Microbial β-glucuronidase genes, gene products and uses thereof

Abstract

Genes encoding microbial β-glucuronidases and proteins and their uses are provided.

Description

TECHNICAL FIELD

The present invention relates generally to microbial β-glucuronidases, and more specifically to secreted forms of β-glucuronidase, and uses of these β-glucuronidases.

BACKGROUND OF THE INVENTION

The enzyme β-glucuronidase (GUS; E.C.3.2.1.31) hydrolyzes a wide variety of glucuronides. Virtually any aglycone conjugated to D-glucuronic acid through a β-O-glycosidic linkage is a substrate for GUS. In vertebrates, glucuronides containing endogenous as well as xenobiotic compounds are generated through a major detoxification pathway and excreted in urine and bile.

Escherichia coli, the major organism resident in the large intestine of vertebrates, utilizes the glucuronides generated in the liver and other organs as an efficient carbon source. Glucuronide substrates are taken up by E. coli via a specific transporter, the glucuronide permease (U.S. Pat. Nos. 5,288,463 and 5,432,081), and cleaved by β-glucuronidase, releasing glucuronic acid residues that are used as a carbon source. In general, the aglycone component of the glucuronide substrate is not used by E. coli and passes back across the bacterial membrane into the gut to be reabsorbed into the bloodstream and undergo glucuronidation in the liver, beginning the cycle again. In E. coli , β-glucuronidase is encoded by the gusA gene (Novel and Novel, Mol. Gen. Genet. 120:319-335, 1973), which is one member of an operon comprising two other protein-encoding genes, gusB encoding a permease (PER) specific for β-glucuronides, and gusC encoding an outer membrane protein (OMP) that facilitates access of glucuronides to the permease located in the inner membrane.

While β-glucuronidase activity is expressed in almost all tissues of vertebrates and their resident intestinal flora, GUS activity is absent in most other organisms. Notably, plants, most bacteria, fungi, and insects are reported to largely, if not completely, lack GUS activity. Thus, GUS is ideal as a reporter molecule in these organisms and has become one of the most widely used reporter systems for these organisms.

In addition, because both endogenous and xenobiotic compounds are generally excreted from vertebrates as glucuronides, β-glucuronidase is widely used in medical diagnostics, such as drug testing. In therapeutics, GUS has been used as an integral component of prodrug therapy. For example, a conjugate of GUS and a targeting molecules, such as an antibody specific for a tumor cell type, is delivered along with a nontoxic prodrug, provided as a glucuronide. The antibody targets the cell and GUS cleaves the prodrug, releasing an active drug at the target site.

Because the E. coli GUS enzyme is much more active and stable than the mammalian enzyme against most biosynthetically derived β-glucuronides (Tomasic and Keglevic, Biochem J 133:789, 1973; Levvy and Conchie, 1966), the E. coli GUS is preferred in both reporter and medical diagnostic systems.

Production of GUS for use in in vitro assays, such as medical diagnostics, however, is costly and requires extensive manipulation as GUS must be recovered from cell lysates. A secreted form of GUS would reduce manufacturing expenses, however, attempts to cause secretion have been largely unsuccessful. In addition, for use in transgenic organisms, the current GUS system has somewhat limited utility because enzymatic activity is detected intracellularly by deposition of toxic colorimetric products during the staining or detection of GUS. Moreover, in cells that do not express a glucuronide permease, the cells must be permeabilized or sectioned to allow introduction of the substrate. Thus, this conventional staining procedure generally results in the destruction of the stained cells. In light of these limitations, a secreted GUS would facilitate development of non-destructive marker systems, especially useful for agricultural field work.

Furthermore, the E. coli enzyme, although more robust than vertebrate GUS, has characteristics that limit its usefulness. For example, it is heat-labile and inhibited by detergents and end product (glucuronic acid). For many applications, a more resilient enzyme would be beneficent.

The present invention provides gene and protein sequences of microbial β-glucuronidases, variants thereof, and use of the proteins as a transformation marker, effector molecule, and component of medical diagnostic and therapeutic systems, while providing other related advantages.

SUMMARY OF INVENTION

In one aspect, an isolated nucleic acid molecule is provided comprising a nucleic acid sequence encoding a microbial of β-glucuronidase, provided that the β-glucuronidase is not from E. coli. Nucleic acid sequences are provided for β-glucuronidases from Thermotoga, Bacillus, Staphylococcus, Salmonella, Enterobacter, and Pseudomonas. In certain embodiments, the nucleic acid molecule encoding β-glucuronidase is derived from a eubacteria, such as purple bacteria, gram(+) bacteria, cyanobacteria, spirochaetes, green sulphur bacteria, bacteroides and flavobacteria, planctomyces, chlamydiae, radioresistant micrococci, and thermotogales.

In another aspect, microbial β-glucuronidases are provided that have enhanced characteristics. In one aspect, thermostable β-glucuronidases and nucleic acids encoding them are provided. In general, a thermostable β-glucuronidase has a half-life of at least 10 min at 65° C. In preferred embodiments, the thermostable β-glucuronidase is from Thermotoga or Bacillus groups. In other embodiments, the β-glucuronidase converts at least 50 nmoles of p-nitrophenyl-glucuronide to p-nitrophenyl per minute, per microgram of protein. In even further embodiments, the β-glucuronidase retains at least 80% of its activity in 10 mM glucuronic acid.

In another aspect, fusion proteins of microbial β-glucuronidase or an enzymatically active portion thereof are provided. In certain embodiments, the fusion partner is an antibody or fragment thereof that binds antigen.

In other aspects, expression vectors comprising a gene encoding a microbial β-glucuronidase or a portion thereof that has enzymatic activity in operative linkage with a heterologous promoter are provided. In such a vector, the microbial β-glucuronidase is not E. coli β-glucuronidase. In the expression vectors, the heterologous promoter is a promoter selected from the group consisting of a developmental type-specific promoter, a tissue type-specific promoter, a cell type-specific promoter and an inducible promoter. The promoter should be functional in the host cell for the expression vector. Examples of cell types include a plant cell, a bacterial cell, an animal cell and a fungal cell. In certain embodiments, the expression vector also comprises a nucleic acid sequence encoding a product of a gene of interest or portion thereof. The gene of interest may be under control of the same or a different promoter.

Isolated forms of recombinant microbial β-glucuronidase are also provided in this invention, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase. The recombinant β-glucuronidases may be from eubacteria, archaea, or eucarya. When eubacteria β-glucuronidases are clones, the eubacteria is selected from purple bacteria, gram(+) bacteria, cyanobacteria, spirochaetes, green sulphur bacteria, bacteroides and flavobacteria, planctomyces, chlamydiae, radioresistant micrococci, and thermotogales and the like.

The present invention also provides methods for monitoring expression of a gene of interest or a portion thereof in a host cell, comprising: (a) introducing into the host cell a vector construct, the vector construct comprising a nucleic acid molecule according to claim 1 and a nucleic acid molecule encoding a product of the gene of interest or a portion thereof; (b) detecting the presence of the microbial β-glucuronidase, thereby monitoring expression of the gene of interest; methods for transforming a host cell with a gene of interest or portion thereof, comprising: (a) introducing into the host cell a vector construct, the vector construct comprising a nucleic acid sequence encoding a microbial β-glucuronidase, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase, and a nucleic acid sequence encoding a product of the gene of interest or a portion thereof, such that the vector construct integrates into the genome of the host cell; and (b) detecting the presence of the microbial β-glucuronidase, thereby establishing that the host cell is transformed.

Methods are also provided for positive selection for a transformed cell, comprising: (a) introducing into a host cell a vector construct, the vector construct comprising nucleic acid sequence encoding a microbial β-glucuronidase, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase; (b) exposing the host cell to the sample comprising a glucuronide, wherein the glucuronide is cleaved by the β-glucuronidase, such that the compound is released, wherein the compound is required for cell growth. In all these methods, a microbial glucuronide permease gene may be also introduced.

Transgenic plants expressing a microbial β-glucuronidase other than E. coli β-glucuronidase are also provided. The present invention also provides seeds of transgenic plants. Transgenic animals, such as aquatic animals are also provided. Methods for identifying a microorganism that secretes β-glucuronidase, are provided comprising: (a) culturing the microorganism in a medium containing a substrate for β-glucuronidase, wherein the cleaved substrate is detectable, and wherein the microorganism is an isolate of a naturally occurring microorganism or a transgenic microorganism; and (b) detecting the cleaved substrate in the medium. In certain embodiments, the microorganism is cultured under specific conditions that are favorable to particular microorganisms.

In another aspect, a method for providing an effector compound to a cell in a transgenic plant is provided. The method comprises (a) growing a transgenic plant that comprises an expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter and a nucleic acid sequence comprising a gene encoding a cell surface receptor for an effector compound and (b) exposing the transgenic plant to a glucuronide, wherein the glucuronide is cleaved by the β-glucuronidase, such that the effector compound is released. This method is especially useful for directing glucuronides to particular and specific cells by further introducing into the transgenic plant a vector construct comprising a nucleic acid sequence that binds the effector compound. The effector compound can then be used to control expression of a gene of interest by linking a gene of interest with the nucleic acid sequence that binds the effector compound.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth below which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents DNA sequence (SEQ ID No: 1) of an approximately 6 kb fragment that encodes β-glucuronidase from Bacillus.

FIG. 2 is a schematic of the DNA sequence of a Bacillus 6 kb fragment showing the location and orientation of the major open reading frames. S-GUS is β-glucuronidase.

FIGS. 3A-B present amino acid sequences (SEQ ID Nos: 2-6) of representative microbial β-glucuronidases.

FIGS. 4A-J present DNA sequences (SEQ ID Nos.: 7, 9-14) and an amino acid sequence ( FIGS. 4A-C ; SEQ ID No.:8) of representative microbial β-glucuronidases.

FIGS. 5A-C present amino acid alignments of Bacillus GUS (BGUS) (SEQ ID Nos: 7-14) E. coli GUS (EGUS) (SEQ ID No: 16) and human GUS (HGUS) (SEQ ID No: 17) (5A). Microbial GUSes (5B) (SEQ ID Nos: 18-23) and nucleotide sequence alignments (SEQ ID Nos: 24-26) of Bacillus, Salmonella, and Pseudomonas β-glucuronidases.

FIG. 6 is a graph showing that Bacillus GUS is secreted in E. coli transformed with an expression vector encoding Bacillus GUS. The secretion index is the percent of total activity in periplasm less the percent of total β-galactosidase activity in periplasm.

FIG. 7 is a graph illustrating the half-life of Bacillus GUS and E. coli GUS at 65° C.

FIG. 8 is a graph showing the turnover number of Bacillus GUS and E. coli GUS enzymes at 37° C.

FIG. 9 is a graph showing the turnover number of Bacillus GUS and E. coli GUS enzymes at room temperature.

FIG. 10 is a graph presenting relative enzyme activity of Bacillus GUS in various detergents.

FIG. 11 is a graph presenting relative enzyme activity of Bacillus GUS in the presence of glucuronic acid.

FIG. 12 is a graph presenting relative enzyme activity of Bacillus GUS in various organic solvents and in salt.

FIGS. 13A-C present a DNA sequence (SEQ ID No.: 27) and amino acid sequence (SEQ ID No.: 28).

FIG. 14 is a schematic of the DNA sequence of Bacillus GUS that is codon-optimized for production in E. coli.

FIG. 15 presents schematics of two expression vectors for use in yeast (upper Afigure) and plants (lower figure).

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms that will be used hereinafter.

