. coli
This example illustrates how it is possible to express an S. pristinaespiralis gene in E. coli so as to be able to identify, purify and study the protein encoded by this gene.
11.1. Construction of Plasmid pVRC706
Expression of the papM gene in E. coli is obtained by placing this gene downstream of the promoter and ribosomes binding site of the lacZ gene of E. coli. The 1.7-kb MluI-StuI fragment was isolated from plasmid pVRC409 described in example 7.8, and then cloned into plasmid pMTl23 (Chambers et al. 1988) cut at the BamHI site subsequently filled in using the Klenow enzyme (Maniatis et al. 1989) and at the MluI site, to give plasmid pVRC706 shown in FIG. 31. Cloning at the MluI site enables an in-frame fusion to be obtained between the first 32 amino acids of .beta.-galactosidase encoded by the lacZ gene of plasmid pMTL23 and the last eleven amino acids of the gene located immediately upstream of papM, thereby making it possible to preserve the translational coupling which appears to exist between the papM gene and this upstream gene in the light of the nucleotide sequence given in Example 7.8. Thus, the expression of the hybrid gene between lacZ and the gene upstream of papM and that of the papM gene is under the control of the expression signals of the lacZ gene.
11.2. Expression in E. coli Strain DH5a of the Product of the papM Gene
Plasmids pVRC706 and pMTL23 were introduced by transformation into E. coli strain DH5.alpha., and the expression of their genes was studied under conditions where the promoter of lacZ gene is induced as already described (Maniatis et al. 1989). The E. coli strains carrying plasmid pVRC706 or the control plasmid pMTL23 were cultured in 500 of LB rich medium containing 100 mg/ml of ampicillin and 1 mM IPTG, permitting induction of the promoter of the lacZ gene. These cultures are sampled when they have reached an optical density at 600 nm in the region of 1, and the protein extracts are prepared as described below.
11.3. Assay of the Activity of the Product of the papM Gene Expressed in E. coli
The activity corresponding to the protein encoded by the papM gene is assayed on the two extracts prepared from E. coli cultures carrying plasmid pVRC706 or plasmid pMTL23 Example 5.7). It was shown that the extract prepared from the strain E. coli::pVRC706 catalyses the methylation of p-aminophenylalanine to p-dimethylaminophenylalanine with an activity of 235 unit/mg, whereas this activity is absent in the extracts of the control strain E. coli::pMTL23 (see Example 5.7.1.C). These results indicate that it is possible to express the papM gene of S. pristinaespiralis in E. coli, and that the corresponding protein is indeed the enzyme catalysing the methylation of p-aminophenylalanine to p-dimethylaminophenylalanine. This example shows that it is possible to express genes for the biosynthesis of streptogramins in heterologous strains (such as E. coli, but also in other microorganisms) using appropriate expression vectors in order to produce precursors of antibiotics or even natural or hybrid antibiotics.
EXAMPLE 12
Demonstration of the Homology of Genes Involved in the Biosynthesis of Streptogramins in Different Streptomyces
This example illustrates how it is possible to demonstrate, by hybridization with total DNAs, the homology existing between different genes involved in the biosynthesis of streptogramins in different strains of Streptomyces producing streptogramins.
12.1. Extraction of Total DNA of Different Streptomyces Producing Streptogramins
This example illustrates how the DNA of different strains producing streptogramins was purified. These strains of Streptomyces were chosen from those described in Table 1:
Streptomyces loidensis
Streptomyces olivaceus
Streptomyces ostreogriseus
Streptomyces virginiae
A strain not producing streptogramins: Streptomyces hygroscopicus, was chosen as negative control.
The extractions of the different total DNAs were carried out from cultures in YEME medium, as described in Example 1.
12.2. Hybridization of Total DNAs of Strains Producing Streptogramins with DNA Fragments Containing Genes Involved in the Biosynthesis of Pristinamycins and Isolated from S. pristinaespiralis Strain SP92
This example illustrates hot it is possible, starting from genes involved in the biosynthesis of pristinamycins and isolated from the strain SP92 as described in the preceding examples, to demonstrate homologous genes by hybridization of the total DNAs of strains producing streptogramins.
The DNA fragments used as a probe were:
The 3.9-kb XhoI--XhoI fragment isolated from pVRC1106 described in Example 6.5, the restriction map of which is presented in FIG. 18. This fragment contains a portion of the gene coding for pristinamycin I synthase II.
The 6-kb BamHI--BamHI fragment isolated from plasmid pXL2045 described in Example 6.3, the map of which is presented in FIG. 16. This fragment contains the structural genes for the two subunits of PIIA synthase.
The total DNAs of the four strains producing streptogramins, the strain S. hygroscopicus and also the strain SP92, were digested with the restriction enzymes BamHI and XhoI. The DNA fragments thereby obtained were separated on 0.7% agarose gel and the DNA was transferred onto a nylon membrane as described by Maniatis et al. (1989). Labelling of the 3.6-kb XhoI--XhoI and 6-kb BamHI--BamHI fragments was carried out by labelling by random priming as described in Example 9.1.2. Hybridization of the membranes was carried out in the presence of formamide at 42.degree. C. as described in Maniatis et al. (1989). Washing of the membranes after hybridization was carried out at 50 and 60.degree. C. in a solution containing SSC (Maniatis et al. (1989) diluted 10-fold and 0.1% SDS.
The following results are demonstrated by these hybridizations:
The strain S. hygroscopicus does not display and hybridization with the two probes used.
The total DNAs (digested with XhoI and BamHI) of the strains S. ostreogirseus, S. olivaceus, S. loidensis and S. virginiae all display hybridization signals of intensity comparable to those observed on the total DNA of the strain SP92 with both probes used.
The total DNA (digested with XhoI and BamHI) of the strain S. virginiae displays signals with both probes used, but their intensity is weaker than that observed in SP92.
This example shows that different strains of Streptomyces producing streptogramins contain genes that hybridize with genes isolated in S. pristinaespiralis SP92 and which are involved in the biosynthesis of streptogramins, as presented in the preceding examples. These hybridizations thus demonstrate the homology existing between the genes involved in the biosynthesis of streptogramins of the strains SP92 and those involved in the biosynthesis of streptogramins of other strains producing streptogramins.
This example hence shows that it is possible, starting from genes isolated from SP92 and involved in the biosynthesis of streptogramins, to isolate by hybridization and cloning the homologous genes present in other strains producing streptogramins.
EXAMPLE 13
Study of the physical binding of the different S. pristinaespiralis SP 92 genes involved in the biosynthesis of pristinamycins I and pristinamycins II
This example illustrates how it is possible to study the physical binding of the S. pristinaespiralis SP 92 genes involved in the biosynthesis of pristinamycins I and II. This study was carried out for the purpose of showing that all these genes are grouped together on the chromosome in a cluster, and that it is hence possible by chromosome walking from the genes already identified to isolate other genes involved in the biosynthesis of pristinamycins I and II. Such an approach mahy be envisaged for the genes involved in the biosynthesis of other streptogramins.
13.1 Restriction enzymes used for pulsed-filed electrophoresis
The S. pristinaespiralis SP92 genome is composed of 70% to 75% of nucleotides containing the basis G and C. To cut its genome into a small number of large fragments, we used enzymes which recognize a sequence rich in AT, such as AseI (AT/TAAT) and SspI (AAT/AAT) but also HindIII (A/AGCTT), EcoRI (G/AATTC), NdeI (CA/TATG) and ClaI (AT/CGAT).
13.2. S. pristinaespiralis strains used for pulsed-field electrophoresis
We used the chromosomal DNA of several strains to study by pulsed-field electrophrosis the physical binding of the genes involved in the biosynthesis of pristinamycins I and pristinamycins II. We prepared inserts as described ibn Example 4.1 of the chromosomal DNA of S. pristinaespiralis strain SP92, and also of the chromosomal DNA of the strains derived from SP92 whose construction is described in Examples 9.1 and 9.4. These are the strain SP92::pVRC505 in which the snaA gene has been disrupted by integration of plasmid pVRC505 (Example 9.1), and the strain SP92::pVRC404 in which the snbA gene has been disrupted by integration of plasmid pVRC404 (Example 9.4). The latter two strains were included in this study since they enabled the snaA and snbB genes to be positioned accurately on the chromosome map by exploiting the presence of sites which rarely cut chromosomal DNA, AseI, SspI, HindIII, EcoRI, NdeI and ClaI, in plasmids pVRC505 and pVRC404.
13.3. DNA probes used for hybridization of the fragments isolated by pulsed-filed electrophoresis
We used different DNA fragments to obtain radioactively labelled probes as is described in Example 9.1, which we hybridized with the fragments separated by pulsed-field electrophoresis after enzymatic cleavage of the chromosomal DNA inserts of the three strains presented above. The probes are as follows: the 3.2-kb EcoRI-BamHI fragment isolated from plasmid pVRC701 carrying the snaB and samS genes (see Example 9.2), the 1.5-kb BamH1-Sst1 fragment isolated from plasmid pVRC1000 carrying a portion of the snaD gene (see Example 6.8), the 1.1-kb XhoI-HindIII fragment isolated from plasmid pVRC402 carrying the snbA gene (see Example 6.1), the 2.4-kb PstI-Pst1 fragment isolated from plasmid pVRC900 carrying papA gene (see Example 6.7) and the 1.5-kb XhoI-PstI fragment isolated from plasmid pVRC509 carrying the snaC gene (see Example 6.9).
13.4. Localization on the chromosome of the different genes involved in the biosynthesis of pristinamycins I and II and study of their physical binding
Hybridization of the chromosomal DNAs of S. pristinaespiralis strains SP92, SP92::pVRC404 and SP92::pVRC505, cut by single digestions and double digestions using the six enzymes mentioned above, with the different probes described above lead to the general map shown in FIG. 32: the position of major sites has been indicated, together with the position and direction of transcription of the genes involved in the biosynthesis of pristinamycins PI and PII. Thus, it is possible to calculate the distance separating the 3 chromosomal regions containing the genes identified, namely that of the snbA, snbR, papA and papM genes (cosmid pIBV2, Example 5.2), that of the snaA, snaB, samS, snaD, snbC, snbD and snbE genes (cosmids pIBV1 and 3, Example 5.1) and lastly that of the snaC gene (cosmid pIBV4, Example 5.6). For example, the distance between the snaA and snbA genes has been evaluated at approximately 160-170 kb. This shows that the genes already identified are all contained in a region covering only 200 kb of the chromosome of the S. pristinaespiralis strain, equivalent to less than 3% of the total length of the genome, which we have been able to estimate at 7500 kb by the pulsed-field electrophoresis technique.
These results show that the genes involved in the biosynthesis of pristinamycins I and II are grouped together on the chromosome in a cluster, and that it is hence possible by chromosome walking from the genes already identified to isolate other genes involved in the biosynthesis of pristinamycins I and pristinamycins II. More generally, it is possible, by chromosome walking from any gene involved in the biosynthesis of streptogramins, to identify the other genes involved in this biosynthesis.
EXAMPLE 14
Isolation of a large fragment of S. virginiae genomic DNA containing the structural genes for virginiamycins S synthases
This example illustrates how it is possible to isolate genes for the biosynthesis of streptogramins other than pristinamycins, by hybridizing the genomic DNA of strains that produce streptogramins, as described in Example 12, with fragments of genomic DNA of the strain SP92, containing genes for the biosynthesis of pristinamycins, which are described in Example 8.
More especially, this example illustrates how, from a library of genomic DNA of S. virginiae, the strain that produces virginiamycins, and using as probe a DNA fragment of the strain SP92 containing the structural gene for pristinamycin I synthase III, it is possible to isolate a large fragment of S. virginiae DNA containing structural genes for virginiamycin S synthases (VS synthases) which display homologies with pristinamycins I synthases (PI synthases).
14.1 Production of a library of S. virginiae genomic DNA
In a first stage, a library of S. virginiae genomic DNA is produced in E. coli HB101 as described in Example 3, by cloning high molecular weight DNA fragments into cosmid pXL667 (Ditta et al. 1980).
Total DNA of the S. virginiae strain was prepared from a culture in YEME medium+glycine according to the protocol described in Example 3. This total DNA was then partially digested with Bg1II in the buffer recommended by the supplier. The amount of enzyme used to obtain DNA fragments of molecular weight between 15 and 25 kb was determined empirically, as described in Example 3. Approximately 100 .mu.g of total DNA are digested in this way, and the DNA fragments between 15 and 25 kb in size are then isolated using a 10-40% sucrose gradient. Their size is verified on a 0.5% agarose gel.
Cosmid pXL667 was extracted as described in Example 2.1, from E. coli HB101.
Approximately 150 ng of cosmid linearized by a BamHI digestion were precipitated using ethanol with 350 ng of fragments of S. virginiae total DNA, prepared as described above. The pellet was taken up in 10 .mu.l of ligation buffer, and 0.5 .mu.l of T4 DNA ligase was added, as described in Example 3. Incubation was carried out overnight at 15.degree. C.
Encapsidation of the hybrid cosmids after ligation was carried out using the Gigapack II Gold kit, as described in Example 3. 2.times.4 .mu.l of ligation mixture, equivalent to 2.times.70 ng of hybrid cosmids, were encapsidated in vitro according to the procedure described by the supplier. The hybrid cosmids were transfected into E. coli HB101, after preparation of the cells, as described in Example 3. The transfectants are then selected on solid LB medium+tetracycline 12 .mu.g/ml. The number of transfectants obtained is approximately 10.sup.3 to 10.sup.4 .mu.g of recombinant cosmid.
After verification of the average size of the fragments inserted into cosmid pXL667, approximately 2000 colonies originating from the transfection carried out with the HB101 strains are subcultured in 96-well microtitration plates containing 200 .mu.l of Hogness medium (described in Example 3)+tetracycline 12 .mu.g/ml. These plates are incubated overnight at 37.degree. C. and then stored at -80.degree. C.
14.2 Isolation of cosmid pIBV30 containing the structural genes for virginiamycin S synthases
This example illustrates how it is possible to obtain a cosmid, as constructed in Example 14.1, containing the structural genes for VS synthases.
The 2000 clones constituting the genomic DNA library wee subcultured, from the 20 microtitration plates, on LB medium containing 12 .mu.g/ml of tetracycline using a replica plater, and the DNA of the colonies was transferred onto Biohylon Z.sup.+ membranes, as described in Example 3.
These membranes were then hybridized with the 3.6-kb XhoI--XhoI probe purified from plasmid pVRC1106 and containing the gene for pristinamycin I synthase III of the strain SP92, described in Example 5.4. 7 clones were hybridized with this probe. Analysis by enzymatic digestion of the cosmids contained in these clones enabled 2 clones having comparable restriction profiles to be distinguished. These two cosmids have in common, in particular, a doublet of BamHI--BamHI DNA bands of approximately 3 kb, hybridizing with the 3.6-kb XhoI--XhoI probe. This doublet is also visible on hybridization of XhoI-digested S. virginiae genomic DNA, as described in Example 12.2. A double digestion with the restriction enzymes BamHI and SacI enables the doublet to be separated into a 3-kb BamHI--BamHI fragment and a 2.2-kb BamHI-SacI fragment, both of them hybridizing with the 3.6-kb XhoI--XhoI probe. One of these cosmids was designated pIBV30 and was studied more particularly. It contains an approximately 22-kb fragment of S. virginiae total DNA. A partial restriction map is presented in FIG. 33. This DNA insert probably originates from the simultaneous ligation of two BglII fragments, one of 1.9-kb and one of approximately 20-kb. The 20-kb fragment contains the 3-kb BamHI--BamHI doublet which hybridizes with the 3.6-kb XhoI--XhoI fragment containing the gene coding for PI synthase III, and hence probably contains one or more genes coding for VS synthases.
14.3 Identification of virginiamycin S synthases on the 20-kb Bg1II--Bg1II fragment
This example illustrates how, from fragment of S. pristinaespiralis DNA containing the structural genes for pristinamycins I synthases II, III and IV described in Examples 5.3, 5.4 and 5.5, it is possible to identify, on the fragment of S. virginiae genomic DNA contained in cosmid pIBV30, the homologous genes coding for VS synthases.
Cosmid pIBV30 was digested with different restriction enzymes, and the DNA fragments thus generated were transferred onto nylon membranes after separation on agarose gel, as described in the previous examples.
Hybridizations were carried out with the following three probes:
Probe 1 (P1): 0.9-kb XhoI--XhoI fragment isolated from pVRC1105 described in Example 6.4. This fragment contains a portion of the structural gene for pristinamycin I synthases II (snbC).
Probe 2 (P2): 3.6-kb XhoI--XhoI fragment isolated from pVRC1106 described in Example 6.5. This fragment contains a portion of the structural gene for pristinamycin I synthases III (snbD).
Probe 3 (P3): 1-kb PstI--PstI fragment isolated from pVRC1104 described in Example 6.6. This fragment contains a portion of the structural gene for pristinamycin I synthase IV (snbE).
These hybridizations enabled thee successive regions, each hybridizing preferentially with one of the three probes (P1, P2, P3) described, to be identified on cosmid pIBV30. These regions are presented in FIG. 33. These hybridization results indicate the presence on the 20-kb fragment contained in cosmid pIBV30 of genes homologous with the snbC, -D and -E genes involved in the synthesis of PI, and a portion of the coding frame of which is contained in the DNA fragments corresponding to the probes P1, P2 and P3, respectively. Furthermore, the organization of these genes is similar to that of the snbC, -D and -E genes in S. pristinaespiralis. In effect, the fragments of cosmid pIBV30 DNA which hybridize with the probes P1, P2 and P3 follow one another in the order corresponding to the order of the snbC, -D and -E genes on cosmid pIBV3, described in Examples 5.1, 5.3, 5.4 and 5.5.
14.4 Cloning of a 2.2-kb BamHI-SacI fragment containing a gene coding for a virginiamycin S synthase
The BamHI-SacI fragment, which was identified as described above by hybridization with the 3.6-kb XhoI-13 XhoI probe carrying the snbD gene, was subcloned into a pUC19. The subcloning was carried out as described in the previous examples. The recombinant plasmid was named pVRC510, and its restriction map is presented in FIG. 34.
The nucleotide sequence of the 2.2-kb BamHI-SacI fragment was determined completely (SEQ ID No. 17) by subcloning into the vectors M13mp18 and M13mp19. The different inserts were sequenced by the chain termination reaction method, using as synthesis primer the universal primer or synthetic oligonucloetides complementary to a sequence of 20 nucleotides of the insert to be sequenced, as described in the previous examples.
Analysis of the nucleotide sequence as described in Example 8 enabled a single open reading frame (orf), spread over the entire length of the 2.2-kb fragment and displaying a codon usage typical of Streptomyces, to be detected. This orf, referred to as orf1, does not contain any stop codon, indicating that it corresponds merely to a portion of a larger open reading frame.
Comparison of the product of this portion of open reading frame with the protein sequences contained in the Genpro bank reveals a 30 to 35% homology with different peptide synthases of different microorganisms. Moreover, the product of orf1 displays 64% homology with the 3.6-kb XhoI--XhoI fragment containing the snbD gene of S. pristinaespiralis.
These homology results confirm the presence on the approximately 20-kb Bg1II--Bg1II fragment of a gene (orf1) coding for a peptide synthase, homologous with different peptide synthases and more especially with pristinamycin I synthase III (SnbD). This peptide synthase probably participates in the syntheses of virginiamycin S, just as SnbD participates in the synthesis of pristinamycin I. Further, hybridizations of cosmid pIBV30 containing this gene with the probes P1, P2 and P3 corresponding, respectively, to the snbC, -D and -E genes of S. pristinaespiralis, coding for PI synthases II, III and VI, indicate that the corresponding genes of S. virginiae are present on the 20-kb Bg1II--Bg1II fragment in a manner identical to the organization observed in S. pristinaespiralis. The presence in S. virginiae of a cluster containing the genes coding for VS synthases, identical to the one present in S. pristinaespiralis, containing PI synthases, enables us to envisage the cloning of the other genes involved in the syntheses of virginiamycins S and M, by chromosome walking, as is the usual custom with Streptomyces, in which the genes involved in a single biosynthesis are physically grouped together on the chromosome.
Although the invention has been described in conjunction with specific embodiments, it is evident that many alternatives and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, the invention is intended to embrace all of the alternatives and variations that fall within the spirit and scope of the appended claims. The above references are hereby incorporated by reference.
TABLE 1______________________________________MICROORGANISMS ANTIBIOTICS______________________________________FUNGIMicromonospora sp. VernamycinsSTREPTOMYCESS. albiorectus VirginiamycinsS. conqanesis (ATCC13528) F1370 A, BS. diastaticus Plauracins, StreptograminsS. graminofasciens StreptograminsS. griseus (NRRL2426) Viridogrisein (Etamycin)S. griseoviridus GriseoviridinS. griseoviridus (FERMP3562) NeoviridogriseinsS. lavendulae EtamycinsS. loidensis (ATCC11415) VernamycinsS. mitakaensis (ATCC15297) MikamycinsS. olivaceus (ATCC12019) Synergistins (PA114 A, B)S. ostreogriseus (ATCC27455) OstreogrycinsS. pristinaespiralis (ATCC25486) PristinamycinsS. virginiae (ATCC13161) Virgiuniamycins (Staphylomycins)ACTINOMYCETESA. auranticolor (ATCC31011) PlauracinsA. azureus (ATCC31157) PlauracinsA. daghestanicus EtamycinA. philippinensis A-2315 A, B, CActinioplanes sp. (ATCC33002) A15104Actinoplanes sp. A17002 A, B, C, FActinomadura flava Madumycins______________________________________Abbreviations used:DNA: deoxyribonucleic acidAMP: adenosine 5'-monophosphateATP: adenosine 5'-triphosphateETB: ethidium bromidebis-tris: (bis[2-hydroxyethyl]iminotris[hydroxymethyl]- methane)bis-tris propane: (1,3-bis[tris(hydroxymethyl)- methylamino]propane)BSA: bovine serum albuminHPLC: high performance liquid chromatographyOD: optical densityDTE: dithioerythritolDTT: dithiothreitolE64: trans-epoxysuccinyl-L-leucylamido-(4- guanidino) butaneEDTA: ethylenediaminetetraacetic acidEGTA: ethylene glycol bis(.beta.-aminoethyl)tetraacetic acidFMN: flavin mononucleotideFMNH.sub.2 : reduced flavin mononucleotideHepes: (N-[2-hydroxyethyl]piperazine-N'-[2- ethanesulphonic acid])IPTG: isopropyl .beta.-D-thiogalactopyranosidekDa: kilodaltonkb: kilobaseLB: Luria broth (rich growth medium for E. coli)NAD: nicotinamide dinucleotideNADH: reduced nicotinamide dinucleotidePAGE: polyacrylamide gel electrophoresisbp: base pairPMSF: phenylmethylsulphonyl fluoridePPi: pyrophosphaterpm: revolutions per minA.S.: ammonium sulphateSAM: S-adenosylmethionineSDS: sodium dodecyl sulphateSTI: soybean trypsin inhibitorTE: buffer comprising 10 mM Tris-HCl, 1 mM EDTA, pH 7.5Tris: 2-amino-2-hydroxymethyl-1,3-propanediolUV: ultraviolet raysXgal: 5-bromo-4-chloro-3-indoyl b-D-galactosideYEME: yeast extract-malt extract medium (rich growth medium for Streptomyces)PEG: Polyethylene glycolLMP: Low melting pointMW: Molecular weight______________________________________
BIBLIOGRAPHY
Anzai H., Murakami T., Imai A., Satoh A., Nagaoka K. and Thompson C. J. (1987) J. Bacteriol., 169: 3482-3488.
Bancroft I. and Wolk C. P. (1989) J. Bacteriol., 171: 5949-5954.
Bibb M. J., Findlay P. R. and Johnson M. W. (1984), Gene, 30: 157-166.
Birnboim H. C. and Doly J. (1979) Nucleic Acids Res., 7: 1513-1523.
Blattner F. R., Williams B. G., Blechl A. E., Denniston-Thompson K., Faber H. E. Furlong L. A., Grunwald D. J., Kiefer D. O., Moore D. D., Schumm J. W., Sheldon E. L. and Smithies O. (1977) Science, 196: 161-169.
Bolivar F., Rodriguez R. L., Greene P. J. Betlach M. C., Heynecker H. L., Boyer H. W., Crosa J. H. and Falkow S. (1977) Gene, 2: 95-113.
Boyer H. W. and Roulland-Dussoix D. (1969) J. Mol. Biol., 41: 459.
Chater K. F. (1990) Bio/Technology, 8: 115-121.
Cocito C. G. (1979) Microbiol. Rev., 43: 145-198.
Cocito C. G. (1983) In Antibiotics, 6: (Ed. F. E. Hahn), 296-332.
Dessen P. C., Fondrat C., Valencien C. et Mugnier C. (1990) Comp. Appl. in Biosciences, 6: 355-356.
Di Giambattista M., Chinali G. and Cocito C. G. (1989) J. Antim. Chemother., 24: 485-507.
Fernandez-Moreno M. A., Caballero J. L., Hopwood D. A. and Malpartida F. (1991) Cell, 66: 769-780.
Gibson T. J. (1984) Ph.D. thesis, Cambridge University, England.
Hallam S. E., Malpartida F. and Hopwood D. A. (1988) Gene, 74: 305-320.
Hames B. D. and Higgins S. J. (1985) IRL Press Ltd., Oxford, U.K.,
Hanahan D. (1983) J. Mol. Biol., 166: 557
Hillemann D., Pulher A. and Wohlleben W. (1991) Nucl. Acids Res., 19: 727-731.
Hohn B. and Collins J. F. (1980) Gene, 11: 291-298.
Hook J. D. and Vining L. C. (1973) J.C.S. Chem. Comm., 185-186.
Hopwood D. A., Bibb M. J., Chater K. F., Kieser T., Bruton C. J., Kieser H. M., Lydiate D. J., Smith C. P., Ward J. M. and Scrempf H. (1985) A laboratory manual., The John Innes Fondation, Norwich, England.
Hopwood D. A., Bibb M. J. Chater K. F., Janssen G. R., Malpartida F. and Smith C. (1986b) In Regulation of gene expression--25 years on (ed. I; A. Booth C; F. Higgins), 251-276.
Hopwood D. A., Malpartida F., Kieser H. M., Ikeda H., Duncan J., Fujii I., Rudd A. M., Floss H. G. and Omura S. (1985a) Nature, 314: 642-644.
Hopwood D. A., Malpartida F. Kieser H. M., Ikeda H. and Omura S. (1985b) In Microbiology (ed S. Silver). American Society for Microbiology, Washington D.C., 409-413.
Hopwood D. A., Malpartida F. and Chater K. F. (1986a) In Regulation of secondary metabolite formation. (eds. H. Kleinkauf, H. von Hohren, H. Dornauer G. Nesemann), 22-33.
Hoshino T., Ikeda T., Tomizuka N. and Furukawa K. (1985) Gene, 37: 131-138.
Hutchinson C. R., Borell C. W., Otten S. L., Stutzman-Engwall K. J. and Wang Y. (1989) J. Med. Chem., 32: 929-937.
Ish-Horowitz D. and Burk J. F. (1981) Nucleic Acids Res., 9: 2989-298.
Kanehisa M. I. (1984) Nucleic Acids Res., 12: 203-215.
Kay R. and McPherson J. (1987) Nucleic Acids Res., 15 (6): 2778.
Khan S. A. and Novick R. (1983) Plasmid, 10: 251-259.
Kingston D. G. I., Kolpak M. X, Lefevre W. and Borup-Grochtmann I. B. (1983) J. Am. Chem. Soc., 105: 5106-5110.
Kyte J. and Doolittle R. (1982) J. Biol. Mol., 157: 105-135.
Low B. (1968) Proc. Nalt. Acad. Sci., 60: 160.
Lydiate D. J., Malpartida F. and Hopwood D. A. (1985) Gene, 35: 223-235.
Maniatis T., Fritsh E. F. and Sambrook J (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor N.Y.,
Meinkoth J. and Wahl G. (1984) Anal. Biochem., 138: 267-284.
Messing J., Crea R. and Seeburg P. H. (1981) Nucleic Acids Res., 9: 309.
Neal R. J. and Chater K. F. (1987) Gene, 58: 229-241.
Ohnuki T., Imanaka T. and Aiba S. (1985) J. Bacteriol., 164: 85-94.
Sawadogo M. and Van Dyke M. W. (1991) Nucl. Acids Res., 19: 674.
Staad J. F., Elkins M. F. and Earhart C. F. (1989) FEMS Microbial. Lett., 59: 15.
Staden R. and McLachlan A. D. (1982) Nucleic Acids Res., 10: 141-156.
Videau D. (1982) Path. Biol., 30: 529-534.
Chambers S. P., Prior S. E., Barstow D. A. and Minton N. P. (1988) Gene, 68:139
Gutierrez S., Diez B., Montenegro E. and Martin J. F. (1991) Journal of bacteriology, 173:2354-2365.
Hori K., Yamamoto Y., Minetoki T., Kurotsu T., Kanda M., Miura S., Okamura K., Kuruyama J. and Saito Y. (1989) J. Biochem., 106: 639-645.
Horikawa S., Ishikawa M., Ozaka H. and Tsukada K. (1989) Eur. J. Biochem., 184: 497-501.
Kaplan, J., Merkel W. and Nichols B. (1985) J. Mol. Biol., 183: 327-340.
Markham G. D., DeParasis J. and Gatmaitan J. (1984) J. Biol Chem., 259: 14505-14507.
Sharka B., Westlake D. W. S. and Vining L. C. (1970) Chem. Zvesti, 24:66-72.
Thomas D., Rothstein R., Rosenberg N. and Surdin-Kerjan Y. (1988) Mol. Cell. Biol., 8:5132-5139.
Turgay K. et al (1992) Molecular Microbiology, 6(4):546.
Weckermann R., FYrbab R. and Marahiel M. A. (1988) Nucl. Acids Res., 16:11841
Yanisch-Perron C., Vieira J. and Messing J. (1985) Gene, 33: 103-119.
Zimmer W., Aparicio C. and Elmerich C. (1991) Mol. Gen. Genet., 229:41-51.
Reed et al. J. Nat. Prod 49 (1986) 626
Molinero et al., J. Nat. Peod 52 (1989) 99
Reed et al. J. Org. Chem. 54 (1989) 1161
Watanabe et al., Mol. Cell. Biochem. 44(1982) 181
Jablonski et al., Biochemistry 16 (1977) 2832
Duane et al., Mol. Cell. Biochem. 6 (1975) 53.
__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 45- (2) INFORMATION FOR SEQ ID NO: 1:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 5392 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: NO- (iii) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: S.pristinaes - #piralis#1: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- GGATCCTGGC GTCCGCCGTC AAGAACTGAA CCGAGGAGAC ACCCACCATG AC - #CGCACCCC 60- GCCGGCGCAT CACCCTCGCC GGCATCATCG ACGGCCCCGG CGGCCATGTG GC - #CGCCTGGC 120- GCCACCCGGC GACCAAGGCG GACGCCCAGC TCGACTTCGA ATTCCACCGC GA - #CAACGCCC 180- GCACCCTCGA ACGCGGCCTG TTCGACGCCG TGTTCATCGC GGACATCGTC GC - #CGTGTGGG 240- GCACCCGCCT GGACTCCCTG TGCCGCACCT CGCGCACCGA GCACTTCGAA CC - #GCTCACCC 300- TGCTCGCCGC CTACGCCGCG GTCACCGAGC ACATCGGCCT GTGCGCCACC GC - #CACCACCA 360- CGTACAACGA ACCGGCGCAC ATCGCCGCCC GCTTCGCCTC CCTCGACCAC CT - #CAGCGGCG 420- GCCGGGCCGG CTGGAACGTC GTCACCTCCG CCGCACCGTG GGAGTCCGCC AA - #CTTCGGCT 480- TCCCCGAGCA CCTGGAGCAC GGCAAACGCT ACGAGCGGGC CGAGGAGTTC AT - #CGACGTCG 540- TCAAAAAACT GTGGGACAGC GACGGCCGCC CCGTCGACCA CCGCGGCACC CA - #CTTCGAGG 600- CCCCCGGCCC GCTCGGGATC GCCCGCCCCC CGCAGGGCCG CCCCGTCATC AT - #CCAGGCCG 660- GCTCCTCGCC GGTGGGACGC GAGTTCGCCG CCCGGCACGC CGAGGTCATC TT - #CACCCGGC 720- ACAACCGGCT CTCCGACGCC CAGGACTTCT ACGGCGACCT CAAGGCACGC GT - #CGCCCGGC 780- ACGGCCGCGA CCCCGAGAAG GTCCTCGTGT GGCCGACCCT CGCGCCGATC GT - #CGCCGCCA 840- CCGACACCGA GGCGAAGCAG CGCCTGCAGG AACTGCAGGA CCTCACCCAC GA - #CCATGTCG 900- CCCTGCGCAC CCTTCAGGAC CACCTCGGCG ACGTCGACCT GAGCGCGTAC CC - #GATCGACG 960- GGCCCGTCCC CGACATCCCG TACACCAACC AGTCCCAGTC GACGACCGAG CG - #GCTGATCG1020- GCCTGGCCAG GCGCGAGAAC CTCAGCATCC GCGAGCTGGC CCTGCGGCTG AT - #GGGCGACA1080- TCGTCGTCGG CACACCGGAG CAGCTCGCCG ACCACATGGA GAGCTGGTTC AC - #CGGCCGCG1140- GCGCCGACGG CTTCAACATC GACTTCCCGT ACCTGCCGGG CTCCGCCGAC GA - #CTTCGTCG1200- ACCACGTGGT GCCCGAACTG CAGCGCCGCG GCCTGTACCG CTCGGGCTAC GA - #GGGCACCA1260- CCCTGCGGGC CAACCTCGGC ATCGACGCCC CCCGGAAGGC AGGTGCAGCG GC - #TTGACTTC1320- CGTCCTAAAG GCGGGGGATT CCAGCGGTCG CCCGCTGGGG TTCCTGCTTC AC - #CGACGACC1380- GCCCCGTCCG GGAGGACTCC CGTTGAGGTC TTATACCGTC TCCACAGGCC GA - #CGCCGCCA1440- GCCCGGCGGC CAGGATGTTG CGTGCCGCAT TCACGTCGCG GTCATGCACA GC - #GCCGCAGT1500- CGCACGTCCA CTCCCGGACG TTCAGCGGCA GCTTCCCGCG GACCGTGCCG CA - #GGTTCCGC1560- ACAGCTTGGA GCTGGGGAAC CAGCGGTCGA TCACGACGAG TTCGCGCCCA TA - #CCAGGCGC1620- ACTTGTACTC CAGCATGGAG CGCAGTTCCG TCCAGGCCGC GTCGGAGATG GC - #GCGCGCGA1680- GCTTGCCGTT CTTCAGCAGG TTGCGGACGG TGAGGTCCTC GATCACGACC GT - #TTGGTTCT1740- CACGGACGAG TCGAGTCGAC AGCTTGTGGA GGAAGTCGCA GCGCCGGTCG GT - #GATCCGGG1800- CGTGGACGCG GGCGACCTTG CGGCGGGCTT TCTTCCGGTT CGCCGACCCC TT - #CGCCTTGC1860- GCGACACGTC CCGCTGAGCC TTCGCGAGGC GGGCGCGGTC ACGGCGCTCG TG - #CTTGGGGT1920- TGGTGATCTT CTCCCCGGTG GACAGGGTCA CCAGGGAGGT GATCCCGGCG TC - #GATGCCGA1980- CGGCCGCCGT GGTGGCGGGC GCGGGGGTGA TGGTGTCCTC GCACAGCAGG GA - #CACGAACC2040- AGCGGCCCGC ACGGTCGCGG GACACGGTCA CCGTCGTCGG CTCCGCCCCT TC - #GGGAAGGG2100- GACGGGACCA GCGGATGTCC AGGGGCTCCG CGGTCTTCGC CAGCGTGAGC TG - #TCCGTTAC2160- GCCACGTGAA GGCGCTGCGG GTGTACTCGG CCGACGCCCT GGACTTTTTC CG - #CGACTTGT2220- ACCGCGGGTA CTTCGACCGC TTGGCGAAGA AGTTGGCGAA CGCCGTCTGC AA - #GTGCCGCA2280- GCGCCTGCTG GAGCGGGACG GAGGACACCT CCGAGAGGAA GGCGAGTTCT TC - #GGTCTTCT2340- TCCACTCCGT CAGCGCGGCG GACGACTGCA CGTAGGAGAC CCGGCGCTGC TC - #GCCGTACC2400- AGGCTCGCGT GCGCCCCTCA AGCGCCTTGT TGTACACGAG GCGGACACAG CC - #GAACGTGC2460- GGGACAGCTC AGCCGCCTGC TCGTCCGTGG GATAAAAGCG GTACTTGAAA GC - #CCGCTTGA2520- CCTGCTGCAT CACGCCTCAC ACGCTATCAG TTCCCGTGTG AGCGGCGGGT GT - #CTGCCGGT2580- GGTTGCAGAC GCCGAACCGC CCTGGCGGCG ATTCGCCCAT CCCTGCCCTG CT - #CCGCAAGA2640- GCTTCGTCTC CTCCCCGGTC TGAAGGCCGG GGTATCCACG AAGGAATTCT GA - #TGACCGCG2700- CCCATCCTCG TCGCCACCCT CGACACCCGC GGCCCCGCCG CCACCCTCGG CA - #CGATCACC2760- CGCGCCGTGC GGGCCGCGGA GGCCGCCGGA TTCGACGCCG TCCTGATCGA CG - #ACCGGGCC2820- GCCGCCGGCG TCCAGGGCCG GTTCGAGACG ACGACGCTGA CCGCCGCGCT GG - #CCGCCGTC2880- ACCGAGCACA TCGGCCTGAT CACCGCCCCG CTCCCGGCCG ACCAGGCCCC CT - #ACCACGTG2940- TCCCGGATCA CCGCCTCGCT CGACCACCTC GCCCACGGCC GCACCGGCTG GC - #TCGCGAGC3000- ACGGACACCA CCGACCCCGA GGGCCGCACC GGCGAACTCA TCGACGTCGT CC - #GCGGCCTG3060- TGGGACAGCT TCGACGACGA CGCCTTCGTC CACGACCGCG CCGACGGCCT GT - #ACTGGCGG3120- CTGCCCGCCG TCCACCAACT CGACCACCAG GGCAGGCACT TCGACGTGGC CG - #GCCCCCTC3180- AACGTCGCCC GCCCGCCGCA GGGCCACCCC GTCGTCGCCG TCACCGGCCC CG - #CCCTCGCC3240- GCGGCCGCCG ACCTCGTCCT GCTCGACGAG GCGGCCGACG CCGCCTCGGT GA - #AGCAGCAG3300- GCACCGCACG CCAAGATCCT CCTGCCGCTG CCCGGCCCGG CCGCCGAACT GC - #CCGCCGAC3360- AGCCCCGCGG ACGGCTTCAC GGTGGCGCTC ACCGGCTCCG ACGACCCGGT CC - #TGGCCGCG3420- CTCGCCGCCC GGCCCGGCCG CCCGGACCGC ACCGCGGCCA CCACCCTGCG CG - #AACGCCTG3480- GGCCTGGCCC GCCCCGAGAG CCGCCACGCC CTCACCACCG CCTGACGACC CG - #TCCGCCCG3540- CTGCTTCCTG GAGAGTCATG TCCCGTCGCC TGTTCACCTC GGAGTCCGTG AC - #CGAGGGCC3600- ACCCCGACAA GATCGCCGAC CAGATCAGTG ACACCGTCCT CGACGCCCTG CT - #GCGCGAGG3660- ACCCCGCCTC ACGCGTCGCG GTCGAGACCC TGATCACCAC CGGCCAGGTC CA - #CATCGCCG3720- GCGAGGTCAC CACCAAGGCG TACGCGCCCA TCGCCCAACT GGTCCGCGAC AC - #GATCCTGG3780- CCATCGGCTA CGACTCGTCC GCCAAGGGCT TCGACGGCGC CTCCTGCGGC GT - #CTCCGTCT3840- CCATCGGCGC GCAGTCCCCG GACATCGCCC AGGGCGTCGA CAGCGCCTAC GA - #GACCCGCG3900- TCGAGGGCGA GGACGACGAG CTCGACCAGC AGGGCGCCGG CGACCAGGGC CT - #GATGTTCG3960- GCTACGCCAC CGACGAGACC CCCTCGCTGA TGCCGCTGCC CATCGAGCTC GC - #CCACCGCC4020- TCTCGCGCCG GCTCACCGAG GTCCGCAAGG ACGGCACCGT CCCCTACCTG CG - #CCCCGACG4080- GCAAGACCCA GGTCACCATC GAGTACCAGG GCAGCCGCCC GGTGCGCCTG GA - #CACCGTCG4140- TCGTCTCCTC CCAGCACGCC GCCGACATCG ACCTCGGCTC CCTGCTCACC CC - #CGACATCC4200- GCGAGCACGT CGTCGAGCAC GTCCTCGCCG CACTCGCCGA GGACGGCATC AA - #GCTCGAGA4260- CGGACAACTA CCGCCTGCTG GTCAACCCGA CCGGCCGTTT CGAGATCGGC GG - #CCCGATGG4320- GCGACGCCGG CCTGACCGGC CGCAAGATCA TCATCGACAC GTACGGCGGC AT - #GGCCCGCC4380- ACGGCGGTGG