As used herein, “β-glucuronidase” refers to an enzyme that catalyzes the hydrolysis of β-glucuronides. Assays and some exemplary substrates for determining β-glucuronidase activity, also known as GUS activity, are provided in U.S. Pat. No. 5,268,463. In assays to detect β-glucuronidase activity, fluorogenic or chromogenic substrates are preferred. Such substrates include, but are not limited to, p-nitrophenyl β-D-glucuronide and 4-methylumbelliferyl β-D-glucuronide.

As used herein, a “secreted form of a microbial β-glucuronidase” refers to a microbial β-glucuronidase that is capable of being localized to an extracellular environment of a cell, including extracellular fluids, periplasm, or membrane bound on the external face of a cell but not membrane bound as an integral membrane protein. Some of the protein may be found intracellularly. The amino acid and nucleotide sequences of an exemplary secreted β-glucuronidase are presented in FIG. 1 and SEQ ID Nos.: 1 and 2. Secreted microbial GUS also encompasses variants of β-glucuronidase. A variant may be a portion of the secreted β-glucuronidase and/or have amino acid substitutions, insertions, and deletions, either found naturally as a polymorphic allele or constructed.

As used herein, “percent sequence identity” is a percentage determined by the number of exact matches of amino acids or nucleotides to a reference sequence divided by the number of residues in the region of overlap. Within the context of this invention, preferred amino acid sequence identity for a variant is at least 75% and preferably greater than 80%, 85%, 90% or 95%. Such amino acid sequence identity may be determined by standard methodologies, including use of the National Center for Biotechnology Information BLAST search methodology (Altschul et al. “Basic local alignment search tool.” J. Mol. Biol. 215:403-410, 1990; Gish et al. “Identification of protein coding regions by database similarity search.” Nature Genet. 3:266-272, 1993; Madden et al. “Applications of network BLAST server” Meth. Enzymol. 266:131-141, 1996; Zhang et al. “PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation.” Genome Res. 7:649-656, 1997). The identity methodologies preferred are non-gapped BLAST. However, those described in U.S. Pat. No. 5,691,179 and Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997), all of which are incorporated herein by reference are also useful. Accordingly, if Gapped BLAST 2.0 is utilized, then it is utilized with default settings. Further, a nucleotide variant will typically be sufficiently similar in sequence to hybridize to the reference sequence under stringent hybridization conditions (for nucleic acid molecules over about 500 bp, stringent conditions include a solution comprising about 1 M Na+ at 25° to 30° C. below the Tm; e.g., 5×SSPE, 0.5% SDS, at 65° C.; see, Ausubel, et al., Current Protocols in Molecular Biology, Greene Publishing, 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989). Some variants may not hybridize to the reference sequence because of codon degeneracy, such as degeneracies introduced for codon optimization in a particular host, in which case amino acid identity may be used to assess similarity of the variant to the reference protein.

As used herein, a “glucuronide” or “β-glucuronide” refers to an aglycone conjugated in a hemiacetal linkage, typically through the hydroxyl group, to the C1 of a free D-glucuronic acid in the β configuration. Glucuronides include, but are not limited to, O-glucuronides linked through an oxygen atom, S-glucuronides, linked through a sulfur atom, N-glucuronides, linked through a nitrogen atom and C-glucuronides, linked through a carbon atom (see, Dutton, Glucuronidation of Drugs and Other Compounds, CRC Press, Inc. Boca Raton, Fla. pp 13-15). β-glucuronides consist of virtually any compound linked to the C1-position of glucuronic acid as a beta anomer, and are typically, though by no means exclusively, found as an O-glycoside. β-glucuronides are produced naturally in most vertebrates through the action of UDP-glucuronyl transferase as a part of the process of solubilizing, detoxifying, and mobilizing both natural and xenobiotic compounds, thus directing them to sites of excretion or activity through the circulatory system.

β-glucuronides in polysaccharide form are also common in nature, most abundantly in vertebrates, where they are major constituents of connective and lubricating tissues in polymeric form with other sugars such as N-acetylglucosamine (e.g., chondroitan sulfate of cartilage, and hyaluronic acid, which is the principle constituent of synovial fluid and mucus). Other polysaccharide sources of β-glucuronides occur in bacterial cell walls, e.g., cellobiuronic acid. β-glucuronides are relatively uncommon or absent in plants. Glucuronides and galacturonides found in plant cell wall components (such as pectin) are generally in the alpha configuration, and are frequently substituted as the 4-O-methyl ether; hence, such glucuronides are not substrates for β-glucuronidase.

An “isolated nucleic acid molecule” refers to a polynucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid construct, that has been separated from its source cell (including the chromosome it normally resides in) at least once in a substantially pure form. Nucleic acid molecules may be comprised of a wide variety of nucleotides, including DNA, RNA, nucleotide analogues, or some combination of these.

Microbial β-glucuronidase Genes

As noted above, this invention provides gene sequences and gene products for microbial β-glucuronidase including secreted β-glucuronidase. As exemplified herein, genes from microorganisms, including a gene from Bacillus that encodes a secreted β-glucuronidase, are identified and characterized biochemically, genetically, and by DNA sequence analysis. Exemplary isolations of β-glucuronidase genes and gene products from several phylogenetic groups, including Bacillus, Thermotoga, Pseudomonas, Salmonella, Staphylococcus, Enterobacter, Arthobacter, and the like, are provided herein. Microbial β-glucuronidases from additional organisms may be identified as described herein or by hybridization of one of the microbial β-glucuronidase gene sequence to genomic or cDNA libraries, by genetic complementation, by function, by amplification, by antibody screening of an expression library and the like (see Sambrook et al., infra Ausubel et al., infra for methods and conditions appropriate for isolation of a β-glucuronidase from other species).

The existence of a microbial β-glucuronidase may be observed by a variety of methods and procedures. Particularly useful screens for identifying β-glucuronidase are biochemical screening and genetic complementation. Test samples containing microbes, may be obtained from sources such as soil, animal or human skin, saliva, mucous, feces, water, and the like. Microbes present in such samples include organisms from the phylogenetic domains, Eubacteria, Archaea, and Eucarya (Woese, Microbiol. Rev. 58: 1-9, 1994), the Eubacteria phyla: purple bacteria (including the α, β, γ, and δ subdivisions), gram (+) bacteria (including the high G+C content, low G+C content, and photosynthetic subdivisions), cyanobacteria, spirochaetes, green sulphur bacteria, bacteroides and flavobacteria, planctomyces and relatives, chlamydiae, radioresistant micrococci and relatives, and thermotogales. It will be appreciated by those in the art that the names and number of the phyla may vary somewhat according to the precise criteria for categorization (see Strunk et al., Electrophoresis 19: 554, 1998). Other microbes include, but are not limited to, entamoebae, fungi, and protozoa.

Colonies of microorganisms are generally obtained by plating on a suitable substrate in appropriate conditions. Conditions and substrates will vary according to the growth requirements of the microorganism. For example, anaerobic conditions, liquid culture or special defined media may be used to grow the microorganisms. Many different selective media have been devised to grow specific microorganisms (see, e.g., Merck Media Handbook). Substrates such as deoxycholate, citrate, etc. may be used to inhibit extraneous and undesired organisms such as gram-positive cocci and spore forming bacilli. Other substances to identify particular microbes (e.g., lactose fermenters, gram positives) may also be used. A glucuronide substrate is added that is readily detectable when cleaved by β-glucuronidase. A microbe that secretes β-glucuronidase should exhibit a diffuse staining (halo) pattern surrounding the colony.

A complementation assay may be additionally performed to verify that the staining pattern is due to expression of a GUS gene or to assist in isolating and cloning the GUS gene. Briefly, in this assay, the candidate GUS gene is transfected into an E. coli strain that is deleted for the GUS operon (e.g., KW1 described herein), and the staining pattern of the transfectant is compared to a mock-transfected host. For cloning by complementation, microbial genomic DNA is digested by e.g., restriction enzyme reaction and ligated to a vector, which ideally is an expression vector. The recombinants are transfected into a host strain, which ideally is deleted for endogenous GUS gene (e.g., KW1). In some cases, the host strain may express GUS gene but preferably not in the compartment to be assayed. If GUS is secreted, the transfectant should exhibit a diffuse staining pattern (halo) surrounding the colony, whereas, the host will not.

The microorganisms can be identified in myriad ways, including morphology, virus sensitivity, sequence similarity, metabolism signatures, and the like. A preferred method is similarity of rRNA sequence determined after amplification of genomic DNA. A region of rRNA is chosen that is flanked by conserved sequences that will anneal amplification primers. The amplification product is subjected to DNA sequence analysis and compared to known rRNA sequences described herein.

In one exemplary screen, a bacterial colony isolated from a soil sample displays a strong, diffuse staining pattern. The bacterium is identified as a Bacillus by sequence determination of 16S rRNA after amplification. A genomic library from this Bacillus is constructed in the vector pBSII KS+. The recombinant plasmids are transfected into KW1, a strain deleted for the β-glucuronidase operon. One resulting colony, containing the plasmid pRAJa17.1, exhibited a strong, diffuse staining pattern similar to the Bacillus isolate.

In other exemplary screens of microorganisms found in soil and in skin samples, numerous microbes exhibit a diffuse staining pattern around the colony or stained blue. The phylogenetic classifications of some of these are determined by sequence analysis of 16S rRNA. At least eight different genera are represented. Genetic complementation assays demonstrate that the staining pattern is most likely due to expression of the GUS gene. Not all complementation assays yield positive results, however, which may be due to the background genotype of the receptor strain or to restriction enzyme digestion within the GUS gene. The DNA sequence and predicted amino acid sequences of the GUS genes from several of these microorganisms found in these screens microorganisms are determined.

A DNA sequence of the GUS gene contained in the insert of pRAJa17.1 is presented in FIG. 1 and as SEQ ID No: 1. A schematic of the insert is presented in FIG. 2 . The β-glucuronidase gene contained in the insert is identified by similarity of the predicted amino acid sequence of an open reading frame to the E. coli and human β-glucuronidase amino acid sequences (FIG. 5 A). Overall, Bacillus β-glucuronidase has approximately 47-49% amino acid identity to E. coli GUS and to human GUS. An open reading frame of Bacillus GUS is 1854 bases, which would result in a protein that is 618 amino acids in length. The first methionine codon, however, is unlikely to encode the initiator methionine. Rather the second methionine codon is most likely the initiator methionine. Such a translated product is 602 amino acids long and is the sequence presented in FIGS. 3A-B and 4 A-I. The assignment of the initiator methionine is based upon a consensus Shine-Dalgamo sequence found upstream of the second Met, but not the first Met, and alignment of the Bacillus, human, and E. coli GUS amino acid sequences. Furthermore, as shown herein, Bacillus GUS gene lacking sequence encoding the 16 amino acids is expressed in E. coli transfectants. In addition, the 16 amino acids (Met-Leu-Ile-Ile-Thr-Cys-Asn-His-Leu-His-Leu-Lys-Arg-Ser-Ala-Ile) SEQ ID No. 29 are not a canonical signal peptide sequence.

There is a single Asn-Asn-Ser sequence (residues 118-120 in FIGS. 3A-B ) that can serve as a site for N-glycosylation in the ER. Furthermore, unlike the E. coli and human β-glucuronidases, which have 9 and 4 cysteines respectively, the Bacillus protein has only a single Cys residue (residue 499 in FIGS. 3 A-B).