CGCGTTCTCC GGCAAGGACC CGTCCAAGGT CGACCGTTCC GC - #CGCGTACG4440- CGATGCGCTG GGTCGCCAAG AACGTCGTCG CCGCGGGCCT CGCCTCCCGC TG - #CGAGGTCC4500- AGGTCGCCTA CGCCATCGGC AAGGCCGAGC CGGTCGGCCT GTTCGTCGAG AC - #GTTCGGCA4560- CCGGCACCGT CGCCCAGGAG CGCATCGAGA AGGCCATCAC CGAGGTCTTC GA - #CCTGCGCC4620- CCGCGGCCAT CATCCGCGAC CTCGACCTGC TGCGGCCCAT CTACGCCGCC AC - #CGCCGCCT4680- ACGGCCACTT CGGCCGCGAA CTGCCCGACT TCACCTGGGA GCGGACCGAC CG - #CGCCCACC4740- GGCTCAAGGC CGCGGCCGGT CTCTGAGCCG GCCGGACCTG TGAGGAGACC TG - #ACGTGCGC4800- ATCGCTGTCA CCGGTTCCAT CGCCACCGAC CATCTGATGG TCTTCCCCGG CC - #GGTTCGCG4860- GATCAGCTGA TCCCCGACCA GCTCGCTCAT GTCTCGCTCT CCTTCCTGGT CG - #ACGCACTC4920- GAGGTGCGCC GGGGCGGAGT GGCGGACAAC GTCGCCTTCG GCCTCGGCGG CC - #TCGGCCTC4980- ACCCCCCAGC TGGTCGGCGC CGTGGGCAGC GACTTCGCCG AGTACGAGGT CT - #GGCTCAAG5040- GAACACGGCG TCGACACCGG CCCCGTCCTG GTCTCCACCG AGCGGCAGAC CG - #CCCGGTTC5100- ATGTGCATCA CCGACCAGGA CTCCAACCAG ATCGCCTCCT TCTACGCGGG CG - #CCATGCAA5160- GAGGCCCGCG ACATCGACCT GTGGCACCTG ACCACCGGCA GCGTCCGCCC CG - #ACCTCGTC5220- CTGGTCTGCC CGAACGACCC GGCGGCGATG CTGCGCCACA CGGGGAGTGC CG - #CGAAACTG5280- GGCCTGCCGT TCGCCGCCGA CCCCTCCCAG CAGCTCGCCC GCCTGGAGGG AG - #GGAGGTAC5340- GCGAACTCGG TCGACGGGGC CCGTTGGTTT TTCACCAACG AAGTACGAGG CC - #5392- (2) INFORMATION FOR SEQ ID NO: 2:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1268 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: NO- (iii) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: S.pristinaes - #piralis- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1268#2: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- ATG ACC GCA CCC CGC CGG CGC ATC ACC CTC GC - #C GGC ATC ATC GAC GGC 48Met Thr Ala Pro Arg Arg Arg Ile Thr Leu Al - #a Gly Ile Ile Asp Gly# 15- CCC GGC GGC CAT GTG GCC GCC TGG CGC CAC CC - #G GCG ACC AAG GCG GAC 96Pro Gly Gly His Val Ala Ala Trp Arg His Pr - #o Ala Thr Lys Ala Asp# 30- GCC CAG CTC GAC TTC GAA TTC CAC CGC GAC AA - #C GCC CGC ACC CTC GAA 144Ala Gln Leu Asp Phe Glu Phe His Arg Asp As - #n Ala Arg Thr Leu Glu# 45- CGC GGC CTG TTC GAC GCC GTG TTC ATC GCG GA - #C ATC GTC GCC GTG TGG 192Arg Gly Leu Phe Asp Ala Val Phe Ile Ala As - #p Ile Val Ala Val Trp# 60- GGC ACC CGC CTG GAC TCC CTG TGC CGC ACC TC - #G CGC ACC GAG CAC TTC 240Gly Thr Arg Leu Asp Ser Leu Cys Arg Thr Se - #r Arg Thr Glu His Phe# 80- GAA CCG CTC ACC CTG CTC GCC GCC TAC GCC GC - #G GTC ACC GAG CAC ATC 288Glu Pro Leu Thr Leu Leu Ala Ala Tyr Ala Al - #a Val Thr Glu His Ile# 95- GGC CTG TGC GCC ACC GCC ACC ACC ACG TAC AA - #C GAA CCG GCG CAC ATC 336Gly Leu Cys Ala Thr Ala Thr Thr Thr Tyr As - #n Glu Pro Ala His Ile# 110- GCC GCC CGC TTC GCC TCC CTC GAC CAC CTC AG - #C GGC GGC CGG GCC GGC 384Ala Ala Arg Phe Ala Ser Leu Asp His Leu Se - #r Gly Gly Arg Ala Gly# 125- TGG AAC GTC GTC ACC TCC GCC GCA CCG TGG GA - #G TCC GCC AAC TTC GGC 432Trp Asn Val Val Thr Ser Ala Ala Pro Trp Gl - #u Ser Ala Asn Phe Gly# 140- TTC CCC GAG CAC CTG GAG CAC GGC AAA CGC TA - #C GAG CGG GCC GAG GAG 480Phe Pro Glu His Leu Glu His Gly Lys Arg Ty - #r Glu Arg Ala Glu Glu145 1 - #50 1 - #55 1 -#60- TTC ATC GAC GTC GTC AAA AAA CTG TGG GAC AG - #C GAC GGC CGC CCC GTC 528Phe Ile Asp Val Val Lys Lys Leu Trp Asp Se - #r Asp Gly Arg Pro Val# 175- GAC CAC CGC GGC ACC CAC TTC GAG GCC CCC GG - #C CCG CTC GGG ATC GCC 576Asp His Arg Gly Thr His Phe Glu Ala Pro Gl - #y Pro Leu Gly Ile Ala# 190- CGC CCC CCG CAG GGC CGC CCC GTC ATC ATC CA - #G GCC GGC TCC TCG CCG 624Arg Pro Pro Gln Gly Arg Pro Val Ile Ile Gl - #n Ala Gly Ser Ser Pro# 205- GTG GGA CGC GAG TTC GCC GCC CGG CAC GCC GA - #G GTC ATC TTC ACC CGG 672Val Gly Arg Glu Phe Ala Ala Arg His Ala Gl - #u Val Ile Phe Thr Arg# 220- CAC AAC CGG CTC TCC GAC GCC CAG GAC TTC TA - #C GGC GAC CTC AAG GCA 720His Asn Arg Leu Ser Asp Ala Gln Asp Phe Ty - #r Gly Asp Leu Lys Ala225 2 - #30 2 - #35 2 -#40- CGC GTC GCC CGG CAC GGC CGC GAC CCC GAG AA - #G GTC CTC GTG TGG CCG 768Arg Val Ala Arg His Gly Arg Asp Pro Glu Ly - #s Val Leu Val Trp Pro# 255- ACC CTC GCG CCG ATC GTC GCC GCC ACC GAC AC - #C GAG GCG AAG CAG CGC 816Thr Leu Ala Pro Ile Val Ala Ala Thr Asp Th - #r Glu Ala Lys Gln Arg# 270- CTG CAG GAA CTG CAG GAC CTC ACC CAC GAC CA - #T GTC GCC CTG CGC ACC 864Leu Gln Glu Leu Gln Asp Leu Thr His Asp Hi - #s Val Ala Leu Arg Thr# 285- CTT CAG GAC CAC CTC GGC GAC GTC GAC CTG AG - #C GCG TAC CCG ATC GAC 912Leu Gln Asp His Leu Gly Asp Val Asp Leu Se - #r Ala Tyr Pro Ile Asp# 300- GGG CCC GTC CCC GAC ATC CCG TAC ACC AAC CA - #G TCC CAG TCG ACG ACC 960Gly Pro Val Pro Asp Ile Pro Tyr Thr Asn Gl - #n Ser Gln Ser Thr Thr305 3 - #10 3 - #15 3 -#20- GAG CGG CTG ATC GGC CTG GCC AGG CGC GAG AA - #C CTC AGC ATC CGC GAG1008Glu Arg Leu Ile Gly Leu Ala Arg Arg Glu As - #n Leu Ser Ile Arg Glu# 335- CTG GCC CTG CGG CTG ATG GGC GAC ATC GTC GT - #C GGC ACA CCG GAG CAG1056Leu Ala Leu Arg Leu Met Gly Asp Ile Val Va - #l Gly Thr Pro Glu Gln# 350- CTC GCC GAC CAC ATG GAG AGC TGG TTC ACC GG - #C CGC GGC GCC GAC GGC1104Leu Ala Asp His Met Glu Ser Trp Phe Thr Gl - #y Arg Gly Ala Asp Gly# 365- TTC AAC ATC GAC TTC CCG TAC CTG CCG GGC TC - #C GCC GAC GAC TTC GTC1152Phe Asn Ile Asp Phe Pro Tyr Leu Pro Gly Se - #r Ala Asp Asp Phe Val# 380- GAC CAC GTG GTG CCC GAA CTG CAG CGC CGC GG - #C CTG TAC CGC TCG GGC1200Asp His Val Val Pro Glu Leu Gln Arg Arg Gl - #y Leu Tyr Arg Ser Gly385 3 - #90 3 - #95 4 -#00- TAC GAG GGC ACC ACC CTG CGG GCC AAC CTC GG - #C ATC GAC GCC CCC CGG1248Tyr Glu Gly Thr Thr Leu Arg Ala Asn Leu Gl - #y Ile Asp Ala Pro Arg# 415# 126 - #8TGLys Ala Gly Ala Ala Ala 420- (2) INFORMATION FOR SEQ ID NO: 3:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 833 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: NO- (iii) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: S.pristinaes - #piralis- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..833#3: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- ATG ACC GCG CCC ATC CTC GTC GCC ACC CTC GA - #C ACC CGC GGC CCC GCC 48Met Thr Ala Pro Ile Leu Val Ala Thr Leu As - #p Thr Arg Gly Pro Ala# 15- GCC ACC CTC GGC ACG ATC ACC CGC GCC GTG CG - #G GCC GCG GAG GCC GCC 96Ala Thr Leu Gly Thr Ile Thr Arg Ala Val Ar - #g Ala Ala Glu Ala Ala# 30- GGA TTC GAC GCC GTC CTG ATC GAC GAC CGG GC - #C GCC GCC GGC GTC CAG 144Gly Phe Asp Ala Val Leu Ile Asp Asp Arg Al - #a Ala Ala Gly Val Gln# 45- GGC CGG TTC GAG ACG ACG ACG CTG ACC GCC GC - #G CTG GCC GCC GTC ACC 192Gly Arg Phe Glu Thr Thr Thr Leu Thr Ala Al - #a Leu Ala Ala Val Thr# 60- GAG CAC ATC GGC CTG ATC ACC GCC CCG CTC CC - #G GCC GAC CAG GCC CCC 240Glu His Ile Gly Leu Ile Thr Ala Pro Leu Pr - #o Ala Asp Gln Ala Pro# 80- TAC CAC GTG TCC CGG ATC ACC GCC TCG CTC GA - #C CAC CTC GCC CAC GGC 288Tyr His Val Ser Arg Ile Thr Ala Ser Leu As - #p His Leu Ala His Gly# 95- CGC ACC GGC TGG CTC GCG AGC ACG GAC ACC AC - #C GAC CCC GAG GGC CGC 336Arg Thr Gly Trp Leu Ala Ser Thr Asp Thr Th - #r Asp Pro Glu Gly Arg# 110- ACC GGC GAA CTC ATC GAC GTC GTC CGC GGC CT - #G TGG GAC AGC TTC GAC 384Thr Gly Glu Leu Ile Asp Val Val Arg Gly Le - #u Trp Asp Ser Phe Asp# 125- GAC GAC GCC TTC GTC CAC GAC CGC GCC GAC GG - #C CTG TAC TGG CGG CTG 432Asp Asp Ala Phe Val His Asp Arg Ala Asp Gl - #y Leu Tyr Trp Arg Leu# 140- CCC GCC GTC CAC CAA CTC GAC CAC CAG GGC AG - #G CAC TTC GAC GTG GCC 480Pro Ala Val His Gln Leu Asp His Gln Gly Ar - #g His Phe Asp Val Ala145 1 - #50 1 - #55 1 -#60- GGC CCC CTC AAC GTC GCC CGC CCG CCG CAG GG - #C CAC CCC GTC GTC GCC 528Gly Pro Leu Asn Val Ala Arg Pro Pro Gln Gl - #y His Pro Val Val Ala# 175- GTC ACC GGC CCC GCC CTC GCC GCG GCC GCC GA - #C CTC GTC CTG CTC GAC 576Val Thr Gly Pro Ala Leu Ala Ala Ala Ala As - #p Leu Val Leu Leu Asp# 190- GAG GCG GCC GAC GCC GCC TCG GTG AAG CAG CA - #G GCA CCG CAC GCC AAG 624Glu Ala Ala Asp Ala Ala Ser Val Lys Gln Gl - #n Ala Pro His Ala Lys# 205- ATC CTC CTG CCG CTG CCC GGC CCG GCC GCC GA - #A CTG CCC GCC GAC AGC 672Ile Leu Leu Pro Leu Pro Gly Pro Ala Ala Gl - #u Leu Pro Ala Asp Ser# 220- CCC GCG GAC GGC TTC ACG GTG GCG CTC ACC GG - #C TCC GAC GAC CCG GTC 720Pro Ala Asp Gly Phe Thr Val Ala Leu Thr Gl - #y Ser Asp Asp Pro Val225 2 - #30 2 - #35 2 -#40- CTG GCC GCG CTC GCC GCC CGG CCC GGC CGC CC - #G GAC CGC ACC GCG GCC 768Leu Ala Ala Leu Ala Ala Arg Pro Gly Arg Pr - #o Asp Arg Thr Ala Ala# 255- ACC ACC CTG CGC GAA CGC CTG GGC CTG GCC CG - #C CCC GAG AGC CGC CAC 816Thr Thr Leu Arg Glu Arg Leu Gly Leu Ala Ar - #g Pro Glu Ser Arg His# 270# 833 CC TGAla Leu Thr Thr Ala 275- (2) INFORMATION FOR SEQ ID NO: 4:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1208 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: NO- (iii) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: S.pristinaes - #piralis- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1208#4: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- ATG TCC CGT CGC CTG TTC ACC TCG GAG TCC GT - #G ACC GAG GGC CAC CCC 48Met Ser Arg Arg Leu Phe Thr Ser Glu Ser Va - #l Thr Glu Gly His Pro# 15- GAC AAG ATC GCC GAC CAG ATC AGT GAC ACC GT - #C CTC GAC GCC CTG CTG 96Asp Lys Ile Ala Asp Gln Ile Ser Asp Thr Va - #l Leu Asp Ala Leu Leu# 30- CGC GAG GAC CCC GCC TCA CGC GTC GCG GTC GA - #G ACC CTG ATC ACC ACC 144Arg Glu Asp Pro Ala Ser Arg Val Ala Val Gl - #u Thr Leu Ile Thr Thr# 45- GGC CAG GTC CAC ATC GCC GGC GAG GTC ACC AC - #C AAG GCG TAC GCG CCC 192Gly Gln Val His Ile Ala Gly Glu Val Thr Th - #r Lys Ala Tyr Ala Pro# 60- ATC GCC CAA CTG GTC CGC GAC ACG ATC CTG GC - #C ATC GGC TAC GAC TCG 240Ile Ala Gln Leu Val Arg Asp Thr Ile Leu Al - #a Ile Gly Tyr Asp Ser# 80- TCC GCC AAG GGC TTC GAC GGC GCC TCC TGC GG - #C GTC TCC GTC TCC ATC 288Ser Ala Lys Gly Phe Asp Gly Ala Ser Cys Gl - #y Val Ser Val Ser Ile# 95- GGC GCG CAG TCC CCG GAC ATC GCC CAG GGC GT - #C GAC AGC GCC TAC GAG 336Gly Ala Gln Ser Pro Asp Ile Ala Gln Gly Va - #l Asp Ser Ala Tyr Glu# 110- ACC CGC GTC GAG GGC GAG GAC GAC GAG CTC GA - #C CAG CAG GGC GCC GGC 384Thr Arg Val Glu Gly Glu Asp Asp Glu Leu As - #p Gln Gln Gly Ala Gly# 125- GAC CAG GGC CTG ATG TTC GGC TAC GCC ACC GA - #C GAG ACC CCC TCG CTG 432Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr As - #p Glu Thr Pro Ser Leu# 140- ATG CCG CTG CCC ATC GAG CTC GCC CAC CGC CT - #C TCG CGC CGG CTC ACC 480Met Pro Leu Pro Ile Glu Leu Ala His Arg Le - #u Ser Arg Arg Leu Thr145 1 - #50 1 - #55 1 -#60- GAG GTC CGC AAG GAC GGC ACC GTC CCC TAC CT - #G CGC CCC GAC GGC AAG 528Glu Val Arg Lys Asp Gly Thr Val Pro Tyr Le - #u Arg Pro Asp Gly Lys# 175- ACC CAG GTC ACC ATC GAG TAC CAG GGC AGC CG - #C CCG GTG CGC CTG GAC 576Thr Gln Val Thr Ile Glu Tyr Gln Gly Ser Ar - #g Pro Val Arg Leu Asp# 190- ACC GTC GTC GTC TCC TCC CAG CAC GCC GCC GA - #C ATC GAC CTC GGC TCC 624Thr Val Val Val Ser Ser Gln His Ala Ala As - #p Ile Asp Leu Gly Ser# 205- CTG CTC ACC CCC GAC ATC CGC GAG CAC GTC GT - #C GAG CAC GTC CTC GCC 672Leu Leu Thr Pro Asp Ile Arg Glu His Val Va - #l Glu His Val Leu Ala# 220- GCA CTC GCC GAG GAC GGC ATC AAG CTC GAG AC - #G GAC AAC TAC CGC CTG 720Ala Leu Ala Glu Asp Gly Ile Lys Leu Glu Th - #r Asp Asn Tyr Arg Leu225 2 - #30 2 - #35 2 -#40- CTG GTC AAC CCG ACC GGC CGT TTC GAG ATC GG - #C GGC CCG ATG GGC GAC 768Leu Val Asn Pro Thr Gly Arg Phe Glu Ile Gl - #y Gly Pro Met Gly Asp# 255- GCC GGC CTG ACC GGC CGC AAG ATC ATC ATC GA - #C ACG TAC GGC GGC ATG 816Ala Gly Leu Thr Gly Arg Lys Ile Ile Ile As - #p Thr Tyr Gly Gly Met# 270- GCC CGC CAC GGC GGT GGC GCG TTC TCC GGC AA - #G GAC CCG TCC AAG GTC 864Ala Arg His Gly Gly Gly Ala Phe Ser Gly Ly - #s Asp Pro Ser Lys Val# 285- GAC CGT TCC GCC GCG TAC GCG ATG CGC TGG GT - #C GCC AAG AAC GTC GTC 912Asp Arg Ser Ala Ala Tyr Ala Met Arg Trp Va - #l Ala Lys Asn Val Val# 300- GCC GCG GGC CTC GCC TCC CGC TGC GAG GTC CA - #G GTC GCC TAC GCC ATC 960Ala Ala Gly Leu Ala Ser Arg Cys Glu Val Gl - #n Val Ala Tyr Ala Ile305 3 - #10 3 - #15 3 -#20- GGC AAG GCC GAG CCG GTC GGC CTG TTC GTC GA - #G ACG TTC GGC ACC GGC1008Gly Lys Ala Glu Pro Val Gly Leu Phe Val Gl - #u Thr Phe Gly Thr Gly# 335- ACC GTC GCC CAG GAG CGC ATC GAG AAG GCC AT - #C ACC GAG GTC TTC GAC1056Thr Val Ala Gln Glu Arg Ile Glu Lys Ala Il - #e Thr Glu Val Phe Asp# 350- CTG CGC CCC GCG GCC ATC ATC CGC GAC CTC GA - #C CTG CTG CGG CCC ATC1104Leu Arg Pro Ala Ala Ile Ile Arg Asp Leu As - #p Leu Leu Arg Pro Ile# 365- TAC GCC GCC ACC GCC GCC TAC GGC CAC TTC GG - #C CGC GAA CTG CCC GAC1152Tyr Ala Ala Thr Ala Ala Tyr Gly His Phe Gl - #y Arg Glu Leu Pro Asp# 380- TTC ACC TGG GAG CGG ACC GAC CGC GCC CAC CG - #G CTC AAG GCC GCG GCC1200Phe Thr Trp Glu Arg Thr Asp Arg Ala His Ar - #g Leu Lys Ala Ala Ala385 3 - #90 3 - #95 4 -#00# 1208Gly Leu- (2) INFORMATION FOR SEQ ID NO: 5:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1879 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: NO- (iii) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: S.pristinaes - #piralis- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 110..1858#5: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- GATCGGCTCC TGACGGAGCG GCGGCGCGCG GGCGCGGCGC ATCAGCGGCG TG - #TCAACGGC 60- GCTGCCGACA CTGGGCGCGA CGCGAGGACG AAGCCGGAAA GGACCAACG ATG - # CTG 115#Met Leu# 1- GAC GGA TGC GTT CCC TGG CCC GAG GAT GTG GC - #C GCG AAG TAC CGG GCG 163Asp Gly Cys Val Pro Trp Pro Glu Asp Val Al - #a Ala Lys Tyr Arg Ala# 15- GCC GGC TAC TGG CGG GGC GAG CCG CTG GGC AT - #G CTG CTG GGC CGC TGG 211Ala Gly Tyr Trp Arg Gly Glu Pro Leu Gly Me - #t Leu Leu Gly Arg Trp# 30- GCG GAG CAG TAC GGC GAG CGG GAG GCG CTG GT - #C GGC GCG GAC GGG TGC 259Ala Glu Gln Tyr Gly Glu Arg Glu Ala Leu Va - #l Gly Ala Asp Gly Cys# 50- TCC CGT GTC ACC TAC CGT GCC CTG GAC CGC TG - #G TGC GAC CGG CTG GCG 307Ser Arg Val Thr Tyr Arg Ala Leu Asp Arg Tr - #p Cys Asp Arg Leu Ala# 65- GCG GGG TTC GCG GCG CGC GGG ATC GGC GCC GG - #C GAG CGG GTG CTG GTG 355Ala Gly Phe Ala Ala Arg Gly Ile Gly Ala Gl - #y Glu Arg Val Leu Val# 80- CAG CTG CCG AAC ACG CCC GAG TTC GTC GCG GT - #G TGC TTC GCG CTG TTC 403Gln Leu Pro Asn Thr Pro Glu Phe Val Ala Va - #l Cys Phe Ala Leu Phe# 95- CGT CTG GGC GCG CTG CCG GTG TTC GCG CTG CC - #C GCG CAC CGT GCC GCC 451Arg Leu Gly Ala Leu Pro Val Phe Ala Leu Pr - #o Ala His Arg Ala Ala# 110- GAG GTG GGG CAC CTG CTC GAG CTG TCC GGC GC - #C GTC GCC CAC ATC CTG 499Glu Val Gly His Leu Leu Glu Leu Ser Gly Al - #a Val Ala His Ile Leu115 1 - #20 1 - #25 1 -#30- CCG GGC ACC GGC ACC GGC TAC GAC CAT GTC GC - #G GCG GCC GTG GAG GCC 547Pro Gly Thr Gly Thr Gly Tyr Asp His Val Al - #a Ala Ala Val Glu Ala# 145- CGT GCC CGC CGT GCC CGC CCG GTG CAG GTG TT - #C GTG GCG GGC GAG GCG 595Arg Ala Arg Arg Ala Arg Pro Val Gln Val Ph - #e Val Ala Gly Glu Ala# 160- CCC GCG GTG CTG CCC GAG GGG TTC ACC GCG CT - #G GCC GAC GTG GAC GGC 643Pro Ala Val Leu Pro Glu Gly Phe Thr Ala Le - #u Ala Asp Val Asp Gly# 175- GAC CCG GTG GCG CCG GCG GAC GTG GAC GCC TT - #C CGA CGT GGC GTC TTC 691Asp Pro Val Ala Pro Ala Asp Val Asp Ala Ph - #e Arg Arg Gly Val Phe# 190- CTG CTG TCC GGG GGG ACG ACC GCG CTG CCG AA - #G CTG ATC CCG CGC ACC 739Leu Leu Ser Gly Gly Thr Thr Ala Leu Pro Ly - #s Leu Ile Pro Arg Thr195 2 - #00 2 - #05 2 -#10- CAC GAC GAC TAC GCC TAC CAG TGC CGG GTC AC - #G GCC GGT ATC TGC GGC 787His Asp Asp Tyr Ala Tyr Gln Cys Arg Val Th - #r Ala Gly Ile Cys Gly# 225- CTG GAC GCG GAC AGT GTC TAT CTG GCG GTG CT - #G CCG GCC GAG TTC AAC 835Leu Asp Ala Asp Ser Val Tyr Leu Ala Val Le - #u Pro Ala Glu Phe Asn# 240- TTC CCC TTC GGC TGC CCG GGC ATC CTG GGC AC - #C CTG CAC GCC GGC GGG 883Phe Pro Phe Gly Cys Pro Gly Ile Leu Gly Th - #r Leu His Ala Gly Gly# 255- CGG GTG GTG TTC GCG CTG TCA CCG CAG CCC GA - #G GAG TGC TTC GCG CTG 931Arg Val Val Phe Ala Leu Ser Pro Gln Pro Gl - #u Glu Cys Phe Ala Leu# 270- ATC GAA CGC GAA CAC GTC ACC TTC ACC TCC GT - #C ATC CCC ACG ATC GTG 979Ile Glu Arg Glu His Val Thr Phe Thr Ser Va - #l Ile Pro Thr Ile Val275 2 - #80 2 - #85 2 -#90- CAC CTG TGG CTG GCG GCC GCC GCA CAA GGC CA - #C GGC CGC GAC CTG GGC1027His Leu Trp Leu Ala Ala Ala Ala Gln Gly Hi - #s Gly Arg Asp Leu Gly# 305- AGC CTT CAG CTG CTG CAG GTC GGC AGC GCC AA - #A CTC CAC GAG GAG CTC1075Ser Leu Gln Leu Leu Gln Val Gly Ser Ala Ly - #s Leu His Glu Glu Leu# 320- GCC GCC CGG ATC GGC CCC GAA CTG GGG GTG CG - #G CTG CAG CAG GTG TTC1123Ala Ala Arg Ile Gly Pro Glu Leu Gly Val Ar - #g Leu Gln Gln Val Phe# 335- GGC ATG GCC GAG GGA CTG CTG ACC TTC ACC CG - #C GAC GAC GAC CCG GCG1171Gly Met Ala Glu Gly Leu Leu Thr Phe Thr Ar - #g Asp Asp Asp Pro Ala# 350- GAC GTG GTG CTG CGC ACC CAG GGC CGG CCG GT - #G TCC GAG GCC GAC GAG1219Asp Val Val Leu Arg Thr Gln Gly Arg Pro Va - #l Ser Glu Ala Asp Glu355 3 - #60 3 - #65 3 -#70- ATA CGC GTC GCC GAC CCC GAC GGC CGG CCC GT - #G CCC CGC GGT GAG ACC1267Ile Arg Val Ala Asp Pro Asp Gly Arg Pro Va - #l Pro Arg Gly Glu Thr# 385- GGT GAA CTG CTC ACC CGC GGC CCC TAC ACG CT - #G CGC GGC TAC TAC CGG1315Gly Glu Leu Leu Thr Arg Gly Pro Tyr Thr Le - #u Arg Gly Tyr Tyr Arg# 400- GCC CCC GAG CAC AAC GCC CGC GCG TTC ACC GA - #G GAC GGC TTC TAC CGC1363Ala Pro Glu His Asn Ala Arg Ala Phe Thr Gl - #u Asp Gly Phe Tyr Arg# 415- AGC GGC GAT CTG GTG CGG CTC ACC GCC GAC GG - #G CAG TTG GTG GTG GAG1411Ser Gly Asp Leu Val Arg Leu Thr Ala Asp Gl - #y Gln Leu Val Val Glu# 430- GGC AGG ATC AAG GAC GTC GTC ATC CGC GGC GG - #C GAC AAG GTC TCC GCG1459Gly Arg Ile Lys Asp Val Val Ile Arg Gly Gl - #y Asp Lys Val Ser Ala435 4 - #40 4 - #45 4 -#50- ACC GAG GTC GAG GGC CAC CTG GGC GCC CAC CC - #C GAC GTC CAG CAG GCC1507Thr Glu Val Glu Gly His Leu Gly Ala His Pr - #o Asp Val Gln Gln Ala# 465- GCC GTC GTC GCC ATG CCC GAC CCG GTG TGG GG - #C GAG AAG GTC TGC GCC1555Ala Val Val Ala Met Pro Asp Pro Val Trp Gl - #y Glu Lys Val Cys Ala# 480- TAC ATC GTG CCC GCA CCC GGC CGT CCC GCA CC - #G CCG ATG GCG GCG CTG1603Tyr Ile Val Pro Ala Pro Gly Arg Pro Ala Pr - #o Pro Met Ala Ala Leu# 495- CGC CGG CTG CTG CGC GCG CGG GGA CTG GCC GA - #C TAC AAG CTT CCC GAC1651Arg Arg Leu Leu Arg Ala Arg Gly Leu Ala As - #p Tyr Lys Leu Pro Asp# 510- CGG GTG GAG GTC GTC GAC GCG TTC CCG CTG AC - #C GGC CTC AAC AAG GTC1699Arg Val Glu Val Val Asp Ala Phe Pro Leu Th - #r Gly Leu Asn Lys Val515 5 - #20 5 - #25 5 -#30- GAC AAG AAG GCC CTG GCG GCC GAC ATC GCC GC - #C AAG ACC GCC CCC ACC1747Asp Lys Lys Ala Leu Ala Ala Asp Ile Ala Al - #a Lys Thr Ala Pro Thr# 545- CGC CCC ACC ACC GCC GGC CAC GGC CCG ACC AC - #G GAC GGC GAT ACG GCC1795Arg Pro Thr Thr Ala Gly His Gly Pro Thr Th - #r Asp Gly Asp Thr Ala# 560- GGT GGG GGT GGG TCC GCG GGC GGG GTG ACG GC - #C GCC GGT GGC GGG CGG1843Gly Gly Gly Gly Ser Ala Gly Gly Val Thr Al - #a Ala Gly Gly Gly Arg# 575# 1879 GAGCGGGCC CGGGCCCGAG GGCGGlu Glu Ala Ala 580- (2) INFORMATION FOR SEQ ID NO: 6:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1833 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: NO- (iii) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: S.pristinaes - #piralis- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 103..1689#6: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- GGATCCCCTC GCCCAGGGCC CTGGCGGGCC CGCCGGGCCG TGGGGGAGGT GC - #GGGGGCCG 60- CGGGCCCCGG CACCGCACGA ACAGAACAAC CGCTCCGGGC CC ATG CGG - # ACT TCA 114# Met Arg Thr Ser# 1- CGG TCC CAC GAC CAG CGG GCC CCT ACC CCC TG - #G AGA CAT CCC TTG CAC 162Arg Ser His Asp Gln Arg Ala Pro Thr Pro Tr - #p Arg His Pro Leu His# 20- AGC ACC CGG CCC GCG CCC GCG GCC GAC CGT GA - #C CCC AGG CGC TGG GTC 210Ser Thr Arg Pro Ala Pro Ala Ala Asp Arg As - #p Pro Arg Arg Trp Val# 35- ATC CTC GGC GTG ATC TGC CTG GCC CAA CTC GT - #C GTC CTG CTC GAC AAC 258Ile Leu Gly Val Ile Cys Leu Ala Gln Leu Va - #l Val Leu Leu Asp Asn# 50- ACC GTC CTC AAC GTC GCC ATC CCG GTG CTC AC - #C ACC GAC CTG GGC GCC 306Thr Val Leu Asn Val Ala Ile Pro Val Leu Th - #r Thr Asp Leu Gly Ala# 65- AGC ACC GCC GAC ATC CAG TGG ATG ATC AAC GC - #C TAC GCG CTC GTG CAG 354Ser Thr Ala Asp Ile Gln Trp Met Ile Asn Al - #a Tyr Ala Leu Val Gln# 80- TCC GGG CTG CTG CTC ACC GCG GGC AGC CTC GC - #G GAC CGC TAC GGC CGC 402Ser Gly Leu Leu Leu Thr Ala Gly Ser Leu Al - #a Asp Arg Tyr Gly Arg#100- AAA CGG CTG CTG ATG CTC GGA CTG GTG CTC TT - #C GGC GCC GGG TCC GCC 450Lys Arg Leu Leu Met Leu Gly Leu Val Leu Ph - #e Gly Ala Gly Ser Ala# 115- TGG GCG GCC TTC GCC CAG GAC TCC GCC CAA CT - #C ATC GCC GCC CGG GCC 498Trp Ala Ala Phe Ala Gln Asp Ser Ala Gln Le - #u Ile Ala Ala Arg Ala# 130- GGC ATG GGC GTG GGC GGG GCG CTG CTG GCG AC - #C ACC ACC CTC GCC GTC 546Gly Met Gly Val Gly Gly Ala Leu Leu Ala Th - #r Thr Thr Leu Ala Val# 145- ATC ATG CAG GTC TTC GAC GAC GAC GAA CGC CC - #C CGG GCG ATC GGC CTG 594Ile Met Gln Val Phe Asp Asp Asp Glu Arg Pr - #o Arg Ala Ile Gly Leu# 160- TGG GGA GCG GCC AGC TCA CTG GGC TTC GCG GC - #C GGC CCG CTG CTC GGC 642Trp Gly Ala Ala Ser Ser Leu Gly Phe Ala Al - #a Gly Pro Leu Leu Gly165 1 - #70 1 - #75 1 -#80- GGC GCC CTC CTC GAC CAC TTC TGG TGG GGC TC - #C ATC TTC CTG ATC AAC 690Gly Ala Leu Leu Asp His Phe Trp Trp Gly Se - #r Ile Phe Leu Ile Asn# 195- CTG CCC GTC GCG CTG CTG GGC CTG CTG GCC GT - #C GCC CGC CTG GTG CCC 738Leu Pro Val Ala Leu Leu Gly Leu Leu Ala Va - #l Ala Arg Leu Val Pro# 210- GAG ACG AAG AAC CCC GAA GGC CGG CGC CCC GA - #C CTG CTC GGC GCC GTG 786Glu Thr Lys Asn Pro Glu Gly Arg Arg Pro As - #p Leu Leu Gly Ala Val# 225- CTC TCC ACC CTC GGC ATG GTC GGC GTC GTC TA - #C GCC ATC ATC TCC GGC 834Leu Ser Thr Leu Gly Met Val Gly Val Val Ty - #r Ala Ile Ile Ser Gly# 240- CCC GAA CAC GGC TGG ACG GCC CCG CAG GTC CT - #C CTG CCG GCC GCC GTC 882Pro Glu His Gly Trp Thr Ala Pro Gln Val Le - #u Leu Pro Ala Ala Val245 2 - #50 2 - #55 2 -#60- GCG GCC GCC GCG CTC ACC GCG TTC GTC CGC TG - #G GAA CTG CAC ACC CCC 930Ala Ala Ala Ala Leu Thr Ala Phe Val Arg Tr - #p Glu Leu His Thr Pro# 275- CAC CCC ATG CTC GAC ATG GGC TTC TTC ACC GA - #C CGG CGC TTC AAC GGG 978His Pro Met Leu Asp Met Gly Phe Phe Thr As - #p Arg Arg Phe Asn Gly# 290- CCG TCG CCG GCG GAG TGC TCG TCG TTC GGC AT - #G GCC GGC TCG CTC TTC1026Pro Ser Pro Ala Glu Cys Ser Ser Phe Gly Me - #t Ala Gly Ser Leu Phe# 305- CTG CTC ACC CAG CAC CTC CAA CTC GTC CTC GG - #C TAC GAC GCC CTG CAG1074Leu Leu Thr Gln His Leu Gln Leu Val Leu Gl - #y Tyr Asp Ala Leu Gln# 320- GCC GGC CTG CGC ACC GCG CCA CTG GCT TTG AC - #G ATC GTC GCC CTC AAC1122Ala Gly Leu Arg Thr Ala Pro Leu Ala Leu Th - #r Ile Val Ala Leu Asn325 3 - #30 3 - #35 3 -#40- CTG GCC GGC CTC GGC GCG AAA CTC CTC GCC GC - #G CTC GGC ACC GCC CGC1170Leu Ala Gly Leu Gly Ala Lys Leu Leu Ala Al - #a Leu Gly Thr Ala Arg# 355- AGC ATC GCC CTG GGC ATG ACA CTG CTG GCC GC - #C GGC CTC AGC GCG GTG1218Ser Ile Ala Leu Gly Met Thr Leu Leu Ala Al - #a Gly Leu Ser Ala Val# 370- GCC GTC GGC GGA TCG GGC CCC GAC GCC GGC TA - #C GGC GGC ATG CTC GCC1266Ala Val Gly Gly Ser Gly Pro Asp Ala Gly Ty - #r Gly Gly Met Leu Ala# 385- GGC CTG CTC CTC ATG GGC GCG GGC ATC GCA CT - #G GCC ATG CCC GCC ATG1314Gly Leu Leu Leu Met Gly Ala Gly Ile Ala Le - #u Ala Met Pro Ala Met# 400- GCC ACC GCC GTG ATG TCC TCC ATC CCG CCC GC - #C AAG GCC GGG GCC GGA1362Ala Thr Ala Val Met Ser Ser Ile Pro Pro Al - #a Lys Ala Gly Ala Gly405 4 - #10 4 - #15 4 -#20- GCG GGC GTG CAG GGC ACC CTG ACC GAG TTC GG - #C GGC GGA CTG GGA GTG1410Ala Gly Val Gln Gly Thr Leu Thr Glu Phe Gl - #y Gly Gly Leu Gly Val# 435- GCG ATC CTC GGC GCC GTC CTC GGC TCC CGC TT - #C GCC TCC CAA CTG CCC1458Ala Ile Leu Gly Ala Val Leu Gly Ser Arg Ph - #e Ala Ser Gln Leu Pro# 450- GCC GCC ATC ACC GGC ACC GGC TCC CTC GAC GA - #G GCA CTG CGC GAC GCC1506Ala Ala Ile Thr Gly Thr Gly Ser Leu Asp Gl - #u Ala Leu Arg Asp Ala# 465- ACA CCC CAA CAG GCC GGG CAG GTC CAC GAC GC - #G TTC GCC GAC GCG GTG1554Thr Pro Gln Gln Ala Gly Gln Val His Asp Al - #a Phe Ala Asp Ala Val# 480- AAC ACC AGC CAA CTC ATC GGC GCC GCC GCC GT - #G TTC ACC GGC GGC CTG1602Asn Thr Ser Gln Leu Ile Gly Ala Ala Ala Va - #l Phe Thr Gly Gly Leu485 4 - #90 4 - #95 5 -#00- CTC GCC GCG CTG CTG CTG CAC CGC GCC GAC CG - #C AAG GCC GCC CCC CAG1650Leu Ala Ala Leu Leu Leu His Arg Ala Asp Ar - #g Lys Ala Ala Pro Gln# 515- CCC ACC GCC CCC ACC CCC GAA CCC ACC ACC AC - #C GCC TGACCCCCGG1696Pro Thr Ala Pro Thr Pro Glu Pro Thr Thr Th - #r Ala# 525- CCCGCCGGGC ACCACACAAC CCACGGCCCC ACCCCTGCGG CTCCCCACCG GG - #ACCCACAG1756- GGGCGGGGCC GTGCCGCTGC CCTGCCCACA CACACAGCCC CCACACACAC AG - #CCCCCGCA1816# 1833 G- (2) INFORMATION FOR SEQ ID NO: 7:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 695 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: NO- (iii) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: S.pristinaes - #piralis- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 212..695#/product= "Gene SnaC"FORMATION:#7: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- CTCGAGCCGC GCCCCCAGGT GCTGGTGTCG CTCGCCGTGG AGAAGGGCGC CG - #ACGGCACC 60- GCGCCGCCGG ACCGGCTGCT GATCCACGAC GGCTTCCCCT GGGGCCGCGC CG - #CCCCGCGC 120- GAAGCGGAGC TGCCCACCGG GCACCGCGCC CTGCCGGCCC TGGCCGGCGC CG - #CCCGCTGA 180#GAC GAC CCG 232G AAGGAGCCCC C GTG ACA GGA GCC# Val - # Thr Gly Ala Asp Asp Pro# 1 5- GCA AGG CCC GCG GTC GGC CCG CAG AGT TTC CG - #A GAC GCG ATG GCG CAG 280Ala Arg Pro Ala Val Gly Pro Gln Ser Phe Ar - #g Asp Ala Met Ala Gln# 20- CTG GCG TCG CCC GTC ACC GTC GTA ACC GTC CT - #C GAC GCG GCC GGA CGC 328Leu Ala Ser Pro Val Thr Val Val Thr Val Le - #u Asp Ala Ala Gly Arg# 35- CGC CAC GGC TTC ACG GCC GGC TCG GTG GTC TC - #T GTG TCG CTG GAC CCG 376Arg His Gly Phe Thr Ala Gly Ser Val Val Se - #r Val Ser Leu Asp Pro# 55- CCG CTG GTG ATG GTC GGC ATC GCG CTC ACC TC - #C AGC TGC CAC ACG GCG 424Pro Leu Val Met Val Gly Ile Ala Leu Thr Se - #r Ser Cys His Thr Ala# 70- ATG GCC GCC GCC GCC GAG TTC TGC GTC AGC AT - #C CTC GGC GAG GAC CAG 472Met Ala Ala Ala Ala Glu Phe Cys Val Ser Il - #e Leu Gly Glu Asp Gln# 85- CGC GCC GTC GCG AAG CGG TGC GCG ACG CAC GG - #C GCC GAC CGG TTC GCG 520Arg Ala Val Ala Lys Arg Cys Ala Thr His Gl - #y Ala Asp Arg Phe Ala# 100- GGC GGC GAG TTC GCC GCC TGG GAC GGT ACG GG - #G GTG CCC TAC CTG CCG 568Gly Gly Glu Phe Ala Ala Trp Asp Gly Thr Gl - #y Val Pro Tyr Leu Pro# 115- GAC GCC AAG GTC GTC CTG CGC TGC CGC ACC AC - #G GAC GTG GTG CGC GCC 616Asp Ala Lys Val Val Leu Arg Cys Arg Thr Th - #r Asp Val Val Arg Ala120 1 - #25 1 - #30 1 -#35- GGC GAC CAC GAC CTG GTG CTC GGC ACG CCC GT - #G GAG ATC CGC ACG GGC 664Gly Asp His Asp Leu Val Leu Gly Thr Pro Va - #l Glu Ile Arg Thr Gly# 150# 695 CA CCC CTG CTG TGG TAC CAsp Pro Ala Lys Pro Pro Leu Leu Trp Tyr# 160- (2) INFORMATION FOR SEQ ID NO: 8:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 640 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: NO- (iii) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: S.pristinaes - #piralis- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..640#/product= "gene SnaD"FORMATION:#8: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- GCG ACC GCC CGG CTC ATC GGC CCG CTG CCG CG - #C CGG CTG GGC CTC CAG 48Ala Thr Ala Arg Leu Ile Gly Pro Leu Pro Ar - #g Arg Leu Gly Leu Gln# 15- GTG CAC CAG GTG ATG ACG GGC GCG TTC GCG CA - #G GCC CTC GCC CGC TGG 96Val His Gln Val Met Thr Gly Ala Phe Ala Gl - #n Ala Leu Ala Arg Trp# 30- CGG GGC AGC CGC GCC GTC ACC TTC GAC GTG GA - #G ACC CAC GGA CGG CAC 144Arg Gly Ser Arg Ala Val Thr Phe Asp Val Gl - #u Thr His Gly Arg His# 45- GGC CGC GAC