The DNA sequences of GUS genes from Staphylococcus homini, Staphylococcus warneri, Thermotoga maritima (TIGR Thermotoga database), Enterobacter, Salmonella, and Pseudomonas are presented in FIGS. 4A-J and (SEQ ID Nos. 7-14). Predicted amino acid sequences are shown in FIGS. 3A-B and (SEQ ID Nos. 2-6). The amino acid sequences are shown in alignment in FIGS. 5A-C . The signature peptide sequences for glycosyl hydrolases (Henrissat, Biochem Soc Trans 26:153, 1998; Henrissat B et al., FEBS Lett 27:425, 1998) are located from amino acids 333 to 358 and from amino acids 406 to 420 (Bacillus numbering in FIGS. 3 A and 5 B). The catalytic nucleophile is Glu 344 (Bacillus numbering) (Wong et al., J. Biol Chem. 18: 34057, 1998). Within these two signature regions, 17/26 and 8/15 residues are identical across the six presented sequences. At the non-identical positions, most of the sequences share an identical residue. Thus, the sequences are highly conserved in these regions (identity between Bacillus and each other GUS gene ranges from 65% to 100% in signature 1 and from 73% to 100% in signature 2) (see FIG. 5 B). In contrast, between Bacillus and β-galactosidase, another glycosyl hydrolase that has signature sequences, identity is 46% in signature 1 and 73% in signature 2.

In addition, portions or fragments of microbial GUS may be isolated or constructed for use in the present invention. For example, restriction fragments can be isolated by well-known techniques from template DNA, e.g., plasmid DNA, and DNA fragments, including restriction fragments, can be generated by amplification. Furthermore, oligonucleotides can be synthesized or isolated from recombinant DNA molecules. One skilled in the art will appreciated that other methods are available to obtain DNA or RNA molecules having at least a portion of a microbial GUS sequence. Moreover, for particular applications, these nucleic acids may be labeled by techniques known in the art, such as with a radiolabel (e.g., 32 P, 33 P, 35 S, 125 I, 131 I, 3 H, 14 C), fluorescent label (e.g., FITC, Cy5, RITC, Texas Red), chemiluminescent label, enzyme, biotin and the like.

In certain aspects, the present invention provides fragments of microbial GUS genes. Fragments may be at least 17 nucleotides long (e.g., at least 20 nt, 25 nt, 30 nt, 40 nt, 50 nt). Fragments may be used in hybridization methods (see, exemplary conditions described infra) or inserted into appropriate vector for expression or production. In certain aspects, the fragments have sequences of one or both of the signatures or have sequence from at least some of the more highly conserved regions of GUS (e.g., from approximately amino acids 272-360 and from amino acids 398-421 or from amino acids 398-545; based on Bacillus numbering in FIG. 5 B). In the various embodiments, useful fragments comprise those nucleic acid sequences which encode at least the active residue at position 344 (Bacillus numbering in FIG. 5B ) and, preferably, comprise nucleic acid sequences 697-1624, 703-1620, 751-1573, 805-1398, 886-1248, 970-1059, and 997-1044 (Bacillus numbering in FIGS. 4 A- 4 C). In other embodiments, oligonucleotides of microbial GUSes are provided especially for use as amplification primers. In such case, the oligonucleotides are at least 12 bases and preferably at least 15 bases (e.g., at least 18, 21, 25, 30 bases) and generally not longer than 35 bases. It will be appreciated that any of these fragments described herein can be double-stranded, single-stranded, derived from coding strand or complementary strand and be exact or mismatched sequence.

Microbial β-glucuronidase Gene Products

The present invention also provides β-glucuronidase gene products in various forms. Forms of the GUS protein include, but are not limited to, secreted forms, membrane-bound forms, cytoplasmic forms, fusion proteins, chemical conjugates of GUS and another molecule, portions of GUS protein, and other variants. GUS protein may be produced by recombinant means, biochemical isolation, and the like.

In certain aspects, variants of secreted microbial GUS are useful within the context of this invention. Variants include nucleotide or amino acid substitutions, deletions, insertions, and chimeras. Typically, when the result of synthesis, amino acid substitutions are conservative, i.e., substitution of amino acids within groups of polar, non-polar, aromatic, charged, etc. amino acids. As will be appreciated by those skilled in the art, a nucleotide sequence encoding microbial GUS may differ from the wild-type sequence presented in the Figures, due to codon degeneracies, nucleotide polymorphisms, or amino acid differences. In certain embodiments, variants preferably hybridize to the wild-type nucleotide sequence at conditions of normal stringency, which is approximately 25-30° C. below Tm of the native duplex (e.g., 1 M Na+ at 65° C.; e.g. 5×SSPE, 0.5% SDS, 5×Denhardt's solution, at 65° C. or equivalent conditions; see generally, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987). Alternatively, the Tm for other than short oligonucleotides can be calculated by the formula Tm=81.5+0.41%(G+C)−log[Na+]. Low stringency hybridizations are performed at conditions approximately 40° C. below Tm, and high stringency hybridizations are performed at conditions approximately 10° C. below Tm.

Variants may be constructed by any of the well known methods in the art (see, generally, Ausubel et al., supra; Sambrook et al., supra). Such methods include site-directed oligonucleotide mutagenesis, restriction enzyme digestion and removal or insertion of bases, amplification using primers containing mismatches or additional nucleotides, splicing of another gene sequence to the reference microbial GUS gene, and the like. Briefly, preferred methods for generating a few nucleotide substitutions utilize an oligonucleotide that spans the base or bases to be mutated and contains the mutated base or bases. The oligonucleotide is hybridized to complementary single stranded nucleic acid and second strand synthesis is primed from the oligonucleotide. Similarly, deletions and/or insertions may be constructed by any of a variety of known methods. For example, the gene can be digested with restriction enzymes and religated such that some sequence is deleted or ligated with an isolated fragment having cohesive ends so that an insertion or large substitution is made. In another embodiment, variants are generated by shuffling of regions (see U.S. Pat. No. 5,605,793). Variant sequences may also be generated by “molecular evolution” techniques (see U. S. Pat. No. 5,723,323). Other means to generate variant sequences may be found, for example, in Sambrook et al. (supra) and Ausubel et al. (supra). Verification of variant sequences is typically accomplished by restriction enzyme mapping, sequence analysis, or probe hybridization, although other methods may be used. The double-stranded nucleic acid is transformed into host cells, typically E. coli, but alternatively, other prokaryotes, yeast, or larger eukaryotes may be used. Standard screening protocols, such as nucleic acid hybridization, amplification, and DNA sequence analysis, can be used to identify mutant sequences.

In addition to directed mutagenesis in which one or a few amino acids are altered, variants that have multiple substitutions may be generated. The substitutions may be scattered throughout the protein or functional domain or concentrated in a small region. For example, a region may be mutagenized by oligonucleotide-directed mutagenesis in which the oligonucleotide contains a string of dN bases or the region is excised and replaced by a string of dN bases. Thus, a population of variants with a randomized amino acid sequence in a region is generated. The variant with the desired properties (e.g., more efficient secretion) is then selected from the population.

In preferred embodiments, the protein and variants are capable of being secreted and exhibit β-glucuronidase activity. A GUS protein is secreted if the amount of secretion expressed as a secretion index is statistically significantly higher for the candidate protein compared to a standard, typically E. coli GUS. Secretion index maybe calculated as the percentage of total GUS activity in periplasm or other extracellular environment less the percentage of total β-galactosidase activity found in the same extracellular environment.

In other preferred embodiments, a microbial GUS or its variant will exhibit one or more of the biochemical characteristics exhibited by Bacillus GUS, such as its increased thermal stability, its higher turnover number, and its activity in detergents, presence of end product, high salt conditions and organic solvents as compared to an E. coli GUS standard.

In certain preferred embodiments, the microbial GUS is thermostable having a half-life of at least 10 minutes at 65° C. (e.g., 14 minutes, 16 minutes, 18 minutes). In other preferred embodiments, GUS protein has a turnover number, expressed as nanomoles of p-nitrophenyl-β-D-glucuronide converted to p-nitrophenol per minute per μg of purified protein, of at least 50 and more preferably at least 60, at least 70, at least 80 and at least 90 nanomoles measured at its temperature optimum. In other preferred embodiments the turnover number is at least 20, at least 30, or at least 40 nanomoles at room temperature. In yet other preferred embodiments, the β-glucuronidase should not be substantially inhibited by the presence of detergents such as SDS (e.g., 0.1%, 1%, 5%), Triton® X-100 (e.g., 0.1%, 1%, 5%), or sarcosyl (e.g., 0.1%, 1%, 5%). In other preferred embodiments, the GUS enzyme is not substantially inhibited (e.g., less than 50% inhibition and more preferably less than 20% inhibition) is by either at 1 mM or as high as 10 mM glucuronic acid. In still other preferred embodiments, GUS retains substantial activity (at least 50% and preferably at least 70%) in organic solvents, such as dimethylformamide, dimethylsulfoxide and in salt (e.g., NaCl).

In other preferred embodiments, GUS and variants thereof are capable of being secreted and exhibit one or more of the biochemical characteristics disclosed herein. In other embodiments, variants of microbial GUS are capable of binding to a hapten, such as biotin, dinitrophenol, and the like.

In other embodiments, variants may exhibit glucuronide binding activity without enzymatic activity or be directed to other cellular compartments, such as membrane or cytoplasm. Membrane-spanning amino acid sequences are generally hydrophobic and many examples of such sequences are well-known. These sequences may be spliced onto microbial secreted GUS by a variety of methods including conventional recombinant DNA techniques. Similarly, sequences that direct proteins to cytoplasm (e.g., Lys-Asp-Glu-Leu) (SEQ ID No: 30) may be added to the reference GUS, typically by recombinant DNA techniques.

In other embodiments, a fusion protein comprising GUS may be constructed from the nucleic acid molecule encoding microbial and another nucleic acid molecule. As will be appreciated, the fusion partner gene may contribute, within certain embodiments, a coding region. In preferred embodiments, microbial GUS is fused to avidin, streptavidin or an antibody. Thus, it may be desirable to use only the catalytic site of GUS (e.g., amino acids 415-508 reference to Bacillus sequence). The choice of the fusion partner depends in part upon the desired application. The fusion partner may be used to alter specificity of GUS, provide a reporter function, provide a tag sequence for identification or purification protocols, and the like. The reporter or tag can be any protein that allows convenient and sensitive measurement or facilitates isolation of the gene product and does not interfere with the function of GUS. For example, green fluorescent protein and β-galactosidase are readily available as DNA sequences. A peptide tag is a short sequence, usually derived from a native protein, which is recognized by an antibody or other molecule. Peptide tags include FLAG®, Glu-Glu tag (Chiron Corp., Emeryville, Calif.), KT3 tag (Chiron Corp.), T7 gene 10 tag (Invitrogen, La Jolla, Calif.), T7 major capsid protein tag (Novagen, Madison, Wis.), His 6 (hexa-His), and HSV tag (Novagen). Besides tags, other types of proteins or peptides, such as glutathione-S-transferase may be used.

In other aspects of the present invention, isolated microbial glucuronidase proteins are provided. In one embodiment, GUS protein is expressed as a hexa-His fusion protein and isolated by metal-containing chromatography, such as nickel-coupled beads. Briefly, a sequence encoding His 6 is linked to a DNA sequence encoding a GUS. Although the His 6 sequence can be positioned anywhere in the molecule, preferably it is linked at the 3′ end immediately preceding the termination codon. The His-GUS fusion may be constructed by any of a variety of methods. A convenient method is amplification of the GUS gene using a downstream primer that contains the codons for His 6 .

In one aspect of the present invention, peptides having microbial GUS sequence are provided. Peptides may be used as immunogens to raise antibodies, as well as other uses. Peptides are generally five to 100 amino acids long, and more usually 10 to 50 amino acids. Peptides are readily chemically synthesized in an automated fashion (e.g., PerkinElmer, ABI Peptide Synthesizer) or may be obtained commercially. Peptides may be further purified by a variety of methods, including high-performance liquid chromatography (HPLC). Furthermore, peptides and proteins may contain amino acids other than the 20 naturally occurring amino acids or may contain derivatives and modification of the amino acids.

β-glucuronidase protein may be isolated by standard methods, such as affinity chromatography using matrices containing saccharose lactone, phenythio-β-glucuronide, antibodies to GUS protein and the like, size exclusion chromatography, ionic exchange chromatography, HPLC, and other known protein isolation methods. (see generally Ausubel et al. supra; Sambrook et al. supra). The protein can be expressed as a hexa-His fusion protein and isolated by metal-affinity chromatography, such as nickel-coupled beads. An isolated purified protein gives a single band on SDS-PAGE when stained with Coomassie brilliant blue.

Antibodies to Microbial GUS

Antibodies to microbial GUS proteins, fragments, or peptides discussed herein may readily be prepared. Such antibodies may specifically recognize reference microbial GUS protein and not a mutant (or variant) protein, mutant (or variant) protein and not wild type protein, or equally recognize both the mutant (or variant) and wild-type forms. Antibodies may be used for isolation of the protein, inhibiting (antagonist) activity of the protein, or enhancing (agonist) activity of the protein.

Within the context of the present invention, antibodies are understood to include monoclonal antibodies, polyclonal antibodies, anti-idiotypic antibodies, antibody fragments (e.g., Fab, and F(ab′) 2 , F v variable regions, or complementarity determining regions). Antibodies are generally accepted as specific against GUS protein if they bind with a K d of greater than or equal to 10 −7 M, preferably greater than of equal to 10 −8 M. The affinity of a monoclonal antibody or binding partner can be readily determined by one of ordinary skill in the art (see Scatchard, Ann. N.Y. Acad. Sci. 51:660-672, 1949).

Briefly, a polyclonal antibody preparation may be readily generated in a variety of warm-blooded animals such as rabbits, mice, or rats. Typically, an animal is immunized with GUS protein or peptide thereof, which may be conjugated to a carrier protein, such as keyhole limpet hemocyanin. Routes of administration include intraperitoneal, intramuscular, intraocular, or subcutaneous injections, usually in an adjuvant (e.g., Freund's complete or incomplete adjuvant). Particularly preferred polyclonal antisera demonstrate binding in an assay that is at least three times greater than background.

Monoclonal antibodies may also be readily generated from hybridoma cell lines using conventional techniques (see U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; see also Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988). Briefly, within one embodiment, a subject animal such as a rat or mouse is injected with GUS or a portion thereof. The protein may be administered as an emulsion in an adjuvant such as Freund's complete or incomplete adjuvant in order to increase the immune response. Between one and three weeks after the initial immunization the animal is generally boosted and may tested for reactivity to the protein utilizing well-known assays. The spleen and/or lymph nodes are harvested and immortalized. Various immortalization techniques, such as mediated by Epstein-Barr virus or fusion to produce a hybridoma, may be used. In a preferred embodiment, immortalization occurs by fusion with a suitable myeloma cell line (e.g., NS-1 (ATCC No. TIB 18), and P3X63-Ag 8.653 (ATCC No. CRL 1580) to create a hybridoma that secretes monoclonal antibody. The preferred fusion partners do not express endogenous antibody genes. Following fusion, the cells are cultured in medium containing a reagent that selectively allows for the growth of fused spleen and myeloma cells such as HAT (hypoxanthine, aminopterin, and thymidine) and are subsequently screened for the presence of antibodies that are reactive against a GUS protein. A wide variety of assays may be utilized, including for example countercurrent immuno-electrophoresis, radioimmunoassays, radioimmunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot assays, western blots, immunoprecipitation, inhibition or competition assays, and sandwich assays (see U.S. Pat. Nos. 4,376,110 and 4,486,530; see also Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988).

Other techniques may also be utilized to construct monoclonal antibodies (see Huse et al., Science 246:1275-1281, 1989; Sastry et al., Proc. Natl. Acad. Sci. USA 86:5728-5732, 1989; Alting-Mees et al., Strategies in Molecular Biology 3:1-9, 1990; describing recombinant techniques). Briefly, RNA is isolated from a B cell population and utilized to create heavy and light chain immunoglobulin cDNA expression libraries in suitable vectors, such as λImmunoZap(H) and λImmunoZap(L). These vectors may be screened individually or co-expressed to form Fab fragments or antibodies (see Huse et al., supra; Sastry et al., supra). Positive plaques may subsequently be converted to a non-lytic plasmid that allows high level expression of monoclonal antibody fragments from E. coli.

Similarly, portions or fragments, such as Fab and Fv fragments, of antibodies may also be constructed utilizing conventional enzymatic digestion or recombinant DNA techniques to yield isolated variable regions of an antibody. Within one embodiment, the genes which encode the variable region from a hybridoma producing a monoclonal antibody of interest are amplified using nucleotide primers for the variable region. These primers may be synthesized by one of ordinary skill in the art, or may be purchased from commercially available sources (e.g., Stratacyte, La Jolla, Calif.) Amplification products are inserted into vectors such as ImmunoZAP™ H or ImmunoZAP™ L (Stratacyte), which are then introduced into E. coli, yeast, or mammalian-based systems for expression. Utilizing these techniques, large amounts of a single-chain protein containing a fusion of the V H and V L domains may be produced (see Bird et al., Science 242:423-426, 1988). In addition, techniques may be utilized to change a “murine” antibody to a “human” antibody, without altering the binding specificity of the antibody.

One of ordinary skill in the art will appreciate that a variety of alternative techniques for generating antibodies exist. In this regard, the following U.S. patents teach a variety of these methodologies and are thus incorporated herein by reference: U.S. Pat. Nos. 5,840,479; 5,770,380; 5,204,244; 5,482,856; 5,849,288; 5,780,225; 5,395,750; 5,225,539; 5,110,833; 5,693,762; 5,693,761; 5,693,762; 5,698,435; and 5,328,834.

Once suitable antibodies have been obtained, they may be isolated or purified by many techniques well known to those of ordinary skill in the art (see Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988). Suitable techniques include peptide or protein affinity columns, HPLC (e.g., reversed phase, size exclusion, ion-exchange), purification on protein A or protein G columns, or any combination of these techniques.

Assays for Function of β-glucuronidase

In preferred embodiments, microbial β-glucuronidase will have at least enzymatic activity and in other preferred embodiments, will also have the capability of being secreted. As noted above, variants of these reference GUS proteins may exhibit altered functional activity and cellular localization. Enzymatic activity may be assessed by an assay such as the ones disclosed herein or in U.S. Pat. No. 5,268,463 (Jefferson). Generally, a chromogenic or fluorogenic substrate is incubated with cell extracts, tissue sections, or purified protein. Cleavage of the substrate is monitored by a method appropriate for the aglycone.

A variety of methods may be used to demonstrate that a β-glucuronidase is secreted. For example, a rapid screening method in which colonies of organisms or cells, such as bacteria, yeast or insect cells, are plated and incubated with a readily visualized glucuronide substrate, such as X-GlcA. A colony with a diffuse staining pattern likely secretes GUS, although such a pattern could indicate that the cell has the ability to pump out the cleaved glucuronide, that the cell has become leaky, or that the enzyme is membrane bound. When test cells express GUS from an introduced vector, a cell that is known to not pump out cleaved substrate and is deleted for endogenous GUS genes is preferably used.

Secretion of the enzyme may be verified by assaying for GUS activity in the extracellular environment. If the cells secreting GUS are gram-positive bacteria, yeasts, molds, plants, or other organisms with cell walls, activity may be assayed in the culture medium and in a cell extract, however, the protein may not be transported through the cell wall. Thus, if no or low activity of a secreted form of GUS is found in the culture medium, protoplasts can be made by osmotic shock or enzymatic digestion of the cell wall or other suitable procedure, and the supernatant assayed for GUS activity. If the cells secreting GUS are gram-negative bacteria, culture supernatant may be tested, but more likely β-glucuronidase will be retained in the periplasmic space between the inner and outer membrane. In this case, spheroplasts may be made by osmotic shock, enzymatic digestion, or other suitable procedure, and the supernatant assayed for GUS activity. Cells without cell walls may be assayed for GUS in cell supernatant and cell extracts. The fraction of activity in each compartment is compared to to the activity of a non-secreted GUS in the same or similar host cells. A β-glucuronidase is secreted if significantly more enzyme activity than E. coli GUS activity is found in extracellular spaces. The amount of secretion is generally normalized to the amount of a non-secreted protein found in extracellular spaces. Less than 10% of E. coli GUS is secreted. Higher amounts of secreted enzyme are preferred (e.g., greater than 20%, 25%, 30%, 40%, 50%).

β-glucuronidases that exhibit specific substrate specificities are also useful within the context of the present invention. As noted above, glucuronides can be linked through an oxygen, carbon, nitrogen or sulfur atom. Glucuronide substrates having each of the linkages may be used in one of the assays described herein. In addition, various glucuronides containing a variety of aglycones may be used.

Common glucuronides include:

Phenyl-β-glucuronide

Phenyl β-D-thio-glucuronide

p-Nitrophenyl-β-glucuronide

4-Methylumbelliferyl-β-glucuronide

p-Aminophenyl-β-D-glucuronide

p-Aminophenyl-1-thio-β-D-glucuronide

Chloramphenicol β-D-glucuronide

8-Hydroxyquinoline β-D-glucuronide

5-Bromo-4-chloro-3-indolyl-β-D-glucuronide (X-GlcA)

5-Bromo-6-chloro-3-indolyI-β-D-glucuronide (Magenta-GlcA)

6-Chloro-3-indolyl-β-D-glucuronide (Salmon-β-D-GlcA)

Indoxyl-β-D-glucuronide (Y-GlcA)

Androsterone-3-β-D-glucuronide

α-Naphthyl-β-D-glucuronide

Estriol-3-β-D-glucuronide

17-β-Estradiol-3-β-D-glucuronide

Estrone-3-β-D-glucuronide

Testosterone-17-β-D-glucuronide

19-nor-Testosterone-17-β-D-glucuronide

Tetrahydrocortisone-3-β-D-glucuronide

Phenolphthalein-β-D-glucuronide

3′-Azido-3′-deoxythymidine-β-D-glucuronide

Methyl-β-D-glucuronide

Morphine-6-β-D-glucuronide

Vectors, Host Cells and Means of Expressing and Producing Protein

Microbial β-glucuronidase may be expressed in a variety of host organisms. For protein production and purification, GUS is preferably secreted and produced in bacteria, such as E. coli, for which many expression vectors have been developed and are available. Other suitable host organisms include other bacterial species (e.g., Bacillus, and eukaryotes, such as yeast (e.g., Saccharomyces cerevisiae ), mammalian cells (e.g., CHO and COS-7), plant cells and insect cells (e.g., Sf9). Vectors for these hosts are well known.