GAA CTG TTC CGT ACC GTC GGC TG - #G TTC ACC TCC ATC CAC 192Gly Arg Asp Glu Leu Phe Arg Thr Val Gly Tr - #p Phe Thr Ser Ile His# 60- CCC GTC GTC CTG GGC GCG GAC CGC TCC GTG CA - #C CCC GAG CAG TAC CTC 240Pro Val Val Leu Gly Ala Asp Arg Ser Val Hi - #s Pro Glu Gln Tyr Leu# 80- GCC CAG ATC GGC GCG GCG CTG ACC GCC GTA CC - #G GAC GGC GGC GTC GGC 288Ala Gln Ile Gly Ala Ala Leu Thr Ala Ala Pr - #o Asp Gly Gly Val Gly# 95- TTC GGC GCC TGC CGC GAG TTC TCC CCG GAC GC - #C GGG CTG CGC ACT CTG 336Phe Gly Ala Cys Arg Glu Phe Ser Pro Asp Al - #a Gly Leu Arg Thr Leu# 110- CTG CGT GAC CTG CCG CCC GCC CTG GTG TGC TT - #C AAC TAC TAC GGT CAG 384Leu Arg Asp Leu Pro Pro Ala Leu Val Cys Ph - #e Asn Tyr Tyr Gly Gln# 125- GCC GAC CAG TTG AGC CCG AAC GGC GGT TTC CG - #T ATG TCG GGC CGT CCC 432Ala Asp Gln Leu Ser Pro Asn Gly Gly Phe Ar - #g Met Ser Gly Arg Pro# 140- ATC CCG CGC GAG CAC TCC GCC CGC TGC GAG CG - #C GTC TAC GGC ATC GAG 480Ile Pro Arg Glu His Ser Ala Arg Cys Glu Ar - #g Val Tyr Gly Ile Glu145 1 - #50 1 - #55 1 -#60- GTG TAC GGC ATC GTC CAC GGC GGC CGC CTG CG - #C ATG GGC CTG ACC TGG 528Val Tyr Gly Ile Val His Gly Gly Arg Leu Ar - #g Met Gly Leu Thr Trp# 175- GTG CCG AGC CCG GCG GAC GGT GTG GAC GAG GC - #C GGC GTC GAC GCG CTC 576Val Pro Ser Pro Ala Asp Gly Val Asp Glu Al - #a Gly Val Asp Ala Leu# 190- GTG GAG CAG ATG AGC TGG GTG CTG GCC ACG CT - #C GCG GGC GCC GAC CCG 624Val Glu Gln Met Ser Trp Val Leu Ala Thr Le - #u Ala Gly Ala Asp Pro# 205# 640 CG GHis Ala Val Thr Pro 210- (2) INFORMATION FOR SEQ ID NO: 9:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 645 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: NO- (iii) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: S.pristinaes - #piralis- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 61..645#/product= "gene papA"FORMATION:#9: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- GGCGTCAAGA ACCTGCCGCT GACCGTACGG CGCGGCTGAC ACAGACAAGG GG - #GCCACCTG 60- GTG CGC ACC GTG CGA ACC CTG CTG ATC GAC AA - #C TAC GAC TCG TTC ACC 108Val Arg Thr Val Arg Thr Leu Leu Ile Asp As - #n Tyr Asp Ser Phe Thr# 15- TAC AAC CTC TTC CAG ATG CTG GCC GAG GTG AA - #C GGC GCC GCT CCG CTC 156Tyr Asn Leu Phe Gln Met Leu Ala Glu Val As - #n Gly Ala Ala Pro Leu# 30- GTC GTC CGC AAC GAC GAC ACC CGC ACC TGG CA - #G GCC CTG GCG CCG GGC 204Val Val Arg Asn Asp Asp Thr Arg Thr Trp Gl - #n Ala Leu Ala Pro Gly# 45- GAC TTC GAC AAC GTC GTC GTC TCA CCC GGC CC - #C GGC CAC CCC GCC ACC 252Asp Phe Asp Asn Val Val Val Ser Pro Gly Pr - #o Gly His Pro Ala Thr# 60- GAC ACC GAC CTG GGC CTC AGC CGC CGG GTG AT - #C ACC GAA TGG GAC CTG 300Asp Thr Asp Leu Gly Leu Ser Arg Arg Val Il - #e Thr Glu Trp Asp Leu# 80- CCG CTG CTC GGG GTG TGC CTG GGC CAC CAG GC - #C CTG TGC CTG CTC GCC 348Pro Leu Leu Gly Val Cys Leu Gly His Gln Al - #a Leu Cys Leu Leu Ala# 95- GGC GCC GCC GTC GTC CAC GCA CCC GAA CCC TT - #T CAC GGC CGC ACC AGC 396Gly Ala Ala Val Val His Ala Pro Glu Pro Ph - #e His Gly Arg Thr Ser# 110- GAC ATC CGC CAC GAC GGG CAG GGC CTG TTC GC - #G AAC ATC CCC TCC CCG 444Asp Ile Arg His Asp Gly Gln Gly Leu Phe Al - #a Asn Ile Pro Ser Pro# 125- CTG ACC GTG GTC CGC TAC CAC TCG CTG ACC GT - #C CGG CAA CTG CCC GCC 492Leu Thr Val Val Arg Tyr His Ser Leu Thr Va - #l Arg Gln Leu Pro Ala# 140- GAC CTG CGC GCC ACC GCC CAC ACC GCC GAC GG - #G CAG CTG ATG GCC GTC 540Asp Leu Arg Ala Thr Ala His Thr Ala Asp Gl - #y Gln Leu Met Ala Val145 1 - #50 1 - #55 1 -#60- GCC CAC CGC CAC CTG CCC CGC TTC GGC GTG CA - #G TTC CAC CCC GAA TCG 588Ala His Arg His Leu Pro Arg Phe Gly Val Gl - #n Phe His Pro Glu Ser# 175- ATC AGC AGC GAA CAC GGC CAC CGG ATG CTC GC - #C AAC TTC CGC GAC CTG 636Ile Ser Ser Glu His Gly His Arg Met Leu Al - #a Asn Phe Arg Asp Leu# 190# 645Ser Leu Arg 195- (2) INFORMATION FOR SEQ ID NO: 10:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1052 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: NO- (iii) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: S.pristinaes - #piralis- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 84..962#/product= "Gene PapM"FORMATION:#ID NO: 10:xi) SEQUENCE DESCRIPTION: SEQ- CTCGAGGACG AGTGGATCGC CTCCGGCGGC GCCCCCGTCC CCACGCCCGT GC - #ACGCGTCC 60- GCGTCCGCGC GGGGGGCCGT GTC GTG ACC GCC GCC GCA CC - #C ACC CTC GCC 110#Thr Leu Ala Thr Ala Ala Ala Pro# 5 1- CAG GCG CTG GAC GAG GCC ACC GGG CAG CTG AC - #C GGC GCC GGG ATC ACC 158Gln Ala Leu Asp Glu Ala Thr Gly Gln Leu Th - #r Gly Ala Gly Ile Thr# 25- GCC GAC GCC GCC CGG GCC GAC ACC CGG CTG CT - #G GCC GCC CAC GCC TGC 206Ala Asp Ala Ala Arg Ala Asp Thr Arg Leu Le - #u Ala Ala His Ala Cys# 40- CAG GTC GCC CCG GGG GAC CTC GAC ACC TGC CT - #G GCC GGC CCG GTG CCG 254Gln Val Ala Pro Gly Asp Leu Asp Thr Cys Le - #u Ala Gly Pro Val Pro# 55- CCC CGG TTC TGG CAC TAC GTC CGG CGC CGT CT - #G ACC CGC GAA CCC GCC 302Pro Arg Phe Trp His Tyr Val Arg Arg Arg Le - #u Thr Arg Glu Pro Ala# 70- GAA CGC ATC GTC GGC CAC GCC TAC TTC ATG GG - #C CAC CGC TTC GAC CTG 350Glu Arg Ile Val Gly His Ala Tyr Phe Met Gl - #y His Arg Phe Asp Leu# 85- GCC CCC GGC GTC TTC GTC CCC AAA CCC GAG AC - #C GAG GAG ATC ACC CGG 398Ala Pro Gly Val Phe Val Pro Lys Pro Glu Th - #r Glu Glu Ile Thr Arg#105- GAC GCC ATC GCC CGC CTG GAG GCC CTC GTC CG - #C CGC GGC ACC ACC GCA 446Asp Ala Ile Ala Arg Leu Glu Ala Leu Val Ar - #g Arg Gly Thr Thr Ala# 120- CCC CTG GTC GTC GAC CTG TGC GCC GGA CCG GG - #C ACC ATG GCC GTC ACC 494Pro Leu Val Val Asp Leu Cys Ala Gly Pro Gl - #y Thr Met Ala Val Thr# 135- CTG GCC CGC CAC GTA CCG GCC GCC CGC GTC CT - #G GGC ATC GAA CTC TCC 542Leu Ala Arg His Val Pro Ala Ala Arg Val Le - #u Gly Ile Glu Leu Ser# 150- CAG GCC GCC GCC CGC GCC GCC CGG CGC AAC GC - #C CGC GGC ACC GGC GCC 590Gln Ala Ala Ala Arg Ala Ala Arg Arg Asn Al - #a Arg Gly Thr Gly Ala# 165- CGC ATC GTG CAG GGC GAC GCC CGC GAC GCC TT - #C CCC GAA CTG AGC GGC 638Arg Ile Val Gln Gly Asp Ala Arg Asp Ala Ph - #e Pro Glu Leu Ser Gly170 1 - #75 1 - #80 1 -#85- ACC GTC GAC CTC GTC GTC ACC AAC CCG CCC TA - #C ATC CCC ATC GGA CTG 686Thr Val Asp Leu Val Val Thr Asn Pro Pro Ty - #r Ile Pro Ile Gly Leu# 200- CGC ACC TCC GCA CCC GAA GTG CTC GAG CAC GA - #C CCG CCG CTG GCC CTG 734Arg Thr Ser Ala Pro Glu Val Leu Glu His As - #p Pro Pro Leu Ala Leu# 215- TGG GCC GGG GAG GAG GGC CTC GGC ATG ATC CG - #C GCC ATG GAA CGC ACC 782Trp Ala Gly Glu Glu Gly Leu Gly Met Ile Ar - #g Ala Met Glu Arg Thr# 230- GCG GCC CGG CTG CTG GCC CCC GGC GGC GTC CT - #G CTC CTC GAA CAC GGC 830Ala Ala Arg Leu Leu Ala Pro Gly Gly Val Le - #u Leu Leu Glu His Gly# 245- TCC TAC CAA CTC GCC TCC GTG CCC GCC CTG TT - #C CGC GCA ACC GGC CGC 878Ser Tyr Gln Leu Ala Ser Val Pro Ala Leu Ph - #e Arg Ala Thr Gly Arg250 2 - #55 2 - #60 2 -#65- TGG AGC CAC GCC TCG TCC CGT CCC ACC TGC AA - #C GAC GGC TGC CTG ACC 926Trp Ser His Ala Ser Ser Arg Pro Thr Cys As - #n Asp Gly Cys Leu Thr# 280- GCC GTA CGC AAC CAC ACC TGC GCA CCG CCC GC - #C TGACACGGCG TCACGGCACG 979Ala Val Arg Asn His Thr Cys Ala Pro Pro Al - #a# 290- GCCGGCCTGT CGGCAACGAC CCTACGCCAT TGACAAACCG ACCGTGCCGT TT - #TTTTAATG1039# 1052- (2) INFORMATION FOR SEQ ID NO: 11:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 227 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: NO- (iii) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: S.pristinaes - #piralis- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 3..227#/product= "Partie du gene SnbC"#11: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- AG ATC TTC GAG CAC AAG ACC GTC GCC CAG CTC - # GCA CCC GTC GCC GAG 47#Leu Ala Pro Val Ala Gluhr Val Ala Gln# 15- ACG CTC GCC GAC ACC ACC CGC GAG GAA CCC GC - #C GCC GTC GCC GCG ACC 95Thr Leu Ala Asp Thr Thr Arg Glu Glu Pro Al - #a Ala Val Ala Ala Thr# 30- GGC GAC GTA CCG CTC ACC CCG ATC ATG CAC TG - #G CTG CGC GAA CGC GGC 143Gly Asp Val Pro Leu Thr Pro Ile Met His Tr - #p Leu Arg Glu Arg Gly# 45- GGC CCC GTC GAC GCG TTC AGC CAG ACG ATG GC - #C GTC ACC GTC CCC GCC 191Gly Pro Val Asp Ala Phe Ser Gln Thr Met Al - #a Val Thr Val Pro Ala# 60# 227GAC CGG GAA CGG CTC GTG GCC GCC CT - #G CAGGly Leu Asp Arg Glu Arg Leu Val Ala Ala Le - #u Gln# 75- (2) INFORMATION FOR SEQ ID NO: 12:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 247 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: NO- (iii) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: S.pristinaes - #piralis- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..247#/product= "Partie du gene SnbC"#12: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- CTC GAG TAC GAC ACC GCC CTG TAC GAG CGG GC - #C ACC GCC GAA GCC CTC 48Leu Glu Tyr Asp Thr Ala Leu Tyr Glu Arg Al - #a Thr Ala Glu Ala Leu# 15- ACC GGC CGG CTG CTG CGG CTC CTC GAC GCC GT - #C GTC ACC GAC CCG CAG 96Thr Gly Arg Leu Leu Arg Leu Leu Asp Ala Va - #l Val Thr Asp Pro Gln# 30- GCG CCG GTC GGC TCC CAC GAC CTC CTC GAA GA - #G GCC GAA CAC GCC CGC 144Ala Pro Val Gly Ser His Asp Leu Leu Glu Gl - #u Ala Glu His Ala Arg# 45- CTG GCA GCC TTC AAC GAC ACC GCC CGG CCC GT - #G CCG CGA GCC GGC CTC 192Leu Ala Ala Phe Asn Asp Thr Ala Arg Pro Va - #l Pro Arg Ala Gly Leu# 60- GCC GAA CTC TTC ACC GCC CAG GCC CGC CGC AC - #C GCC GAT GCG GTC GCC 240Ala Glu Leu Phe Thr Ala Gln Ala Arg Arg Th - #r Ala Asp Ala Val Ala# 80# 247Val Val- (2) INFORMATION FOR SEQ ID NO: 13:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 192 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: NO- (iii) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: S.pristinaes - #piralis- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 3..192#/product= "Partie du gene SnbD"#13: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- GC ATG CCC CCC GTC ACC CCC TAC CGC GCC TAC - # CTG GCC CAC CTC GCC 47#Tyr Leu Ala His Leu Alaro Tyr Arg Ala# 15- GGC CGT GAC GAC GAC GCC GCC CGC GCC GCG TG - #G CGG ACC GCC CTC GCG 95Gly Arg Asp Asp Asp Ala Ala Arg Ala Ala Tr - #p Arg Thr Ala Leu Ala# 30- GAC CTG GAG GAG CCG AGC CTC GTC GCG GGC GC - #C GGA GCA GGC CGC GGC 143Asp Leu Glu Glu Pro Ser Leu Val Ala Gly Al - #a Gly Ala Gly Arg Gly# 45- GCC GCC GAC GGC TCC GCC CTG CCC GGC CAG AT - #C CCC GGT TAC CGA GCT C 192Ala Ala Asp Gly Ser Ala Leu Pro Gly Gln Il - #e Pro Gly Tyr Arg Ala# 60- (2) INFORMATION FOR SEQ ID NO: 14:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 474 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: NO- (iii) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: S.pristinaes - #piralis- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..474#/product= "Partie du gene SnbD"#14: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- CTG CAG GTC GAG GGC CGG CCC GCG CAC CTG GA - #A CTG CCC TGC GAC CAC 48Leu Gln Val Glu Gly Arg Pro Ala His Leu Gl - #u Leu Pro Cys Asp His# 15- CCC CGG CCC GCC GTC GCC ACC CAC CGC GGC GC - #C ACC GTG CCC TTC CAC 96Pro Arg Pro Ala Val Ala Thr His Arg Gly Al - #a Thr Val Pro Phe His# 30- ATC GAC GCC GGC CTC CAC GAG AAG CTG ACC GC - #G CTC TCC AAG GCC TGC 144Ile Asp Ala Gly Leu His Glu Lys Leu Thr Al - #a Leu Ser Lys Ala Cys# 45- GAC AGC AGC CTG TTC ATG GTG CTC CAG GCC GC - #G GTC GCC GCC CTG CTC 192Asp Ser Ser Leu Phe Met Val Leu Gln Ala Al - #a Val Ala Ala Leu Leu# 60- ACC CGG CAC GGC GCC GGC ACC GAC ATC CCC GT - #C GGC AGC CCC GTC GCC 240Thr Arg His Gly Ala Gly Thr Asp Ile Pro Va - #l Gly Ser Pro Val Ala# 80- GGC CGC ACC GAC GAC GCC CTC GAC GAC CTG GT - #G GGC TTC TTC GTC AAC 288Gly Arg Thr Asp Asp Ala Leu Asp Asp Leu Va - #l Gly Phe Phe Val Asn# 95- ACC CTC GTC CTG CGC ACC GAC ACC TCC GGC GA - #C CCC ACC TTC CGC GAA 336Thr Leu Val Leu Arg Thr Asp Thr Ser Gly As - #p Pro Thr Phe Arg Glu# 110- CTC GTC GCA CGC GTG CGG CAG TTC GAC CTC GC - #C GCC TAC ACG CAC CAG 384Leu Val Ala Arg Val Arg Gln Phe Asp Leu Al - #a Ala Tyr Thr His Gln# 125- GAC ATG CCG TTC GAA AAG CTC GTC GAA GAG GT - #C AAC CCC GAG CGC TCC 432Asp Met Pro Phe Glu Lys Leu Val Glu Glu Va - #l Asn Pro Glu Arg Ser# 140- CTG GCC CGC AAC CCG CTC TTC CAG GTC GTC CT - #G GCG CTG CAG# 474Leu Ala Arg Asn Pro Leu Phe Gln Val Val Le - #u Ala Leu Gln145 1 - #50 1 - #55- (2) INFORMATION FOR SEQ ID NO: 15:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 485 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: NO- (iii) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: S.pristinaes - #piralis- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 3..485#/product= "Partie du gene SnbE"#15: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- GC ATG CCG CGC TCC CTC GAC CTG TAC GTC GCA - # CTG CTC GCC GTC CTC 47#Ala Leu Leu Ala Val Leusp Leu Tyr Val# 15- AAG ACC GGC GCC GCC TAC CTG CCC GTC GAC AT - #C TCC TAC CCG GCC GAA 95Lys Thr Gly Ala Ala Tyr Leu Pro Val Asp Il - #e Ser Tyr Pro Ala Glu# 30- CGC ATC GCG TTC ATG ATC GAG GAC GCC CGC CC - #G GTG ACC GTC CTC GAC 143Arg Ile Ala Phe Met Ile Glu Asp Ala Arg Pr - #o Val Thr Val Leu Asp# 45- CGC CTG CCC GAC GAC CTG GGC GCC TAC CGG GA - #C ACC GAC CTC ACC GAC 191Arg Leu Pro Asp Asp Leu Gly Ala Tyr Arg As - #p Thr Asp Leu Thr Asp# 60- GCC GAC CGC ACG GCG CCG CTA CGG CCC GAA CA - #C CCG GCG TAC GTC ATC 239Ala Asp Arg Thr Ala Pro Leu Arg Pro Glu Hi - #s Pro Ala Tyr Val Ile# 75- CAC ACC TCC GGC TCC ACC GGC ACC CCC AAG GC - #C GTC GTC ATG CCC CAC 287His Thr Ser Gly Ser Thr Gly Thr Pro Lys Al - #a Val Val Met Pro His# 95- GCC GGC CTG GTC AAC CTG CTG ACC TGG CAC GC - #C CGC CGC TTC CCC GGC 335Ala Gly Leu Val Asn Leu Leu Thr Trp His Al - #a Arg Arg Phe Pro Gly# 110- GGC ACC GGG GTG CGC ACC GCC CAG TTC ACC GC - #C ATC GGC TTC GAC TTC 383Gly Thr Gly Val Arg Thr Ala Gln Phe Thr Al - #a Ile Gly Phe Asp Phe# 125- TCG GTG CAG