A DNA sequence encoding microbial β-glucuronidase is introduced into an expression vector appropriate for the host. The sequence is derived from an existing clone or synthesized. As described herein, a fragment of the coding region may be used, but if enzyme activity is desired, the catalytic region should be included. A preferred means of synthesis is amplification of the gene from cDNA, genomic DNA, or a recombinant clone using a set of primers that flank the coding region or the desired portion of the protein. Restriction sites are typically incorporated into the primer sequences and are chosen with regard to the cloning site of the vector. If necessary, translational initiation and termination codons can be engineered into the primer sequences. The sequence of GUS can be codon-optimized for expression in a particular host. For example, a secreted form of β-glucuronidase isolated from a bacterial species that is expressed in a fungal host, such as yeast, can be altered in nucleotide sequence to use codons preferred in yeast. Codon-optimization may be accomplished by methods such as splice overlap extension, site-directed mutagenesis, automated synthesis, and the like.

At minimum, the vector must contain a promoter sequence. Other regulatory sequences may be included. Such sequences include a transcription termination signal sequence, secretion signal sequence, origin of replication, selectable marker, and the like. The regulatory sequences are operationally associated with one another to allow transcription or translation.

Expression in Bacteria

The plasmids used herein for expression of secreted GUS include a promoter designed for expression of the proteins in a bacterial host. Suitable promoters are widely available and are well known in the art. Inducible or constitutive promoters are preferred. Such promoters for expression in bacteria include promoters from the T7 phage and other phages, such as T3, T5, and SP6, and the trp, lpp, and lac operons. Hybrid promoters (see, U.S. Pat. No. 4,551,433), such as tac and trc, may also be used. Promoters for expression in eukaryotic cells include the P10 or polyhedron gene promoter of baculovirus/insect cell expression systems (see, e.g., U.S. Pat. Nos. 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784), MMTV LTR, RSV LTR, SV40, metallothionein promoter (see, e.g., U.S. Pat. No. 4,870,009) and other inducible promoters. For expression of the proteins, a promoter is inserted in operative linkage with the coding region for β-glucuronidase.

The promoter controlling transcription of β-glucuronidase may be controlled by a repressor. In some systems, the promoter can be derepressed by altering the physiological conditions of the cell, for example, by the addition of a molecule that competitively binds the repressor, or by altering the temperature of the growth media. Preferred repressor proteins include, but are not limited to the E. coli lacI repressor responsive to IPTG induction, the temperature sensitive λcI857 repressor, and the like. The E. coli lacI repressor is preferred.

In other preferred embodiments, the vector also includes a transcription terminator sequence. A “transcription terminator region” has either a sequence that provides a signal that terminates transcription by the polymerase that recognizes the selected promoter and/or a signal sequence for polyadenylation.

Preferably, the vector is capable of replication in bacterial cells. Thus, the vector preferably contains a bacterial origin of replication. Preferred bacterial origins of replication include the f1-ori and col E1 origins of replication, especially the ori derived from pUC plasmids.

The plasmids also preferably include at least one selectable marker that is functional in the host. A selectable marker gene includes any gene that confers a phenotype on the host that allows transformed cells to be identified and selectively grown. Suitable selectable marker genes for bacterial hosts include the ampicillin resistance gene (Amp r ), tetracycline resistance gene (Tc r ) and the kanamycin resistance gene (Kan r ). Suitable markers for eukaryotes usually require a complementary deficiency in the host (e.g., thymidine kinase (tk) in tk- hosts). However, drug markers are also available (e.g., G418 resistance and hygromycin resistance).

The sequence of nucleotides encoding β-glucuronidase may also include a classical secretion signal, whereby the resulting peptide is a precursor protein processed and secreted. The resulting processed protein may be recovered from the periplasmic space or the fermentation medium. Secretion signals suitable for use are widely available and are well known in the art (von Heijne, J. Mol. Biol. 184:99-105, 1985). Prokaryotic and eukaryotic secretion signals that are functional in E. coli (or other host) may be employed. The presently preferred secretion signals include, but are not limited to pelB, matα, extensin and glycine-rich protein.

One skilled in the art appreciates that there are a wide variety of suitable vectors for expression in bacterial cells and which are readily obtainable. Vectors such as the pET series (Novagen, Madison, Wis.) and the tac and trc series (Pharmacia, Uppsala, Sweden) are suitable for expression of a β-glucuronidase. A suitable plasmid is ampicillin resistant, has a colEI origin of replication, lacI q gene, a lac/trp hybrid promoter in front of the lac Shine-Dalgarno sequence, a hexa-his coding sequence that joins to the 3′ end of the inserted gene, and an rrnB terminator sequence.

The choice of a bacterial host for the expression of a β-glucuronidase is dictated in part by the vector. Commercially available vectors are paired with suitable hosts. The vector is introduced in bacterial cells by standard methodology. Typically, bacterial cells are treated to allow uptake of DNA (for protocols, see generally, Ausubel et al., supra; Sambrook et al., supra). Alternatively, the vector may be introduced by electroporation, phage infection, or another suitable method.

Expression in Plant Cells

As noted above, the present invention provides vectors capable of expressing microbial secreted β-glucuronidase and secreted microbial β-glucuronidases. For agricultural applications, the vectors should be functional in plant cells. Vectors and procedures for cloning and expression in E. coli and animal cells are discussed herein and, for example, in Sambrook et al (supra) and in Ausubel et al (supra). Suitable plants include, but are not limited to, wheat, rice, corn, soybeans, lupins, vegetables, potatoes, canola, nut trees, coffee, alfalfa and other forage plants, cereals, legumes and the like. In one preferred embodiment, rice is a host for GUS gene expression.

Vectors that are functional in plants are preferably binary plasmids derived from Agrobacterium plasmids. Such vectors are capable of transforming plant cells. These vectors contain left and right border sequences that are required for integration into the host (plant) chromosome. At minimum, between these border sequences is the gene to be expressed under control of a promoter. In preferred embodiments, a selectable marker and a reporter gene are also included. The vector also preferably contains a bacterial origin of replication.

A gene for microbial β-glucuronidase should be in operative linkage with a promoter that is functional in a plant cell. Typically, the promoter is derived from a host plant gene, but promoters from other plant species and other organisms, such as insects, fungi, viruses, mammnals, and the like, may also be suitable, and at times preferred. The promoter may be constitutive or inducible, or may be active in a certain tissue or tissues (tissue type-specific promoter), in a certain cell or cells (cell-type specific promoter), of at a particular stage or stages of development (development-type specific promoter). The choice of a promoter depends at least in part upon the application. Many promoters have been identified and isolated (see, generally, GenBank and EMBL databases). Other promoters may be isolated by well-known methods. For example, a genomic clone for a particular gene can be isolated by probe hybridization. The coding region is mapped by restriction mapping, DNA sequence analysis, RNase probe protection, or other suitable method. The genomic region immediately upstream of the coding region comprises a promoter region and is isolated. Generally, the promoter region is located in the first 200 bases upstream, but may extend to 500 or more bases. The candidate region is inserted in a suitable vector in operative linkage with a reporter gene, such as in pBII21 in place of the CaMV 35S promoter, and the promoter is tested by assaying for the reporter gene after transformation into a plant cell. (see, generally, Ausubel et al., supra; Sambrook et al., supra; Methods in Plant Molecular Biology and Biotechnology, Ed. Glick and Thompson, CRC Press, 1993.)

Preferably, the vector contains a selectable marker for identifying transformants. The selectable marker preferably confers a growth advantage under appropriate conditions. Generally, selectable markers are drug resistance genes, such as neomycin phosphotransferase. Other drug resistance genes are known to those in the art and may be readily substituted. Selectable markers for bacteria include, ampicillin resistance, tetracycline resistance, kanamycin resistance, chloramphenicol resistance, and the like. The selectable marker also preferably has a linked constitutive or inducible promoter and a termination sequence, including a polyadenylation signal sequence.

Additionally, a bacterial origin of replication and a selectable marker for bacteria are preferably included in the vector. Of the various origins (e.g., colEI, fd phage), a colEI origin of replication is preferred. Most preferred is the origin from the pUC plasmids, which allow high copy number.

The sequence of nucleotides encoding β-glucuronidase may also include a classical secretion signal, whereby the resulting peptide is a precursor protein processed and secreted. Suitable signal sequences of plant genes include, but are not limited to the signal sequences from glycine-rich protein and extensin. In addition, a glucuronide permease gene may be co-transfected either from the same vector containing microbial GUS or from a separate expression vector.

A general vector suitable for use in the present invention is based on pBI121 (U.S. Pat. No. 5,432,081) a derivative of pBIN19. Other vectors have been described (U.S. Pat. No. 4,536,475) or may be constructed based on the guidelines presented herein. The plasmid pBII21 contains a left and right border sequence for integration into a plant host chromosome and also contains a bacterial origin of replication and selectable marker. These border sequences flank two genes. One is a kanamycin resistance gene (neomycin phosphotransferase) driven by a nopaline synthase promoter and using a nopaline synthase polyadenylation site. The second is the E. coli GUS gene (reporter gene) under control of the CaMV 35S promoter and polyadenlyated using a nopaline synthase polyadenylation site. The E. coli GUS gene is replaced with a gene encoding a secreted form of β-glucuronidase. If appropriate, the CaMV 35S promoter is replaced by a different promoter. Either one of the expression units described above is additionally inserted or is inserted in place of the CaMV promoter and GUS gene.

Plants may be transformed by any of several methods. For example, plasmid DNA may be introduced by Agrobacterium co-cultivation or bombardment. Other transformation methods include electroporation, CaPO 4 -mediated transfection, gene transfer to protoplasts, microinjection, and the like (see, Gene Transfer to Plants, Ed. Potrykus and Spangenberg, Springer, 1995, for procedures). Preferably, vector DNA is first transfected into Agrobacterium and subsequently introduced into plant cells. Most preferably, the infection is achieved by co-cultivation. In part, the choice of transformation methods depends upon the plant to be transformed. For example, monocots may be refractory to transformation by Agrobacterium. Tissues can alternatively be efficiently infected by Agrobacterium utilizing a projectile or bombardment method. Projectile methods are generally used for transforming sunflowers and soybean. Bombardment is used when naked DNA, typically Agrobacterium binary plasmids or pUC-based plasmids, is used for transformation or transient expression.

Briefly, co-cultivation is performed by first transforming Agrobacterium by freeze-thaw method (Holsters et al., Mol. Gen. Genet. 163: 181-187, 1978) or by other suitable methods (see, Ausubel, et al. supra; Sambrook et al., supra). A culture of Agrobacterium containing the plasmid is incubated with leaf disks, protoplasts or meristematic tissue to generate transformed plants (Bevan, Nucl. Acids. Res. 12:8711, 1984).

Briefly, for microprojectile bombardment, seeds are surface sterilized in bleach solution and rinsed with distilled water. Seeds are then imbibed in distilled water, and the cotyledons are broken off to produce a clean fracture at the plane of the embryonic axis. Explants are then bisected longitudinally between the primordial leaves and placed cut surface up on medium with growth regulating hormones, minerals and vitamin additives. Explants from other tissues or methods of preparation may alternatively be used. Explants are bombarded with gold or tungsten microprojectiles by a particle acceleration device. Freshly bombarded explants are placed in a suspension of transformed Agrobacterium transferred to medium with the cut surfaces down for 3 days with an 18 hr light cycle. Explants are transferred to medium lacking growth regulators but containing drug for selection and grown for 2-5 weeks. A positive selection system, such as using cellobiuronic acid and culture medium lacking a carbon source, is preferably used (see, co-pending application Ser. No. 09/130,695 now U.S. Pat No. 6,268,493). After 1-2 weeks more without drug selection, leaf samples from green, drug-resistant shoots are grafted to in vitro grown rootstock and transferred to soil.