GAG ATC CTC TCC CCG CTC GTC AT - #G GGC AAG ACC CTC GCC 431Ser Val Gln Glu Ile Leu Ser Pro Leu Val Me - #t Gly Lys Thr Leu Ala# 140- GTG CCC TCG GAA GAG GTC CGC CAC AGC GCC GA - #A CTG CTG GCC GGC TGG 479Val Pro Ser Glu Glu Val Arg His Ser Ala Gl - #u Leu Leu Ala Gly Trp# 155# 485Leu Glu160- (2) INFORMATION FOR SEQ ID NO: 16:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 291 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: NO- (iii) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: S.pristinaes - #piralis- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..291#/product= "Partie du gene SnbE"#16: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- CTG CAG GCC GAG GGC GCC GAA GTG AGC CTG CT - #G GCC GTC CTC GAC GGC 48Leu Gln Ala Glu Gly Ala Glu Val Ser Leu Le - #u Ala Val Leu Asp Gly# 15- TAC CCC GAC GCC TAC GAC GGC ACC GAG CAC GA - #G GTC GGC GAG GAA CAG 96Tyr Pro Asp Ala Tyr Asp Gly Thr Glu His Gl - #u Val Gly Glu Glu Gln# 30- GTC CTG GCG ATC CTC CTC AAC GCC GCC GGC GT - #C GAC CGG GCC CAG GCC 144Val Leu Ala Ile Leu Leu Asn Ala Ala Gly Va - #l Asp Arg Ala Gln Ala# 45- TTC GGC GAC GCC CCC CTC CAA CGG GCC GCC GT - #G CTC GAG AAG CTG CGC 192Phe Gly Asp Ala Pro Leu Gln Arg Ala Ala Va - #l Leu Glu Lys Leu Arg# 60- GAC AGC GGC AGC GCC CTG GGC AAC CTC GAC GA - #C GAC GCG GTC GGC CGC 240Asp Ser Gly Ser Ala Leu Gly Asn Leu Asp As - #p Asp Ala Val Gly Arg# 80- ATG GTC ACC GTC TTC CTC AAC AAC ACG CGC CT - #C ATC CAG AAC TTC CGG 288Met Val Thr Val Phe Leu Asn Asn Thr Arg Le - #u Ile Gln Asn Phe Arg# 95# 291Pro- (2) INFORMATION FOR SEQ ID NO:17:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 2219 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: genomic DNA- (iii) HYPOTHETICAL: NO- (iii) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: s. virgin - #iae- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..2219#/product = "virginiamycin s synthase gene"- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 17:- GG ATC CGT ACC GTC CTG GGT GCC GAG ATC GCG - # GTC CGT GAC CTG TTC 47#Ala Val Arg Asp Leu Phely Ala Glu Ile# 15- GAG GCA CCC ACC GTC GAG GCC CTC GCC GAA AC - #C CTG GAA GAG GCC CGC 95Glu Ala Pro Thr Val Glu Ala Leu Ala Glu Th - #r Leu Glu Glu Ala Arg# 30- GAG GTC CGC CCC GCC CTG CGC GCC GCC GAC CG - #C CCC GAA CAC GTC CCG 143Glu Val Arg Pro Ala Leu Arg Ala Ala Asp Ar - #g Pro Glu His Val Pro# 45- CTG TCC TTC GCC CAG CGG CGC CTG TGG TTC CT - #C GAC CGC CTG GAA GGA 191Leu Ser Phe Ala Gln Arg Arg Leu Trp Phe Le - #u Asp Arg Leu Glu Gly# 60- CCC AAC TCC ACC TAC AAC ATC CCG CTC GCC CT - #G CGC CTG CGC GGC GAG 239Pro Asn Ser Thr Tyr Asn Ile Pro Leu Ala Le - #u Arg Leu Arg Gly Glu# 75- CTG GAC CGA CCG GCG CTG CAA CAG GCC CTC AC - #C GAC CTG ACG CAC CGC 287Leu Asp Arg Pro Ala Leu Gln Gln Ala Leu Th - #r Asp Leu Thr His Arg#95- CAC GAA AGC CTG CGC ACC GTC TAC CCG AGC GC - #C GAC GGC CGG CCC TAC 335His Glu Ser Leu Arg Thr Val Tyr Pro Ser Al - #a Asp Gly Arg Pro Tyr# 110- CAG CAC GTC CTC GCA CCG CAC GAG GCC GAG CC - #C GGC CTC GTC GTC GTC 383Gln His Val Leu Ala Pro His Glu Ala Glu Pr - #o Gly Leu Val Val Val# 125- CCC GCC GAC GAG GCC GGA CTC GCC GAG ATG CT - #G GCC GAG GCC GCC CGC 431Pro Ala Asp Glu Ala Gly Leu Ala Glu Met Le - #u Ala Glu Ala Ala Arg# 140- CAC GAG TTC GAC GTC ACC TCC GAA CCG CCG CT - #G CGG GTC TCC CTG TTC 479His Glu Phe Asp Val Thr Ser Glu Pro Pro Le - #u Arg Val Ser Leu Phe# 155- ACC CTC GCA CCG GAC GAG CAC GTC CTG CTC CT - #G CTG CTG CAC CAC ATC 527Thr Leu Ala Pro Asp Glu His Val Leu Leu Le - #u Leu Leu His His Ile160 1 - #65 1 - #70 1 -#75- GCC GGC GAC GGC TGG TCG CTC GCA CCA CTC AC - #C CGC GAC CTC ACC CGC 575Ala Gly Asp Gly Trp Ser Leu Ala Pro Leu Th - #r Arg Asp Leu Thr Arg# 190- GCC TAC ACC GCC CGC CGG GAC GGC GCC GCC CC - #C GAC TGG GAG CCC CTC 623Ala Tyr Thr Ala Arg Arg Asp Gly Ala Ala Pr - #o Asp Trp Glu Pro Leu# 205- CCG GTC CAG TAC GCC GAC TAC ACC CTC TGG CA - #G CAG GAG ATG CTC GGC 671Pro Val Gln Tyr Ala Asp Tyr Thr Leu Trp Gl - #n Gln Glu Met Leu Gly# 220- TCG CCG GAC GAC CCC GAC AGC CTC GGC GCC CG - #C CAG CTC GAC CAC TGG 719Ser Pro Asp Asp Pro Asp Ser Leu Gly Ala Ar - #g Gln Leu Asp His Trp# 235- GCC CGG TCC CTG GCC GGC GCC CCC GAG CAA CT - #G GAA CTG CCC ACC GAC 767Ala Arg Ser Leu Ala Gly Ala Pro Glu Gln Le - #u Glu Leu Pro Thr Asp240 2 - #45 2 - #50 2 -#55- CAC AAC CGG CCG GCC GCC GCC GGC CAC CAC GG - #C CGC ACC GTC CCC TTC 815His Asn Arg Pro Ala Ala Ala Gly His His Gl - #y Arg Thr Val Pro Phe# 270- CAC CTG GAG CCC GAG CTG CAC GAG CGG CTC AG - #C GCC CTG GCC AGG TCC 863His Leu Glu Pro Glu Leu His Glu Arg Leu Se - #r Ala Leu Ala Arg Ser# 285- TGC GAC GCC AGC CTG TTC ATG GTC CTG CAC GC - #C GCG TTC GCC GCG CTG 911Cys Asp Ala Ser Leu Phe Met Val Leu His Al - #a Ala Phe Ala Ala Leu# 300- CTC ACC AAG CAC GGT GCC GGC ACC GAC ATC CC - #G ATC GGC AGC CCC ATC 959Leu Thr Lys His Gly Ala Gly Thr Asp Ile Pr - #o Ile Gly Ser Pro Ile# 315- GCC GGC CGC ACC GAC GAG GCC CTC GAC GAT CT - #G GTC GGG TTC TTC GTC1007Ala Gly Arg Thr Asp Glu Ala Leu Asp Asp Le - #u Val Gly Phe Phe Val320 3 - #25 3 - #30 3 -#35- AAC ACC CTG GTC CTG CGC ACC GAC ACC TCC GG - #C GAT CCG ACC TTC CGC1055Asn Thr Leu Val Leu Arg Thr Asp Thr Ser Gl - #y Asp Pro Thr Phe Arg# 350- GAA CTC GTG GCA CGC ACC CGC GCC ACC GAC CT - #G GCC GCA TAC GCA CAC1103Glu Leu Val Ala Arg Thr Arg Ala Thr Asp Le - #u Ala Ala Tyr Ala His# 365- CAG GAC CTG CCC TTC GAG AAG CTC GTC GAG AC - #T CTC AAC CCG CAG CGC1151Gln Asp Leu Pro Phe Glu Lys Leu Val Glu Th - #r Leu Asn Pro Gln Arg# 380- TCG CTC GCC CGC AAC CCG CTG TTC CAG GTA CT - #G CTG GCC TTC CAG AGC1199Ser Leu Ala Arg Asn Pro Leu Phe Gln Val Le - #u Leu Ala Phe Gln Ser# 395- ATG CCC ACG GCA CAG CCC GTG CTG CCC GGC CT - #C GAC GTC GTC CAC GAG1247Met Pro Thr Ala Gln Pro Val Leu Pro Gly Le - #u Asp Val Val His Glu400 4 - #05 4 - #10 4 -#15- CCG GTC CGC GTC GGA TTC GCC AAG TTC GAC CT - #G GCC CTG GCC GTG GCC1295Pro Val Arg Val Gly Phe Ala Lys Phe Asp Le - #u Ala Leu Ala Val Ala# 430- GAG GAA CGG CAC GCC GAC GGC CGC CGG TCG CT - #G CGC GGC GAC TGG GAG1343Glu Glu Arg His Ala Asp Gly Arg Arg Ser Le - #u Arg Gly Asp Trp Glu# 445- TTC AGC ACC GAC CTG TTC GAG CAG GCC ACC GT - #G GAG GCC CTC GGG GCC1391Phe Ser Thr Asp Leu Phe Glu Gln Ala Thr Va - #l Glu Ala Leu Gly Ala# 460- AGG CTC ACC GCC CTG CTG GCG TCG GTC GCC GC - #C GAC CCC GAC CAG CCG1439Arg Leu Thr Ala Leu Leu Ala Ser Val Ala Al - #a Asp Pro Asp Gln Pro# 475- ATC GGA CGG GTG GGC ATC CTC GAC CCG GCC GA - #A CGC CAC CGC ATC CTC1487Ile Gly Arg Val Gly Ile Leu Asp Pro Ala Gl - #u Arg His Arg Ile Leu480 4 - #85 4 - #90 4 -#95- CAC ACC TGG AAC GAC ACC TCC CGC CCC GGC GC - #G GAC GCC ACC TGG CCG1535His Thr Trp Asn Asp Thr Ser Arg Pro Gly Al - #a Asp Ala Thr Trp Pro# 510- GAG CTG TTC CAG GCC CGC GCC GCC GAG CAC CC - #C GAC GCC GTC GCC CTG1583Glu Leu Phe Gln Ala Arg Ala Ala Glu His Pr - #o Asp Ala Val Ala Leu# 525- GTC CAG GAG GGC ACC GAG ACC GGC TAC GCC GA - #C CTG AAC ACC CGG GCC1631Val Gln Glu Gly Thr Glu Thr Gly Tyr Ala As - #p Leu Asn Thr Arg Ala# 540- AAC CGG CTC GCC CGG CTG CTC CGC GCA CAG GG - #C ATC GGC CCG GAG CAG1679Asn Arg Leu Ala Arg Leu Leu Arg Ala Gln Gl - #y Ile Gly Pro Glu Gln# 555- GTG GTG GCC CTG TCG CTG CCC CGC TCC GCA GA - #C CTG ATC GTC TCC GTC1727Val Val Ala Leu Ser Leu Pro Arg Ser Ala As - #p Leu Ile Val Ser Val560 5 - #65 5 - #70 5 -#75- CTC GCC GTG CTG AAG ACC GGC GCC GCC TAC CT - #G CCG GTC GAT CCC GCC1775Leu Ala Val Leu Lys Thr Gly Ala Ala Tyr Le - #u Pro Val Asp Pro Ala# 590- TAC CCG GCC GAG CGG ATC GCG TAC CTG CTC CA - #G GAC GGC GCC CCC GCG1823Tyr Pro Ala Glu Arg Ile Ala Tyr Leu Leu Gl - #n Asp Gly Ala Pro Ala# 605- CTC GTC CTC ACC CAC ACC TCC GTC GCG GCC GG - #C CTG CCC GGC GGC GTA1871Leu Val Leu Thr His Thr Ser Val Ala Ala Gl - #y Leu Pro Gly Gly Val# 620- CCG CAG CTG CTG GTC GAC CAG GTC GGC CTC GA - #C GAT GTC CCC GGC CAC1919Pro Gln Leu Leu Val Asp Gln Val Gly Leu As - #p Asp Val Pro Gly His# 635- GAC CTC ACC GAC GCC GAG CGC ACC ACG CCC CT - #G CAC CCG CTG CAC CCC1967Asp Leu Thr Asp Ala Glu Arg Thr Thr Pro Le - #u His Pro Leu His Pro640 6 - #45 6 - #50 6 -#55- GCC TAC GTC ATC TAC ACC TCC GGC TCC ACC GG - #C CTG CCC AAG GGC GTG2015Ala Tyr Val Ile Tyr Thr Ser Gly Ser Thr Gl - #y Leu Pro Lys Gly Val# 670- CCC GTC CCG CAC CGC AGC GTG GCG TCC GTT CT - #C GTC CCC CTG ATC GAG2063Pro Val Pro His Arg Ser Val Ala Ser Val Le - #u Val Pro Leu Ile Glu# 685- GAG TTC GGC CTC GGC CCC GGC AGC AGG GTC CT - #G CAG TTC GCC TCG ATC2111Glu Phe Gly Leu Gly Pro Gly Ser Arg Val Le - #u Gln Phe Ala Ser Ile# 700- AGC TTC GAC GCC GCC CTG TGG GAG ATC ACC CT - #C GCC CTG CTG TCC GGC2159Ser Phe Asp Ala Ala Leu Trp Glu Ile Thr Le - #u Ala Leu Leu Ser Gly# 715- GCC ACC CTC GTG GTC GCA CCC GCC GAG CAG CT - #T CAG CCC GGC CCC GCG2207Ala Thr Leu Val Val Ala Pro Ala Glu Gln Le - #u Gln Pro Gly Pro Ala720 7 - #25 7 - #30 7 -#35# 2219Leu Ala Glu Leu- (2) INFORMATION FOR SEQ ID NO: 18:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 422 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear#18: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Met Thr Ala Pro Arg Arg Arg Ile Thr Leu Al - #a Gly Ile Ile Asp Gly# 15- Pro Gly Gly His Val Ala Ala Trp Arg His Pr - #o Ala Thr Lys Ala Asp# 30- Ala Gln Leu Asp Phe Glu Phe His Arg Asp As - #n Ala Arg Thr Leu Glu# 45- Arg Gly Leu Phe Asp Ala Val Phe Ile Ala As - #p Ile Val Ala Val Trp# 60- Gly Thr Arg Leu Asp Ser Leu Cys Arg Thr Se - #r Arg Thr Glu His Phe# 80- Glu Pro Leu Thr Leu Leu Ala Ala Tyr Ala Al - #a Val Thr Glu His Ile# 95- Gly Leu Cys Ala Thr Ala Thr Thr Thr Tyr As - #n Glu Pro Ala His Ile# 110- Ala Ala Arg Phe Ala Ser Leu Asp His Leu Se - #r Gly Gly Arg Ala Gly# 125- Trp Asn Val Val Thr Ser Ala Ala Pro Trp Gl - #u Ser Ala Asn Phe Gly# 140- Phe Pro Glu His Leu Glu His Gly Lys Arg Ty - #r Glu Arg Ala Glu Glu145 1 - #50 1 - #55 1 -#60- Phe Ile Asp Val Val Lys Lys Leu Trp Asp Se - #r Asp Gly Arg Pro Val# 175- Asp His Arg Gly Thr His Phe Glu Ala Pro Gl - #y Pro Leu Gly Ile Ala# 190- Arg Pro Pro Gln Gly Arg Pro Val Ile Ile Gl - #n Ala Gly Ser Ser Pro# 205- Val Gly Arg Glu Phe Ala Ala Arg His Ala Gl - #u Val Ile Phe Thr Arg# 220- His Asn Arg Leu Ser Asp Ala Gln Asp Phe Ty - #r Gly Asp Leu Lys Ala225 2 - #30 2 - #35 2 -#40- Arg Val Ala Arg His Gly Arg Asp Pro Glu Ly - #s Val Leu Val Trp Pro# 255- Thr Leu Ala Pro Ile Val Ala Ala Thr Asp Th - #r Glu Ala Lys Gln Arg# 270- Leu Gln Glu Leu Gln Asp Leu Thr His Asp Hi - #s Val Ala Leu Arg Thr# 285- Leu Gln Asp His Leu Gly Asp Val Asp Leu Se - #r Ala Tyr Pro Ile Asp# 300- Gly Pro Val Pro Asp Ile Pro Tyr Thr Asn Gl - #n Ser Gln Ser Thr Thr305 3 - #10 3 - #15 3 -#20- Glu Arg Leu Ile Gly Leu Ala Arg Arg Glu As - #n Leu Ser Ile Arg Glu# 335- Leu Ala Leu Arg Leu Met Gly Asp Ile Val Va - #l Gly Thr Pro Glu Gln# 350- Leu Ala Asp His Met Glu Ser Trp Phe Thr Gl - #y Arg Gly Ala Asp Gly# 365- Phe Asn Ile Asp Phe Pro Tyr Leu Pro Gly Se - #r Ala Asp Asp Phe Val# 380- Asp His Val Val Pro Glu Leu Gln Arg Arg Gl - #y Leu Tyr Arg Ser Gly385 3 - #90 3 - #95 4 -#00- Tyr Glu Gly Thr Thr Leu Arg Ala Asn Leu Gl - #y Ile Asp Ala Pro Arg# 415- Lys Ala Gly Ala Ala Ala 420- (2) INFORMATION FOR SEQ ID NO: 19:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 277 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear#19: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Met Thr Ala Pro Ile Leu Val Ala Thr Leu As - #p Thr Arg Gly Pro Ala# 15- Ala Thr Leu Gly Thr Ile Thr Arg Ala Val Ar - #g Ala Ala Glu Ala Ala# 30- Gly Phe Asp Ala Val Leu Ile Asp Asp Arg Al - #a Ala Ala Gly Val Gln# 45- Gly Arg Phe Glu Thr Thr Thr Leu Thr Ala Al - #a Leu Ala Ala Val Thr# 60- Glu His Ile Gly Leu Ile Thr Ala Pro Leu Pr - #o Ala Asp Gln Ala Pro# 80- Tyr His Val Ser Arg Ile Thr Ala Ser Leu As - #p His Leu Ala His Gly# 95- Arg Thr Gly Trp Leu Ala Ser Thr Asp Thr Th - #r Asp Pro Glu Gly Arg# 110- Thr Gly Glu Leu Ile Asp Val Val Arg Gly Le - #u Trp Asp Ser Phe Asp# 125- Asp Asp Ala Phe Val His Asp Arg Ala Asp Gl - #y Leu Tyr Trp Arg Leu# 140- Pro Ala Val His Gln Leu Asp His Gln Gly Ar - #g His Phe Asp Val Ala145 1 - #50 1 - #55 1 -#60- Gly Pro Leu Asn Val Ala Arg Pro Pro Gln Gl - #y His Pro Val Val Ala# 175- Val Thr Gly Pro Ala Leu Ala Ala Ala Ala As - #p Leu Val Leu Leu Asp# 190- Glu Ala Ala Asp Ala Ala Ser Val Lys Gln Gl - #n Ala Pro His Ala Lys# 205- Ile Leu Leu Pro Leu Pro Gly Pro Ala Ala Gl - #u Leu Pro Ala Asp Ser# 220- Pro Ala Asp Gly Phe Thr Val Ala Leu Thr Gl - #y Ser Asp Asp Pro Val225 2 - #30 2 - #35 2 -#40- Leu Ala Ala Leu Ala Ala Arg Pro Gly Arg Pr - #o Asp Arg Thr Ala Ala# 255- Thr Thr Leu Arg Glu Arg Leu Gly Leu Ala Ar - #g Pro Glu Ser Arg His# 270- Ala Leu Thr Thr Ala 275- (2) INFORMATION FOR SEQ ID NO: 20:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 402 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear#20: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Met Ser Arg Arg Leu Phe Thr Ser Glu Ser Va - #l Thr Glu Gly His Pro# 15- Asp Lys Ile Ala Asp Gln Ile Ser Asp Thr Va - #l Leu Asp Ala Leu Leu# 30- Arg Glu Asp Pro Ala Ser Arg Val Ala Val Gl - #u Thr Leu Ile Thr Thr# 45- Gly Gln Val His Ile Ala Gly Glu Val Thr Th - #r Lys Ala Tyr Ala Pro# 60- Ile Ala Gln Leu Val Arg Asp Thr Ile Leu Al - #a Ile Gly Tyr Asp Ser# 80- Ser Ala Lys Gly Phe Asp Gly Ala Ser Cys Gl - #y Val Ser Val Ser Ile# 95- Gly Ala Gln Ser Pro Asp Ile Ala Gln Gly Va - #l Asp Ser Ala Tyr Glu# 110- Thr Arg Val Glu Gly Glu Asp Asp Glu Leu As - #p Gln Gln Gly Ala Gly# 125- Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr As - #p Glu