Activity of secreted GUS is assayed in whole plants or in selected tissues using a glucuronide substrate that is readily detected upon cleavage. Glucuronide substrates that are calorimetric are preferred. Field testing of plants may be performed by spraying a plant with the glucuronide substrate and observing color formation of the cleaved product.

Expression in Other Organisms

A variety of other organisms are suitable for use in the present invention. For example, various fungi, including yeasts, molds, and mushrooms, insects, especially vectors for diseases and pathogens, and other animals, such as cows, mice, goats, birds, aquatic animals (e.g., shrimp, turtles, fish, lobster and other crustaceans), amphibians and reptiles and the like, may be transformed with a GUS transgene.

The principles that guide vector construction for bacteria and plants, as discussed above, are applicable to vectors for these organisms. In general, vectors are well known and readily available. Briefly, the vector should have at least a promoter functional in the host in operative linkage with GUS. Usually, the vector will also have one or more selectable markers, an origin of replication, a polyadenylation signal and transcription terminator.

The sequence of nucleotides encoding β-glucuronidase may also include a classical secretion signal, whereby the resulting peptide is a precursor protein processed and secreted. Suitable secretion signals may be obtained from a variety of genes, such as mat-alpha or invertase genes. In addition, a permease gene may be co-transfected.

One of ordinary skill in the art will appreciate that a variety of techniques for producing transgenic animals exist. In this regard, the following U.S. patents teach such methodologies and are thus incorporated herein by reference: U.S. Pat. Nos. 5,162,215; 5,545,808; 5,741,957; 4,873,191; 5,780,009; 4,736,866; 5,567,607; and 5,633,076.

Uses of Microbial β-glucuronidase

As noted above, microbial β-glucuronidase may be used in a variety of applications. In general, microbial β-glucuronidase can be used as a reporter/effector molecule and as a diagnostic tool. As taught herein, microbial β-glucuronidase that is secretable is preferred as an in vivo reporter/effector molecule, whereas, in in vitro diagnostic applications, the biochemical characteristics of the β-glucuronidase disclosed herein (e.g., thermal stability, high turnover number) may provide preferred advantages.

Microbial GUS, either secreted or non-secreted, can be used as a marker for transgenic constructions. In a certain embodiments, the transgenic host is a plant, such as rice, corn, wheat, or an aquatic animal. The transgenic GUS may be used in at least three ways: one in a method of positive selection, obviating the need for drug resistance selection, a second as a system to target molecules to specific cells, and a third as a means of detecting and tracking linked genes.

For positive selection, a host cell, (e.g., plant cells) is transformed with a GUS (preferably secretable GUS) transgene. Selection is achieved by providing the cells with a glucuronidated form of a required nutrient. For example, all cells require a carbon source, such as glucose. In one embodiment, glucose is provided as glucuronyl glucose (cellobiuronic acid), which is cleaved by GUS into glucose plus glucuronic acid. The glucose would then bind to receptors and be taken up by cells. The glucuronide may be any required compound, including without limitation, a cytokinin, auxin, vitamin, carbohydrate, nitrogen-containing compound, and the like. It will be appreciated that this positive selection method can be used for cells and tissues derived from diverse organisms, such as animal cells, insect cells, fungi, and the like. The choice of glucuronide will depend in part upon the requirements of the host cell.

As a marker/effector molecule, secreted GUS (s-GUS) is preferred because it is non-destructive, that is, the host does not need to be destroyed in order to assay enzyme activity. A non-destructive marker has special utility as a tool in plant breeding. The GUS enzyme can be used to detect and track linked endogenous or exogenously introduced genes. GUS may also be used to generate sentinel plants that serve as bioindicators of environmental status. Plant pathogen invasion can be monitored if GUS is under control of a pathogen promoter. In addition, such transgenic plants may serve as a model system for screening inhibitors of pathogen invasion. In this system, GUS is expressed if a pathogen invades. In the presence of an effective inhibitor, GUS activity will not be detectable. In certain embodiments, GUS is co-transfected with a gene encoding a glucuronide permease.

Preferred transgenes for introduction into plants encode proteins that affect fertility, including male sterility, female fecundity, and apomixis; plant protection genes, including proteins that confer resistance to diseases, bacteria, fungus, nematodes, viruses and insects; genes and proteins that affect developmental processes or confer new phenotypes, such as genes that control meristem development, timing of flowering, and the like.

Insect and disease resistance genes are well known. Some of these genes are present in the genome of plants and have been genetically identified. Others of these genes have been found in bacteria and are used to confer resistance.

Particularly well known insect resistance genes are the crystal genes of Bacillus thuringiensis. The crystal genes are active against various insects, such as lepidopterans, Diptera, Hemiptera and Coleoptera. Many of these genes have been cloned. For examples, see, GenBank Accession Nos. X96682, X96684; M76442, M90843, M89794, M22472, M37207, D17518, L32019, M97880, L32020, M64478, M11250, M13201, D00117, M73319, X17123, X86902, X06711, X13535, X54939, X54159, X13233, X54160, X56144, X58534, X59797, X75019, X62821, Z46442, U07642, U35780, U43605, U43606, U10985; U.S. Pat. Nos. 5,317,096; 5,254,799; 5,460,963; 5,308,760, 5,466,597, 5,2187,091, 5,382,429, 5,164,180, 5,206,166, 5,407,825, 4,918,066; PCT Applications WO 95/30753, WO 94/24264; AU 9062083; EP 408403 B1, EP 142924 B1, EP 256,553 B1, EP 192,741 B1; JP 62-56932; . Gene sequences for these and related proteins may be obtained by standard and routine technologies, such as probe hybridization of a B. thuringiensis library or amplification (see generally, Sambrook et al., supra, Ausubel et al. supra). The probes and primers may be synthesized based on publicly available sequence information.

Other resistance genes to Sclerotinia, cyst nematodes, tobacco mosaic virus, flax and crown rust, rice blast, powdery mildew, verticillum wilt, potato beetle, aphids, as well as other infections, are useful within the context of this invention. Examples of such disease resistance genes may be isolated from teachings in the following references: isolation of rust disease resistance gene from flax plants (WO 95/29238); isolation of the gene encoding Rps2 protein from Arabidopsis thaliana that confers disease resistance to pathogens carrying the avrRpt2 avirulence gene (WO 95/28478); isolation of a gene encoding a lectin-like protein of kidney bean confers insect resistance (JP 71-32092); isolation of the Hm1 disease resistance gene to C. carbonum from maize (WO 95/07989); for examples of other resistance genes, see WO 95/05743; U.S. Pat. Nos. 5,496,732; 5,349,126; EP 616035; EP 392225; WO 94/18335; JP 43-20631; EP 502719; WO 90/11770; U.S. Pat. Nos. 5,270,200; 5,218,104 and 5,306,863). In addition, general methods for identification and isolation of plant disease resistance genes are disclosed (WO 95/28423). Any of these gene sequences suitable for insertion in a vector according to the present invention may be obtained by standard recombinant technology techniques, such as probe hybridization or amplification. When amplification is performed, restriction sites suitable for cloning are preferably inserted. Nucleotide sequences for other transgenes, such as controlling male fertility, are found in U.S. Pat. No. 5,478,369, references therein, and Mariani et al., Nature 347:737, 1990.

In similar fashion, microbial GUS, preferably secreted, can be used to generate transgenic insects for tracking insect populations or facilitate the development of a bioassay for compounds that affect molecules critical for insect development (e.g., juvenile hormone). Secreted GUS may also serve as a marker for beneficial fungi destined for release into the environment. The non-destructive marker is useful for detecting persistence and competitive advantage of the released organisms.

In animal systems, secreted GUS may be used to achieve extracellular detoxification of glucuronides (e.g, toxin glucuronide) and examine conjugation patterns of glucuronides. Furthermore, as discussed above, secreted GUS may be used as a transgenic marker to track cells or as a positive selection system, or to assist in development of new bioactive GUS substrates that do not need to be transported across membrane. Aquatic animals are suitable hosts for GUS transgene. GUS may be used in these animals as a marker or effector molecule.

Within the context of this invention, GUS may also be used in a system to target molecules to cells. This system is particularly useful when the molecules are hydrophobic and thus, not readily delivered. These molecules can be useful as effectors (e.g., inducers) of responsive promoters. For example, molecules such as ecdysone are hydrophobic and not readily transported through phloem in plants. When ecdysone is glucuronidated it becomes amphipathic and can be delivered to cells by way of phloem. Targeting of compounds such as ecdysone-glucuronic acid to cells is accomplished by causing cells to express receptor for ecdysone. As ecdysone receptor is naturally only expressed in insect cells, however a host cell that is transgenic for ecdysone receptor will express it. The glucuronide containing ecdysone then binds only to cells expressing the receptor. If these cells also express GUS, ecdysone will be released from the glucuronide and able to induce expression from an ecdysone-responsive promoter. Plasmids containing ecdysone receptor genes and ecdysone responsive promoter can be obtained from Invitrogen (Carlsbad, Calif.). Other ligand-receptors suitable for use in this system include glucocorticoids/glucocorticoid receptor, estrogen/estrogen receptor and the like (see also U.S. Pat. Nos. 5,693,769 and 5,612,317).

In another aspect, purified microbial β-glucuronidase is used in medical applications. For these applications, secretion is not a necessary characteristic although it may be desirable a characteristic for production and purification. The biochemical attributes, such as increased stability and enzymatic activity disclosed herein are preferred characteristics. The microbial glucuronidase preferably has one or more of the disclosed characteristics.

For the majority of drug or pharmaceutical analysis, the compounds in urine, blood, saliva, or other bodily fluids are de-glucuronidated prior to analysis. Such a procedure is undertaken because compounds are often, if not nearly always, detoxified by glucuronidation in vertebrates. Thus, drugs that are in circulation and have passed through a site of glucuronidation (e.g., liver) are found conjugated to glucuronic acid. Such glucuronides yield a complex pattern upon analysis by, for example, HPLC. However, after the aglycone (drug) is cleaved from the glucuronic acid, a spectrum can be compared to a reference spectrum. Currently, E. coli GUS is utilized, but as shown herein, microbial GUS, e.g. Bacillus GUS has superior qualities.

The microbial GUS enzymes disclosed herein may be used in traditional medical diagnostic assays, such as described above for drug testing, pharmacokinetic studies, bioavailability studies, diagnosis of diseases and syndromes, following progression of disease or its response to therapy and the like (see U.S. Pat. Nos. 5,854,009, 4,450,239, 4,274,832, 4,473,640, 5,726,031, 4,939,264, 4,115,064, 4,892,833). These β-glucuronidase enzymes may be used in place of other traditional enzymes (e.g., alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like) and compounds (e.g., green fluorescent protein, radionuclides) that serve as visualizing agents. Microbial GUS has qualities advantageous for use as a visualizing agent: it is highly specific for the substrate, water soluble and the substrates are stable. Thus, microbial GUS is suitable for use in Southern analysis of DNA, Northern analysis, ELISA, and the like.

In preferred embodiments, microbial GUS binds a hapten, either as a fusion protein with a partner protein that binds the hapten (e.g., avidin that binds biotin, antibody) or alone. If used alone, microbial GUS can be mutagenized and selected for hapten-binding abilities. Mutagenesis and binding assays are well known in the art. In addition, microbial GUS can be conjugated to avidin, streptavidin, antibody or other hapten binding protein and used as a reporter in the myriad assays that currently employ enzyme-linked binding proteins. Such assays include immunoassays, Western blots, in situ hybridizations, HPLC, high-throughput binding assays, and the like (see, for examples, U.S. Pat. Nos. 5,328,985 and 4,839,293, which teach avidin and streptavidin fusion proteins and U.S. Pat. No. 4,298,685, Diamandis and Christopoulos, Clin. Chem. 37:625, 1991; Richards, Methods Enzymol. 184:3, 1990; Wilchek and Bayer, Methods Enzymol. 184:467, 1990; Wilchek and Bayer, Methods Enzymol. 184:5, 1990; Wilchek and Bayer, Methods Enzymol. 184:14, 1990; Dunn, Methods Mol. Biol 32:227, 1994; Bloch, J. Hitochem. Cytochem. 41:1751, 1993; Bayer and Wilchek J. Chromatogr. 510:3, 1990, which teach various applications of enzyme-linked technologies and methods).