Thr Pro Ser Leu# 140- Met Pro Leu Pro Ile Glu Leu Ala His Arg Le - #u Ser Arg Arg Leu Thr145 1 - #50 1 - #55 1 -#60- Glu Val Arg Lys Asp Gly Thr Val Pro Tyr Le - #u Arg Pro Asp Gly Lys# 175- Thr Gln Val Thr Ile Glu Tyr Gln Gly Ser Ar - #g Pro Val Arg Leu Asp# 190- Thr Val Val Val Ser Ser Gln His Ala Ala As - #p Ile Asp Leu Gly Ser# 205- Leu Leu Thr Pro Asp Ile Arg Glu His Val Va - #l Glu His Val Leu Ala# 220- Ala Leu Ala Glu Asp Gly Ile Lys Leu Glu Th - #r Asp Asn Tyr Arg Leu225 2 - #30 2 - #35 2 -#40- Leu Val Asn Pro Thr Gly Arg Phe Glu Ile Gl - #y Gly Pro Met Gly Asp# 255- Ala Gly Leu Thr Gly Arg Lys Ile Ile Ile As - #p Thr Tyr Gly Gly Met# 270- Ala Arg His Gly Gly Gly Ala Phe Ser Gly Ly - #s Asp Pro Ser Lys Val# 285- Asp Arg Ser Ala Ala Tyr Ala Met Arg Trp Va - #l Ala Lys Asn Val Val# 300- Ala Ala Gly Leu Ala Ser Arg Cys Glu Val Gl - #n Val Ala Tyr Ala Ile305 3 - #10 3 - #15 3 -#20- Gly Lys Ala Glu Pro Val Gly Leu Phe Val Gl - #u Thr Phe Gly Thr Gly# 335- Thr Val Ala Gln Glu Arg Ile Glu Lys Ala Il - #e Thr Glu Val Phe Asp# 350- Leu Arg Pro Ala Ala Ile Ile Arg Asp Leu As - #p Leu Leu Arg Pro Ile# 365- Tyr Ala Ala Thr Ala Ala Tyr Gly His Phe Gl - #y Arg Glu Leu Pro Asp# 380- Phe Thr Trp Glu Arg Thr Asp Arg Ala His Ar - #g Leu Lys Ala Ala Ala385 3 - #90 3 - #95 4 -#00- Gly Leu- (2) INFORMATION FOR SEQ ID NO: 21:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 582 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear#21: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:# Met Leu# 1- Asp Gly Cys Val Pro Trp Pro Glu Asp Val Al - #a Ala Lys Tyr Arg Ala# 15- Ala Gly Tyr Trp Arg Gly Glu Pro Leu Gly Me - #t Leu Leu Gly Arg Trp# 30- Ala Glu Gln Tyr Gly Glu Arg Glu Ala Leu Va - #l Gly Ala Asp Gly Cys# 50- Ser Arg Val Thr Tyr Arg Ala Leu Asp Arg Tr - #p Cys Asp Arg Leu Ala# 65- Ala Gly Phe Ala Ala Arg Gly Ile Gly Ala Gl - #y Glu Arg Val Leu Val# 80- Gln Leu Pro Asn Thr Pro Glu Phe Val Ala Va - #l Cys Phe Ala Leu Phe# 95- Arg Leu Gly Ala Leu Pro Val Phe Ala Leu Pr - #o Ala His Arg Ala Ala# 110- Glu Val Gly His Leu Leu Glu Leu Ser Gly Al - #a Val Ala His Ile Leu115 1 - #20 1 - #25 1 -#30- Pro Gly Thr Gly Thr Gly Tyr Asp His Val Al - #a Ala Ala Val Glu Ala# 145- Arg Ala Arg Arg Ala Arg Pro Val Gln Val Ph - #e Val Ala Gly Glu Ala# 160- Pro Ala Val Leu Pro Glu Gly Phe Thr Ala Le - #u Ala Asp Val Asp Gly# 175- Asp Pro Val Ala Pro Ala Asp Val Asp Ala Ph - #e Arg Arg Gly Val Phe# 190- Leu Leu Ser Gly Gly Thr Thr Ala Leu Pro Ly - #s Leu Ile Pro Arg Thr195 2 - #00 2 - #05 2 -#10- His Asp Asp Tyr Ala Tyr Gln Cys Arg Val Th - #r Ala Gly Ile Cys Gly# 225- Leu Asp Ala Asp Ser Val Tyr Leu Ala Val Le - #u Pro Ala Glu Phe Asn# 240- Phe Pro Phe Gly Cys Pro Gly Ile Leu Gly Th - #r Leu His Ala Gly Gly# 255- Arg Val Val Phe Ala Leu Ser Pro Gln Pro Gl - #u Glu Cys Phe Ala Leu# 270- Ile Glu Arg Glu His Val Thr Phe Thr Ser Va - #l Ile Pro Thr Ile Val275 2 - #80 2 - #85 2 -#90- His Leu Trp Leu Ala Ala Ala Ala Gln Gly Hi - #s Gly Arg Asp Leu Gly# 305- Ser Leu Gln Leu Leu Gln Val Gly Ser Ala Ly - #s Leu His Glu Glu Leu# 320- Ala Ala Arg Ile Gly Pro Glu Leu Gly Val Ar - #g Leu Gln Gln Val Phe# 335- Gly Met Ala Glu Gly Leu Leu Thr Phe Thr Ar - #g Asp Asp Asp Pro Ala# 350- Asp Val Val Leu Arg Thr Gln Gly Arg Pro Va - #l Ser Glu Ala Asp Glu355 3 - #60 3 - #65 3 -#70- Ile Arg Val Ala Asp Pro Asp Gly Arg Pro Va - #l Pro Arg Gly Glu Thr# 385- Gly Glu Leu Leu Thr Arg Gly Pro Tyr Thr Le - #u Arg Gly Tyr Tyr Arg# 400- Ala Pro Glu His Asn Ala Arg Ala Phe Thr Gl - #u Asp Gly Phe Tyr Arg# 415- Ser Gly Asp Leu Val Arg Leu Thr Ala Asp Gl - #y Gln Leu Val Val Glu# 430- Gly Arg Ile Lys Asp Val Val Ile Arg Gly Gl - #y Asp Lys Val Ser Ala435 4 - #40 4 - #45 4 -#50- Thr Glu Val Glu Gly His Leu Gly Ala His Pr - #o Asp Val Gln Gln Ala# 465- Ala Val Val Ala Met Pro Asp Pro Val Trp Gl - #y Glu Lys Val Cys Ala# 480- Tyr Ile Val Pro Ala Pro Gly Arg Pro Ala Pr - #o Pro Met Ala Ala Leu# 495- Arg Arg Leu Leu Arg Ala Arg Gly Leu Ala As - #p Tyr Lys Leu Pro Asp# 510- Arg Val Glu Val Val Asp Ala Phe Pro Leu Th - #r Gly Leu Asn Lys Val515 5 - #20 5 - #25 5 -#30- Asp Lys Lys Ala Leu Ala Ala Asp Ile Ala Al - #a Lys Thr Ala Pro Thr# 545- Arg Pro Thr Thr Ala Gly His Gly Pro Thr Th - #r Asp Gly Asp Thr Ala# 560- Gly Gly Gly Gly Ser Ala Gly Gly Val Thr Al - #a Ala Gly Gly Gly Arg# 575- Glu Glu Ala Ala 580- (2) INFORMATION FOR SEQ ID NO: 22:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 528 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear#22: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:# Met Arg Thr Se - #r# 1- Arg Ser His Asp Gln Arg Ala Pro Thr Pro Tr - #p Arg His Pro Leu His# 20- Ser Thr Arg Pro Ala Pro Ala Ala Asp Arg As - #p Pro Arg Arg Trp Val# 35- Ile Leu Gly Val Ile Cys Leu Ala Gln Leu Va - #l Val Leu Leu Asp Asn# 50- Thr Val Leu Asn Val Ala Ile Pro Val Leu Th - #r Thr Asp Leu Gly Ala# 65- Ser Thr Ala Asp Ile Gln Trp Met Ile Asn Al - #a Tyr Ala Leu Val Gln# 80- Ser Gly Leu Leu Leu Thr Ala Gly Ser Leu Al - #a Asp Arg Tyr Gly Arg#100- Lys Arg Leu Leu Met Leu Gly Leu Val Leu Ph - #e Gly Ala Gly Ser Ala# 115- Trp Ala Ala Phe Ala Gln Asp Ser Ala Gln Le - #u Ile Ala Ala Arg Ala# 130- Gly Met Gly Val Gly Gly Ala Leu Leu Ala Th - #r Thr Thr Leu Ala Val# 145- Ile Met Gln Val Phe Asp Asp Asp Glu Arg Pr - #o Arg Ala Ile Gly Leu# 160- Trp Gly Ala Ala Ser Ser Leu Gly Phe Ala Al - #a Gly Pro Leu Leu Gly165 1 - #70 1 - #75 1 -#80- Gly Ala Leu Leu Asp His Phe Trp Trp Gly Se - #r Ile Phe Leu Ile Asn# 195- Leu Pro Val Ala Leu Leu Gly Leu Leu Ala Va - #l Ala Arg Leu Val Pro# 210- Glu Thr Lys Asn Pro Glu Gly Arg Arg Pro As - #p Leu Leu Gly Ala Val# 225- Leu Ser Thr Leu Gly Met Val Gly Val Val Ty - #r Ala Ile Ile Ser Gly# 240- Pro Glu His Gly Trp Thr Ala Pro Gln Val Le - #u Leu Pro Ala Ala Val245 2 - #50 2 - #55 2 -#60- Ala Ala Ala Ala Leu Thr Ala Phe Val Arg Tr - #p Glu Leu His Thr Pro# 275- His Pro Met Leu Asp Met Gly Phe Phe Thr As - #p Arg Arg Phe Asn Gly# 290- Pro Ser Pro Ala Glu Cys Ser Ser Phe Gly Me - #t Ala Gly Ser Leu Phe# 305- Leu Leu Thr Gln His Leu Gln Leu Val Leu Gl - #y Tyr Asp Ala Leu Gln# 320- Ala Gly Leu Arg Thr Ala Pro Leu Ala Leu Th - #r Ile Val Ala Leu Asn325 3 - #30 3 - #35 3 -#40- Leu Ala Gly Leu Gly Ala Lys Leu Leu Ala Al - #a Leu Gly Thr Ala Arg# 355- Ser Ile Ala Leu Gly Met Thr Leu Leu Ala Al - #a Gly Leu Ser Ala Val# 370- Ala Val Gly Gly Ser Gly Pro Asp Ala Gly Ty - #r Gly Gly Met Leu Ala# 385- Gly Leu Leu Leu Met Gly Ala Gly Ile Ala Le - #u Ala Met Pro Ala Met# 400- Ala Thr Ala Val Met Ser Ser Ile Pro Pro Al - #a Lys Ala Gly Ala Gly405 4 - #10 4 - #15 4 -#20- Ala Gly Val Gln Gly Thr Leu Thr Glu Phe Gl - #y Gly Gly Leu Gly Val# 435- Ala Ile Leu Gly Ala Val Leu Gly Ser Arg Ph - #e Ala Ser Gln Leu Pro# 450- Ala Ala Ile Thr Gly Thr Gly Ser Leu Asp Gl - #u Ala Leu Arg Asp Ala# 465- Thr Pro Gln Gln Ala Gly Gln Val His Asp Al - #a Phe Ala Asp Ala Val# 480- Asn Thr Ser Gln Leu Ile Gly Ala Ala Ala Va - #l Phe Thr Gly Gly Leu485 4 - #90 4 - #95 5 -#00- Leu Ala Ala Leu Leu Leu His Arg Ala Asp Ar - #g Lys Ala Ala Pro Gln# 515- Pro Thr Ala Pro Thr Pro Glu Pro Thr Thr Th - #r Ala# 525- (2) INFORMATION FOR SEQ ID NO: 23:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 161 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear#23: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:#Val Thr Gly Ala Asp Asp Pro# 1 5- Ala Arg Pro Ala Val Gly Pro Gln Ser Phe Ar - #g Asp Ala Met Ala Gln# 20- Leu Ala Ser Pro Val Thr Val Val Thr Val Le - #u Asp Ala Ala Gly Arg# 35- Arg His Gly Phe Thr Ala Gly Ser Val Val Se - #r Val Ser Leu Asp Pro# 55- Pro Leu Val Met Val Gly Ile Ala Leu Thr Se - #r Ser Cys His Thr Ala# 70- Met Ala Ala Ala Ala Glu Phe Cys Val Ser Il - #e Leu Gly Glu Asp Gln# 85- Arg Ala Val Ala Lys Arg Cys Ala Thr His Gl - #y Ala Asp Arg Phe Ala# 100- Gly Gly Glu Phe Ala Ala Trp Asp Gly Thr Gl - #y Val Pro Tyr Leu Pro# 115- Asp Ala Lys Val Val Leu Arg Cys Arg Thr Th - #r Asp Val Val Arg Ala120 1 - #25 1 - #30 1 -#35- Gly Asp His Asp Leu Val Leu Gly Thr Pro Va - #l Glu Ile Arg Thr Gly# 150- Asp Pro Ala Lys Pro Pro Leu Leu Trp Tyr# 160- (2) INFORMATION FOR SEQ ID NO: 24:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 213 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear#24: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Ala Thr Ala Arg Leu Ile Gly Pro Leu Pro Ar - #g Arg Leu Gly Leu Gln# 15- Val His Gln Val Met Thr Gly Ala Phe Ala Gl - #n Ala Leu Ala Arg Trp# 30- Arg Gly Ser Arg Ala Val Thr Phe Asp Val Gl - #u Thr His Gly Arg His# 45- Gly Arg Asp Glu Leu Phe Arg Thr Val Gly Tr - #p Phe Thr Ser Ile His# 60- Pro Val Val Leu Gly Ala Asp Arg Ser Val Hi - #s Pro Glu Gln Tyr Leu# 80- Ala Gln Ile Gly Ala Ala Leu Thr Ala Ala Pr - #o Asp Gly Gly Val Gly# 95- Phe Gly Ala Cys Arg Glu Phe Ser Pro Asp Al - #a Gly Leu Arg Thr Leu# 110- Leu Arg Asp Leu Pro Pro Ala Leu Val Cys Ph - #e Asn Tyr Tyr Gly Gln# 125- Ala Asp Gln Leu Ser Pro Asn Gly Gly Phe Ar - #g Met Ser Gly Arg Pro# 140- Ile Pro Arg Glu His Ser Ala Arg Cys Glu Ar - #g Val Tyr Gly Ile Glu145 1 - #50 1 - #55 1 -#60- Val Tyr Gly Ile Val His Gly Gly Arg Leu Ar - #g Met Gly Leu Thr Trp# 175- Val Pro Ser Pro Ala Asp Gly Val Asp Glu Al - #a Gly Val Asp Ala Leu# 190- Val Glu Gln Met Ser Trp Val Leu Ala Thr Le - #u Ala Gly Ala Asp Pro# 205- His Ala Val Thr Pro 210- (2) INFORMATION FOR SEQ ID NO: 25:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 195 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear#25: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Val Arg Thr Val Arg Thr Leu Leu Ile Asp As - #n Tyr Asp Ser Phe Thr# 15- Tyr Asn Leu Phe Gln Met Leu Ala Glu Val As - #n Gly Ala Ala Pro Leu# 30- Val Val Arg Asn Asp Asp Thr Arg Thr Trp Gl - #n Ala Leu Ala Pro Gly# 45- Asp Phe Asp Asn Val Val Val Ser Pro Gly Pr - #o Gly His Pro Ala Thr# 60- Asp Thr Asp Leu Gly Leu Ser Arg Arg Val Il - #e Thr Glu Trp Asp Leu# 80- Pro Leu Leu Gly Val Cys Leu Gly His Gln Al - #a Leu Cys Leu Leu Ala# 95- Gly Ala Ala Val Val His Ala Pro Glu Pro Ph - #e His Gly Arg Thr Ser# 110- Asp Ile Arg His Asp Gly Gln Gly Leu Phe Al - #a Asn Ile Pro Ser Pro# 125- Leu Thr Val Val Arg Tyr His Ser Leu Thr Va - #l Arg Gln Leu Pro Ala# 140- Asp Leu Arg Ala Thr Ala His Thr Ala Asp Gl - #y Gln Leu Met Ala Val145 1 - #50 1 - #55 1 -#60- Ala His Arg His Leu Pro Arg Phe Gly Val Gl - #n Phe His Pro Glu Ser# 175- Ile Ser Ser Glu His Gly His Arg Met Leu Al - #a Asn Phe Arg Asp Leu# 190- Ser Leu Arg 195- (2) INFORMATION FOR SEQ ID NO: 26:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 292 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear#26: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:# Val Thr Ala Ala Ala - # Pro Thr Leu Ala# 5 1- Gln Ala Leu Asp Glu Ala Thr Gly Gln Leu Th - #r Gly Ala Gly Ile Thr# 25- Ala Asp Ala Ala Arg Ala Asp Thr Arg Leu Le - #u Ala Ala His Ala Cys# 40- Gln Val Ala Pro Gly Asp Leu Asp Thr Cys Le - #u Ala Gly Pro Val Pro# 55- Pro Arg Phe Trp His Tyr Val Arg Arg Arg Le - #u Thr Arg Glu Pro Ala# 70- Glu Arg Ile Val Gly His Ala Tyr Phe Met Gl - #y His Arg Phe Asp Leu# 85- Ala Pro Gly Val Phe Val Pro Lys Pro Glu Th - #r Glu Glu Ile Thr Arg#105- Asp Ala Ile Ala Arg Leu Glu Ala Leu Val Ar - #g Arg Gly Thr Thr Ala# 120- Pro Leu Val Val Asp Leu Cys Ala Gly Pro Gl - #y Thr Met Ala Val Thr# 135- Leu Ala Arg His Val Pro Ala Ala Arg Val Le - #u Gly Ile Glu Leu Ser# 150- Gln Ala Ala Ala Arg Ala Ala Arg Arg Asn Al - #a Arg Gly Thr Gly Ala# 165- Arg Ile Val Gln Gly Asp Ala Arg Asp Ala Ph - #e Pro Glu Leu Ser Gly170 1 - #75 1 - #80 1 -#85- Thr Val Asp Leu Val Val Thr Asn Pro Pro Ty - #r Ile Pro Ile Gly Leu# 200- Arg Thr Ser Ala Pro Glu Val Leu Glu His As - #p Pro Pro Leu Ala Leu# 215- Trp Ala Gly Glu Glu Gly Leu Gly Met Ile Ar - #g Ala Met Glu Arg Thr# 230- Ala Ala Arg Leu Leu Ala Pro Gly Gly Val Le - #u Leu Leu Glu His Gly# 245- Ser Tyr Gln Leu Ala Ser Val Pro Ala Leu Ph - #e Arg Ala Thr Gly Arg250 2 - #55 2 - #60 2 -#65- Trp Ser His Ala Ser Ser Arg Pro Thr Cys As - #n Asp Gly Cys Leu Thr# 280- Ala Val Arg Asn His Thr Cys Ala Pro Pro Al - #a# 290- (2) INFORMATION FOR SEQ ID NO: 27:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 75 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear#27: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:#Gln Leu Ala Pro Val Ala Glur Val Ala# 15- Thr Leu Ala Asp Thr Thr Arg Glu Glu Pro Al - #a Ala Val Ala Ala Thr# 30- Gly Asp Val Pro Leu Thr Pro Ile Met His Tr - #p Leu Arg Glu Arg Gly# 45- Gly Pro Val Asp Ala Phe Ser Gln Thr Met Al - #a Val Thr Val Pro Ala# 60- Gly Leu Asp Arg Glu Arg Leu Val Ala Ala Le - #u Gln# 75- (2) INFORMATION FOR SEQ ID NO: 28:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 82 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear#28: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Leu Glu Tyr Asp Thr Ala Leu Tyr Glu Arg Al - #a Thr Ala Glu Ala Leu# 15- Thr Gly Arg Leu Leu Arg Leu Leu Asp Ala Va - #l Val Thr Asp Pro Gln# 30- Ala Pro Val Gly Ser His Asp Leu Leu Glu Gl - #u Ala Glu His Ala Arg# 45- Leu Ala Ala Phe Asn Asp Thr Ala Arg Pro Va - #l Pro Arg Ala Gly Leu# 60- Ala Glu Leu Phe Thr Ala Gln Ala Arg Arg Th - #r Ala Asp Ala Val Ala# 80- Val Val- (2) INFORMATION FOR SEQ ID NO: 29:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 63 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear#29: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:#Ala Tyr Leu Ala His Leu Alao Tyr Arg# 15- Gly Arg Asp Asp Asp Ala Ala Arg Ala Ala Tr - #p Arg Thr Ala Leu Ala# 30- Asp Leu Glu Glu Pro Ser Leu Val Ala Gly Al - #a Gly Ala Gly Arg Gly# 45- Ala Ala Asp Gly Ser Ala Leu Pro Gly Gln Il - #e Pro Gly Tyr Arg Ala# 60- (2) INFORMATION FOR SEQ ID NO: 30:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 158 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear#30: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Leu Gln Val Glu Gly Arg Pro Ala His Leu Gl - #u Leu Pro Cys Asp His# 15- Pro Arg Pro Ala Val Ala Thr His Arg Gly Al - #a Thr Val Pro Phe His# 30- Ile Asp Ala Gly Leu His Glu Lys Leu Thr Al - #a Leu Ser Lys Ala Cys# 45- Asp Ser Ser Leu Phe Met Val Leu Gln Ala Al - #a Val Ala Ala Leu Leu# 60- Thr Arg His Gly Ala Gly Thr Asp Ile Pro Va - #l Gly Ser Pro Val Ala# 80- Gly Arg Thr Asp Asp Ala Leu Asp Asp Leu Va - #l Gly Phe Phe Val Asn# 95- Thr Leu Val Leu Arg Thr Asp Thr Ser Gly As - #p Pro Thr Phe Arg Glu# 110- Leu Val Ala Arg Val Arg Gln Phe Asp Leu Al - #a Ala Tyr Thr His Gln# 125- Asp Met Pro Phe Glu Lys Leu Val Glu Glu Va - #l Asn Pro Glu Arg Ser# 140- Leu Ala Arg Asn Pro Leu Phe Gln Val Val Le - #u Ala Leu Gln145 1 - #50 1 - #55- (2) INFORMATION FOR SEQ ID NO: 31:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 161 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear#31: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:#Ala Leu Leu Ala Val Leu Asp Leu Tyr Val# 15- Lys Thr Gly Ala Ala Tyr Leu Pro Val Asp Il - #e Ser Tyr Pro Ala Glu# 30- Arg Ile Ala Phe Met Ile Glu Asp Ala Arg Pr - #o Val Thr Val Leu Asp# 45- Arg Leu Pro Asp Asp Leu Gly Ala Tyr Arg As - #p Thr Asp Leu Thr Asp# 60- Ala Asp Arg Thr Ala Pro Leu Arg Pro Glu Hi - #s Pro Ala Tyr Val Ile# 75- His Thr Ser Gly Ser Thr Gly Thr Pro Lys Al - #a Val Val Met Pro His# 95- Ala Gly Leu Val Asn Leu Leu Thr Trp His Al - #a Arg Arg Phe Pro Gly# 110- Gly Thr Gly Val Arg Thr Ala Gln Phe Thr Al - #a Ile Gly Phe Asp Phe# 125- Ser Val Gln Glu Ile Leu Ser Pro Leu Val Me - #t Gly Lys Thr Leu Ala# 140- Val Pro Ser Glu Glu Val Arg His Ser Ala Gl - #u Leu Leu Ala Gly Trp# 155- Leu Glu160- (2) INFORMATION FOR SEQ ID NO: 32:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 97 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear#32: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Leu Gln Ala Glu Gly Ala Glu Val Ser Leu Le - #u Ala Val Leu Asp Gly# 15- Tyr Pro Asp Ala Tyr Asp Gly Thr Glu His Gl - #u Val Gly Glu Glu Gln# 30- Val Leu Ala Ile Leu Leu Asn Ala Ala Gly Va - #l Asp Arg Ala Gln Ala# 45- Phe Gly Asp Ala Pro Leu Gln Arg Ala Ala Va - #l Leu Glu Lys Leu Arg# 60- Asp Ser Gly Ser Ala Leu Gly Asn Leu Asp As - #p Asp Ala Val Gly Arg# 80- Met Val Thr Val Phe Leu Asn Asn Thr Arg Le - #u Ile Gln Asn Phe Arg# 95- Pro- (2) INFORMATION FOR SEQ ID NO: 33:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 739 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 33:- Ile Arg Thr Val Leu Gly Ala Glu Ile Ala Va - #l Arg Asp Leu Phe Glu# 15- Ala Pro Thr Val Glu Ala Leu Ala Glu Thr Le - #u Glu Glu Ala Arg Glu# 30- Val Arg Pro Ala Leu Arg Ala Ala Asp Arg Pr - #o Glu His Val Pro Leu# 45- Ser Phe Ala Gln Arg Arg Leu Trp Phe Leu As - #p Arg Leu Glu Gly Pro# 60- Asn Ser Thr Tyr Asn Ile Pro Leu Ala Leu Ar - #g Leu Arg Gly Glu Leu# 80- Asp Arg Pro Ala Leu Gln Gln Ala Leu Thr As - #p Leu Thr His Arg His# 95- Glu Ser Leu Arg Thr Val Tyr Pro Ser Ala As - #p Gly Arg Pro Tyr Gln# 110- His Val Leu Ala Pro His Glu Ala Glu Pro Gl - #y Leu Val Val Val Pro# 125- Ala Asp Glu Ala Gly Leu Ala Glu Met Leu Al - #a Glu Ala Ala Arg His# 140- Glu Phe Asp Val Thr Ser Glu Pro Pro Leu Ar - #g Val Ser Leu Phe Thr145 1 - #50 1 - #55 1 -#60- Leu Ala Pro Asp Glu His Val Leu Leu Leu Le - #u Leu His His Ile Ala# 175- Gly Asp Gly Trp Ser Leu Ala Pro Leu Thr Ar - #g Asp Leu Thr Arg Ala# 190- Tyr Thr Ala Arg Arg Asp Gly Ala Ala Pro As - #p Trp Glu Pro Leu Pro# 205- Val Gln Tyr Ala Asp Tyr Thr Leu Trp Gln Gl - #n Glu Met Leu Gly Ser# 220- Pro Asp Asp Pro Asp Ser Leu Gly Ala Arg Gl - #n Leu Asp His Trp Ala225 2 - #30 2 - #35 2 -#40- Arg Ser Leu Ala Gly Ala Pro Glu Gln Leu Gl - #u Leu Pro Thr Asp His# 255- Asn Arg Pro Ala Ala Ala Gly His His Gly Ar - #g Thr Val Pro Phe His# 270- Leu Glu Pro Glu Leu His Glu Arg Leu Ser Al - #a Leu Ala Arg Ser Cys# 285- Asp Ala Ser Leu Phe Met Val Leu His Ala Al - #a Phe Ala Ala Leu Leu# 300- Thr Lys His Gly Ala Gly Thr Asp Ile Pro Il - #e Gly Ser Pro Ile Ala305 3 - #10 3 - #15 3 -#20- Gly Arg Thr Asp Glu Ala Leu Asp Asp Leu Va - #l Gly Phe Phe Val Asn# 335- Thr Leu Val Leu Arg Thr Asp Thr Ser Gly As - #p Pro Thr Phe Arg Glu# 350- Leu Val Ala Arg Thr Arg Ala Thr Asp Leu Al - #a Ala Tyr Ala His Gln# 365- Asp Leu Pro Phe Glu Lys Leu Val Glu Thr Le - #u Asn Pro Gln Arg Ser# 380- Leu Ala Arg Asn Pro Leu Phe Gln Val Leu Le - #u Ala Phe Gln Ser Met385 3 - #90 3 - #95 4 -#00- Pro Thr Ala Gln Pro Val Leu Pro Gly Leu As - #p Val Val His Glu Pro# 415- Val Arg Val Gly Phe Ala Lys Phe Asp Leu Al - #a Leu Ala Val Ala Glu# 430- Glu Arg His Ala Asp Gly Arg Arg Ser Leu Ar - #g Gly Asp Trp Glu Phe# 445- Ser Thr Asp Leu Phe Glu Gln Ala Thr Val Gl - #u Ala Leu Gly Ala Arg# 460- Leu Thr Ala Leu Leu Ala Ser Val Ala Ala As - #p Pro Asp Gln Pro Ile465 4 - #70 4 - #75 4 -#80- Gly Arg Val Gly Ile Leu Asp Pro Ala Glu Ar - #g His Arg Ile Leu His# 495- Thr Trp Asn Asp Thr Ser Arg Pro Gly Ala As - #p Ala Thr Trp Pro Glu# 510- Leu Phe Gln Ala Arg Ala Ala Glu His Pro As - #p Ala Val Ala Leu Val# 525- Gln Glu Gly Thr Glu Thr Gly Tyr Ala Asp Le - #u Asn Thr Arg Ala Asn# 540- Arg Leu Ala Arg Leu Leu Arg Ala Gln Gly Il - #e Gly Pro Glu Gln Val545 5 - #50 5 - #55 5 -#60- Val Ala Leu Ser Leu Pro Arg Ser Ala Asp Le - #u Ile Val Ser Val Leu# 575- Ala Val Leu Lys Thr Gly Ala Ala Tyr Leu Pr - #o Val Asp Pro Ala Tyr# 590- Pro Ala Glu Arg Ile Ala Tyr Leu Leu Gln As - #p Gly Ala Pro Ala Leu# 605- Val Leu Thr His Thr Ser Val Ala Ala Gly Le - #u Pro Gly Gly Val Pro# 620- Gln Leu Leu Val Asp Gln Val Gly Leu Asp As - #p Val Pro Gly His Asp625 6 - #30 6 - #35 6 -#40- Leu Thr Asp Ala Glu Arg Thr Thr Pro Leu Hi - #s Pro Leu His Pro Ala# 655- Tyr Val Ile Tyr Thr Ser Gly Ser Thr Gly Le - #u Pro Lys Gly Val Pro# 670- Val Pro His Arg Ser Val Ala Ser Val Leu Va - #l Pro Leu Ile Glu Glu# 685- Phe Gly Leu Gly Pro Gly Ser Arg Val Leu Gl - #n Phe Ala Ser Ile Ser# 700- Phe Asp Ala Ala Leu Trp Glu Ile Thr Leu Al - #a Leu Leu Ser Gly Ala705 7 - #10 7 - #15 7 -#20- Thr Leu Val Val Ala Pro Ala Glu Gln Leu Gl - #n Pro Gly Pro Ala Leu# 735- Ala Glu Leu- (2) INFORMATION FOR SEQ ID NO: 34:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 23 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 34:# 23AY CTS CCS GG- (2) INFORMATION FOR SEQ ID NO: 35:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 27 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 35:# 27 CN TTC GTS CAY GAC- (2) INFORMATION FOR SEQ ID NO: 36:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 33 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 36:# 33G CCS GAG GAC GTS GCS GCS AAG TA - #C- (2) INFORMATION FOR SEQ ID NO: 37:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 44 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 37:- GAG GTS GAG GGS CAC CTS GGS GCS CAC CCS GA - #C GTS CAG CAG GC# 44- (2) INFORMATION FOR SEQ ID NO: 38:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 20 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 38:- Val Pro Ala Ala Phe Val Pro Leu Asp Ala Le - #u Pro Leu Thr Gly Asn# 15- Gly Val Leu Asp 20- (2) INFORMATION FOR SEQ ID NO: 39:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 26 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 39:# 26 AC ACS GCS CGS CC- (2) INFORMATION FOR SEQ ID NO: 40:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 26 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 40:# 26 AC GCS CTS CCS CT- (2) INFORMATION FOR SEQ ID NO: 41:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 21 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 41:#21 GS GCS TAC- (2) INFORMATION FOR SEQ ID NO: 42:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 26 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 42:# 26 AG AAC TTC CGB CC- (2) INFORMATION FOR SEQ ID NO: 43:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 26 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 43:# 26 TG GCS CAG CTS GC- (2) INFORMATION FOR SEQ ID NO: 44:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 38 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 44:# 38S GGS GGS GAG TTC GCS GCS TGG GAC GG - #C ACC GG- (2) INFORMATION FOR SEQ ID NO: 45:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 32 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 45:# 32 AAG CCS CCS CTS CTS TGG TAC CG - #__________________________________________________________________________
Claims
  • 1. A purified nucleotide sequence selected from the group consisting of orf1 gene of Streptomyces pristinaespiralis, and a nucleotide sequence which hybridizes to said orf1 gene in the presence of formamide at 42.degree. C. with washes at 50.degree. and 60.degree. C., wherein said purified nucleotide sequence encodes Orf1.
  • 2. A recombinant DNA sequence comprising a gene selected from the group consisting of orf1 of S. pristinaespiralis, and a nucleotide sequence which hybridizes to said orf1 gene in the presence of formamide at 42.degree. C. with washes at 50.degree. and 60.degree. C., wherein said recombinant DNA encodes Orf1.
  • 3. A recombinant DNA sequence selected from the group consisting of cosmid pIBV30 (FIG. 33), and a DNA sequence which hybridizes to said cosmid in the presence of formamide at 42.degree. C. with washes at 50.degree. and 60.degree. C., wherein said recombinant DNA encodes Orf1.
  • 4. A purified nucleotide sequence selected from the group consisting of:
  • (a) the orf1 (SEQ ID NO: 17) gene,
  • (b) the sequences which hybridize in the presence of formamide at 42.degree. C. with washes at 50.degree. and 60.degree. C. with the orf1 gene, wherein said sequences encode Orf1, and
  • (c) sequences which encode the polypeptides encodes by (a) and (b), and which differ from the sequences (a) and (b) owing to the degeneracy of the genetic code.
  • 5. An autonomously replicating or integrative expression vector, characterized in that it comprises a nucleotide sequence or a recombinant DNA according to any one of claims 1, 2, 3, or 4.
  • 6. A vector consisting of plasmid pVRC510 (FIG. 34).
  • 7. A recombinant cell containing a nucleotide sequence, a recombinant DNA, or an expression vector according to any one of claims 1, 2, 3, or 4.
  • 8. A recombinant cell containing an expression vector according to claim 5.
  • 9. A recombinant cell containing a vector according to claim 6.
  • 10. A method for producing a Orf1 polypeptide, comprising culturing a recombinant cell according to claim 8 and recovering the polypeptide.
  • 11. A method for producing a polypeptide involved in the biosynthesis pathway of streptogramins, wherein a recombinant cell according to claim 8 is cultured and the resulting polypeptide is recovered.
  • 12. A method for producing a polypeptide involved in the biosynthesis pathway of streptogramins, wherein a recombinant cell according to claim 9 is cultured and the resulting polypeptide is recovered.
Priority Claims (1)
Number Date Country Kind
92 11441 Sep 1992 FRX
RELATED APPLICATION INFORMATION

This application is a continuation-in-part of U.S. application Ser. No. 08/403,852, filed May 10, 1995 entitled "Polypeptides Involved in the Biosynthesis of Streptogramins, Nucleotide Sequences Coding for These Polypeptides and Their Use", which is a national filing of PCT/FR93/00923, filed Sep. 25, 1993 (WO 94/08014) which is pending, hereby incorporated by reference.

US Referenced Citations (2)
Number Name Date Kind
5252474 Gewain et al. Oct 1993
5591614 Blanche et al. Jan 1997
Foreign Referenced Citations (1)
Number Date Country
59-59198 Apr 1984 JPX
Non-Patent Literature Citations (9)
Entry
Biot et al., Virginiamycin: Properties, Biosynthesis, and Fermentation Drugs and the Pharmaceutical Sciences 22:695-720 (1984).
Blumauerova et al., "Physiological and Genetic Aspects of Vriginiamycin Production," Folia Microbiologica, Prague 35(6)494 (1990).
Chater et al., "The improving prospects for yield increase by genetic engineering in antibiotic-producing streptomycetes," Biotechnology 8:115-121 (1990).
Kazumi et al., "Isolation and properties of IM factor (an inducer of secondary metabolite production) deficient mutants of Streptomyces virgineae MAFF 10-06014," Chemical Abstracts 115(23) :251978n (1991).
Hallam et al., "Nucleotide sequence, transcription and deduced function of a gene involved in polyketide antiobiotic synthesis in Streptomyces coelicolor," Gene 74:305-320 (1988).
Paquet et al., "Induction of Pristinamycins Production in Streptomyces pristinaespiralis," Biotechnology Letters 14 (11) :1065-1070 (1992).
Prikrylova et al., "Strain Development in Streptomyces virginiae, a Producer of Virginiamycyn," Biotechnology and Bioindustry 2:20-22 (1988).
Francis et al. "Biosynthesis of chloramphenicol in Streptomyces species 3022a: the nature of arylamine synthase system" Can J. Microbiol. 25, 1408-1415, 1979.
Spyrou et al. "Characterization of the flavin reductase gene (fre) of Escherichia coli and construction of a plasmid for overproduction of the enzyme" J. Bacteriol. 173, 3673-3679, Jun. 1991.
Continuation in Parts (1)
Number Date Country
Parent 403852 May 1995