Microbial GUSes can also be used in therapeutic methods. By glucuronidating compounds such as drugs, the compound is inactivated. When a glucuronidase is expressed or targeted to the site for delivery, the glucuronide is cleaved and the compound delivered. For these purposes, GUS may be expressed as a transgene or delivered, for example, coupled to an antibody specific for the target cell (see e.g., U.S. Pat. Nos. 5,075,340, 4,584,368, 4,481,195, 4,478,936, 5,760,008, 5,639,737, 4,588,686).

The present invention also provides kits comprising microbial GUS protein or expression vectors containing microbial GUS gene. One exemplary type of kit is a dipstick test. Such tests are widely utilized for establishing pregnancy, as well as other conditions. Generally, these dipstick tests assay the glucuronide form, but it would be advantageous to use reagents that detect the aglycone form. Thus, GUS may be immobilized on the dipstick adjacent to or mixed in with the detector molecule (e.g., antibody). The dipstick is then dipped in the test fluid (e.g., urine) and as the compounds flow past GUS, they are cleaved into aglycone and glucuronic acid. The aglycone is then detected. Such a setup may be extremely useful for testing compounds that are not readily detectable as glucuronides.

In a variation of this method, the microbial GUS enzyme is engineered to bind a glucuronide but lacks enzymatic activity. The enzyme will then bind the glucuronide and the enzyme is detected by standard methodology. Alternatively, GUS is fused to a second protein, either as a fusion protein or as a chemical conjugate, that binds the aglycone. The fusion is incubated with the test substance and an indicator substrate is added. This procedure may be used for ELISA, Northern, Southern analysis and the like.

The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES

Example 1

Identification of Microbes that Express β-Glucuronidase

Skin microbes are obtained using cotton swabs immersed in 0.1% Triton® X-100 and rubbing individual arm pits or by dripping the solution directly into arm pits and recovering it with a pipette. Seven individuals are sampled. Dilutions (1:100, 1:1000) of arm pit swabs are plated on 0.1× and 0.5×TSB (Tryptone Soy Broth, Difco) agar containing 50 μg/mL X-GlcA (5-bromo-4-chloro-3-indolyl β-D-glucuronide), an indicator substrate for β-glucuronidase. This substrate gives a blue precipitate at the site of enzyme activity (see U.S. Pat. No. 5,268,463). TSB is a rich medium which promotes growth of a wide range of microorganisms. Plates are incubated at 37° C.

Soil samples (ca. 1 g) are obtained from an area in Canberra, ACT, Australia (10 samples) and from Queanbeyan, NSW, Australia (12 samples). Although only one of the ten samples from Canberra is intentionally taken from an area of pigeon excrement, most isolates displaying β-glucuronidase activity are in the genera Enterobacter or Salmonella. Soil samples are shaken in 1-2 mL of water; dilutions of the supernatant are treated as for skin samples, except that incubation is at 30° C. and 1.0×TSB plates are used rather than diluted TSB. Some bacteria lose vitality if maintained on diluted medium, although the use of full-strength TSB usually delays, but does not prevent, the onset of indigo from X-GlcA hydrolysis.

Microbes that secrete β-glucuronidase have a strong, diffuse staining pattern (halo) surrounding the colony. The appearance of blue colonies varies in time, from one to several days. Under these conditions (aerobic atmosphere and rich medium) many microorganisms grow. Of these, approximately 0.1-1% display β-glucuronidase phenotype, with the secretory phenotype being less common than the non-secretory phenotype.

Colonies that exhibit a strong, diffuse staining pattern are selected for further purification, which consists of two or more restreaking of those colonies. Occasionally segregation of color production can be observed after the purification procedure. In Table 1 below, a summary of the findings is presented. Some strains are listed as GUS secretion-negative because a later repetition of the halo test was negative, showing that the phenotype can vary, possibly because of growth conditions.

Phylogenetic Analysis

For phylogenetic identification of the microbes, a variable region of 16S rDNA is amplified using primers, P3-16SrDNA and P4-16SrDNA (see Table 2), derived from two conserved regions within stem-loop structures of the rRNA. The amplified region corresponds to nucleotides 361 to 705 of E. coli rRNA, including the primers. Amplification conditions for 16S rDNA are 94° C. for 2 min; followed by 35 cycles of 94° C. for 20 sec, 48° C. for 40 sec, 72° C. for 1.5 min; followed by incubation at 72° C. for 5 min.

Amplified fragments are separated by electrophoresis on TAE agarose gels (approximately 1.2%), excised and extracted by freeze-fracture and phenol treatment. Fragments are further purified using Qiagen (Clifton Hill, Vic, Australia) silica-based membranes in microcentrifuge tubes. Purified DNA fragments are sequenced using the amplification primers in combination with BigDye™ Primer Cycle Sequencing Kit from Perkin-Elmer ABI (fluorescent dye termal cycling sequencing) (Foster City, Calif.). Cycling conditions for DNA sequence reactions are: 2 min at 94° C., followed by 30 cycles of 94° C. for 30 sec, 50° C. for 15 sec, and 60° C. for 2 min. A 10 μL reaction uses 4 μL of BigDye™ Terminator mix, 1 μL of 10 μM primer, and 200-500 ng of DNA. The reaction products are precipitated with ethanol or iso-propanol, resuspended and subjected to gel separation and nucleotide analysis.

The ribosomal sequences are aligned and assigned to phylogenetic placement using the facilities of the Ribosomal Database Project of Michigan State University version 7.1, which contains more than 10,000 16S rRNA sequences (Maidak et al., Nucl. Acids Res. 27:171-173; 1999), Maidak et al., Nucleic Acids Res. 28:173-174, 2000. Phylogenetic placement is used to select strains for further study.

	GUS	GUS
Strain	Secretion	Amplif.	Genus
Skin
EH2	+	Yes	Staphylococcus warneri
EH4	+	Yes	Staphylococcus warneri
EH4-110A	−	Yes	Staphylococcus warneri
LS-B	+	Yes	Staphylococcus haemolyticus/homini
PG-3A	+	No	Staphylococcus homini/warneri
SH1B	+	No	Staphylococcus warneri/aureus
SH1C	+	Yes	Staphylococcus warneri/aureus
CRA1	+	No	Staphylococcus warneri
CRA2	+	No	Staphylococcus warneri

As can be observed from the table above, all GUS expressing skin isolates belong to the genus Staphylococcus and to a limited number of species, Staphlococcus warneri and Staphlococcus homini or haemolyticus. The Canberra soil samples all belonged to the genera Salmonella or Enterobacter/Salmonella. In contrast, a higher degree of microbial diversity was found in the Queanbeyan strains.

The presence of GUS genes is established by amplification using degenerate oligonucleotides derived from a conserved region of the GUS gene. A pair of oligonucleotides is designed-using an alignment of E. coli gusA and human GUS sequences. The primer T3-GUS-2F covers E. coli GUS amino acids 163-168 (DFFNYA) (SEQ ID No: 31), while T7-GUS-5B covers the complementary sequence to amino acids 549-153 (WNFAD) (SEQ ID No: 31). The full length of E. coli GUS is 603 amino acids. As shown in Table 1, amplification is not always successful, likely due to mismatching of the primers with template. Thus, a negative amplification does not necessarily signify that the microorganism lacks a GUS gene.

Example 2

Cloning of GUS Genes by Genetic Complementation

Genomic DNA of several candidate strains is isolated and digested with one of the following enzymes, EcoR I, BamH I, Hind III, Pst I. Digested DNA fragments are ligated into the corresponding site of plasmid vector pBluescript II SK (+), and the ligation mix is electroporated into E. coli KW1, which is a strain deleted for the complete GUS operon. Colonies are plated on LB-X-GlcA plates and assayed for blue color. Halo formation is not used as a criterium, because behavior of the GUS gene in a different genetic background is unknown. In general though, halo formation is obtained in KW1.

Isolated plasmids from GUS+transformants are retransformed into KW1 and also into DH5α to demonstrate that the GUS gene is contained within the construct. In all cases, retransformant colonies stained blue with X-GlcA.

Example 3

DNA Sequence Analysis of GUS Genes Isolated by Complementation

DNA sequence is determined for the isolates that amplified from the primers T3 and T7, which flank the pBS polylinker. Cyclic thermal sequencing was done as above, except that elongation time is increased to 4 min to allow for longer sequence determinations.

The sequence information is used to design new oligonucleotides to obtain the full-length sequence of the clones.

DNA sequences are obtained for GUS genes from seven different genera: Bacillus (see, Example 4), Enterobacter/Salmonella, Pseudomonas, Salmonella, Staphylococcus, and Thermotga (see, The Institute for Genomic Research, Rockville Md.) (FIGS. 4 A-J). Predicted amino acids translations are presented in FIGS. 3A-B . In addition to the biochemical analysis and amplification using GUS primers, confirmation that the isolates contain GUS gene is obtained from the DNA and amino acid sequences. Amino acid alignment of Bacillus GUS with human (HGUS) and E. coli (EGUS) reveal extensive sequence identity and similarity. Likewise, alignment using Clustal W program of Bacillus, Staphylococcus homini, Staphlyococcus warneri, Thermotoga maritima, Enterobacter/Salmonella and E. coli. show considerable amino acid identity and conversation (FIG. 5 B). The darker the shading, the higher the conservation among all GUSes. As seen in FIG. 5B , the region containing the critical catalytic residue (E344 using Bacillus numbering) is highly conserved. This region extends over amino acids ca. 250—ca. 360 and ca. 400—ca. 535. Within these regions there are pockets of nearly complete identity among six sequences. When constructing variants, in general, the regions of highest identity are not altered.

Two additional sequences from Salmonella and Pseudomonas are presented in nucleotide alignment with Bacillus. Significant sequence identity among the three sequences indicates that the Salmonella and Pseudomonas sequences are β-glucuronidase coding sequences.

TABLE 1
			SEQ ID
PRIMER	BASES	SEQUENCE	NO.
T3-GUS-2F	36	AAT TAA CCC TCA CTA AAC GG/A YTT	33
		YTT YAA YTA YGC
T7-GUS-5B	39	GTA ATA CGA CTC ACT ATA GGG/GAA	34
		RTC IGC RAA RTT CCA
CSW-RTSHY (F)	17	ATC GCA CGT CCC ACT AC	35
CSW-RTSHY (R)	18	CGT GCG ATA GGA GTT AGC	36
EH-FRTSHY (F)	22	ATT TAG AAC ATC TCA TTA TCC C	37
EH-FRTSHY (R)	23	TGA GAT GTT CTA AAT GAA TTA GC	38
LSB-KRPVT (R)	17	ATC GTG ACC GGA CGC TT	39
CBP-QAYDE	17	GCG CGT AAT CTT CCT GG	40
NG-RP1L	18	TAG C(GA)C CTT CGC TTT CGG	41
NG-RP1R	20	ATC ATG TTT ACA GAG TAT GG	42
P3-16SrDNA	21	GGA ATA TTG CAC AAT GGG CGC	43
P4-16SrDNA	23	GAT CTC TAC GCA TTT CAC CGC TA	44
Tm-MVRPQRN	17	ATG GTA AGA CCG CAA CG	45
Tm-Nco-MVRPQRN	25	TAA AAA CCA TGG TAA GAC CGC AAC G	46
Tm-RRLWSE (R)	20	CCT CAC TCC ACA GTC TTC TC	47
Tm-RRL WSE (R)-	30	AGA CCG CTA GCC TCA CTC CAC AGT CTT	48
Nhe		CTC
Ps-FDFFNYA (F)	22	TTT GAC TTT TTC AAC TAT GCA G	49
Ps-DFFNYA (R)	23	AAT TCT GCA TAG TTG AAA AAG TC	50

Example 4

Isolation of a Gene from Bacillus Encoding a Secreted β-Glucuronidase

Soil samples are placed in broth and plated for growth of bacterial colonies on agar plates containing 50 μg/mL X-GlcA. Bacteria that secrete β-glucuronidase have a strong, diffuse staining pattern surrounding the colony.

On bacterial colony that exhibited this type of staining pattern is chosen. The bacterium is identified as a Bacillus based on amplification of 16S rRNA, and is most likely in the Bacillus pseudomigaterium group. Oligonucleotide sequences derived from areas exhibiting a high degree of similarity between E. coli and human β-glucoronidases are used in amplification reactions on Bacillus and E. coli DNA. A fragment is observed using Bacillus DNA, which is the same size as the E. coli fragment.

Bacillus DNA is digested with Hind III and ligated to Hind III-digested pBSII-KS plasmid vector. The recombinant plasmid is transfected into KW1, an E. coli strain that is deleted for the GUS operon. Cells are plated on X-GlcA plates, and one colony exhibited strong, diffuse staining pattern, suggesting that this clone encoded a secreted β-glucuronidase enzyme. The plasmid, pRAJa17.1, is isolated and subjected to analysis.

The DNA sequence of part of the insert of pRAJa17.1 is shown in FIG. 1. A schematic of the 6029 bp fragment is shown in FIG. 2 . The fragment contains four large open reading frames. The open reading frame proposed as Bacillus GUS (BoGUS) begins at nucleotide 162 and extends to 1907 (FIG. 1 ). The predicted translate is shown in FIG. 3 A and its alignment with E. coli and human β-glucuronidase is presented in FIG. 5 A. BoGUS is 47.2% identical to E. coli GUS, which is about the same identity as human GUS and E. coli GUS (49.1%). Thus, GUS from Bacillus is about as related to another bacterium as to human. One striking difference in sequence among the proteins is the number of cysteine residues. Whereas, both human and E. coli GUS have 4 and 9 cysteines, respectively, BoGUS has only one cysteine.

The secreted GUS protein is 602 amino acids long and does not appear to have a canonical leader peptide. A prototypic leader sequence has an amino-terminal positively charged region, a central hydrophobic region, and a more polar carboxy-terminal region (see, von Heijne, J. Membrane Biol. 115:195-201, 1990) and is generally about 20 amino acids long. However, in both mammalian and bacterial cells, proteins without canonical or identifiable secretory sequences have been found in extracellular or periplasmic spaces.

Example 5

Properties of Secreted β-Glucuronidase

Although the screen described above suggests that the Bacillus GUS is secreted, the cellular localization of BoGUS is further examined. Cellular fractions (e.g., periplasm, spheroplast, supernatant, etc.) are prepared from KW1 cells transformed with pRAJa17.1 or a subfragment that contains the GUS gene and from E. coli cells that express β-glucuronidase. GUS activity and β-galactosidase (β-gal) activity is determined for each fraction. The percent of total activity in the periplasm fraction for GUS and β-gal (a non-secreted protein) are calculated; the amount of β-gal activity is considered background and thus is subtracted from the amount of β-glucuronidase activity. In FIG. 6 , the relative activities of BoGUS and E. coli GUS in the periplasm fraction are plotted. As shown, approximately 50% of BoGUS activity is found in the periplasm, whereas less than 10% of E. coli GUS activity is present.

The thermal stability of BoGUS and E. coli GUS enzymes are determined at 65° C., using a substrate that can be measured by spectrophotometry, for example. One such substrate is p-nitrophenyl β-D-glucuronide (pNPG), which when cleaved by GUS releases the chromophore p-nitrophenol. At a pH greater than its pKa (approximately 7.15), the ionized chromophore absorbs light at 400-420 nm, therefore appears in the yellow range of visible light. Briefly, reactions are performed in 50 mM Na 3 PO 4 pH 7.0, 10 mM 2-ME, 1 mM EDTA, 1 mM pNPG, and 0.1% Triton® X-100 at 37° C. The reactions are terminated by the addition of 0.4 ml of 2-amino-2-methylpropanediol, and absorbance measured at 415 nm against a substrate blank. Under these conditions, the molar extinction coefficient of p-nitrophenol is assumed to be 14,000. One unit is defined as the amount of enzyme that produces 1 nmole of product/min at 37° C.

As shown in FIG. 7 , BoGUS has a half-life of approximately 16 min, while E. coli GUS has a half-life of less than 2 min. Thus, BoGUS is at least 8 times more stable than the E. coli GUS. In addition, the catalytic properties of BoGUS are substantially better than the E. coli enzyme: The Km is half and the Vmax is 2.5 times greater.

	TABLE 3
	BoGUS	E.coli GUS
	Km	70 μM pNPG	150 μM pNPG
	Vmax	90 nmoles/min/μg	35 nmoles/min/μg

The turnover number of BoGUS is 2.5 to 5 times higher than E. coli GUS at either 37° C. or at room temperature (FIGS. 8 and 9 ). A turnover number is calculated as nmoles of pNPG converted to p-nitrophenol per min per μg of purified protein.

BoGUS enzyme activity is also resistant to inhibition by detergents. Enzyme activity assays are measured in the presence of varying amounts of SDS, Triton® X-100, or sarcosyl. As presented in FIG. 10 , BoGUS was not inhibited or only slightly inhibited (<20% inhibition) in Triton® X-100 and Sarcosyl. In SDS, the enzyme still had substantial activity (60-75% activity). In addition, BoGUS is not inhibited by the end product of the reaction. Activity is determined normally or in the presence of 1 or 10 mM glucuronic acid. No inhibition is seen at either 1 or 10 mM glucuronic acid (FIG. 11 ). The enzyme is also assayed in the presence of organic solvents, dimethylformamide (DMF) and dimethylsulfoxide (DMSO), and high concentrations of NaCl (FIG. 12 ). Only at the highest concentrations of DMF and DMSO (20%) does BoGUS demonstrate inhibition, approximately 40% inhibited. In lesser concentrations of organic solvent and in the presence of 1 M NaCl, BoGUS retains essentially complete activity.

The Bacillus β-glucuronidase is secreted in E. coli when introduced in an expression plasmid as evidenced by approximately half of the enzyme activity being detected in the periplasm. In contrast, less than 10% of E. coli β-glucuronidase is found in periplasm. Secreted microbial GUS is also more stable than E. coli GUS (FIG. 7 ), has a higher turnover number at both 37° C. and room temperature (FIGS. 8 and 9 ), and unlike E. coli GUS, it is not substantially inhibited by detergents ( FIG. 10 ) or by glucuronic acid ( FIG. 11 ) and retains activity in high salt conditions and organic solvents (FIG. 12 ).

As shown herein, multiple mutations at residues Val 128, Leu 141, Tyr 204 and Thr 560 ( FIGS. 3A-B ) result in a non-functional enzyme. Thus, at least one of these amino acids is critical to maintaining enzyme activity. A mutein Bacillus GUS containing the amino acid alterations of Val 128→Ala, Leu 141→His, Tyr 204→Asp and Thr 560→Ala is constructed and exhibits little enzymatic activity. As shown herein, the residue alteration that most directly affected activity is Leu 141. In addition, three residues have been identified as likely contact residues important for catalysis in human GUS (residues Glu 451, Glu 540, and Tyr 504) (Jain et al., Nature Struct. Biol. 3: 375, 1996). Based on alignment with Bacillus GUS, the corresponding residues are Glu 415, Glu 508, and Tyr 471. By analogy with human GUS, Asp 165 may also be close to the reaction center and likely forms a salt bridge with Arg 566. Thus, in embodiments where it is desirable to retain enzymatic activity of GUS, the residues corresponding to Leu 141, Glu 415, Glu 508, Tyr 471, Asp 165, and Arg 566 in Bacillus GUS are preferably unaltered.

Example 6

Construction of a Codon Optimized Secreted β-Glucuronidase

The Bacillus GUS gene is codon-optimized for expression in E. coli and in rice. Codon frequencies for each codon are determined by back translation using ecohigh codons for highly expressed genes of enteric bacteria. These ecohigh codon usages are available from GCG. The most frequently used codon for each amino acid is then chosen for synthesis. In addition, the polyadenylation signal, AATAAA, splice consensus sequences, ATTTA AGGT, and restriction sites that are found in polylinkers are eliminated. Other changes may be made to reduce potential secondary structure. To facilitate cloning in various vectors, four different 5′ ends are synthesized: the first, called A0 (GT CGA C CC ATG G T A GAT CT G ACT AGT CTG TAC CCG) (SEQ ID No: 51) uses a sequence comprising an Nco I (underlined), Bgl II (double underlined), and Spe I (italicized) sites. The Leu (CTG) codon is at amino acid 2 in FIGS. 3A-B . The second variant, called AI (GTC GAC AGG AGT GCT ATC ATG CTG TAC CCG), adds the native Shine/Dalgarno sequence 5′ of the initiator Met (ATG) codon; the third, called AII, (GTC GAC AGGAGT GCT A CC ATG G TG TAC CCG) adds a modified Shine/Dalgarno sequence 5′ of the initiator Met codon such that a Nco I site is added; the fourth one, called AIII (GTC GAC AGG AGT GCT A CC ATG G T A GAT CTG TAC CCG) adds a modified Shine/Dalgarno sequence 5′ of the Leu (CTG) codon (residue 2) and Nco I and Bgl II sites. All of these new 5′ sequences contain a Sal I site at the extreme 5′ end to facilitate construction and cloning. In certain embodiments, to facilitate protein purification, a sequence comprising a Nhe I, Pml I, and BstE II sites (underlined) and encoding hexa-His amino acids joined at the 3′ (COOH-terminus) of the gene.

GCTAGC CATCACCATCACCAT CACGTG TGAATT GGTGACC G

SerSerHisHisHisHisHisHisVal*

Nucleotide and amino acid sequences of one engineered secretable microbial GUS are shown in FIGS. 13A-C , and a schematic is shown in FIG. 14 . The coding sequence for this protein is assembled in pieces. The sequence is dissected into four fragments, A (bases 1-457); B (bases 458-1012); C (bases 1013-1501); and D (bases 1502-1875). Oligonucleotides (Table 4) that are roughly 80 bases (range 36-100 bases) are synthesized to overlap and create each fragment. The fragments are each cloned separately and the DNA sequence verified. Then, the four fragments are excised and assembled in pLITMUS 39 (New England Biolabs, Beverley, Mass.), which is a small, high copy number cloning plasmid.

TABLE 1
			SEQ ID
Oligonucleotide	Size	Sequence	NO
BoGUS A-1-80T	80	TCGACCCATGGTAGATCTGACTAGTCTGTACCCGATCAACACCG	57
		AGACCCGTGGCGTCTTCGACCTCAATGGCGTCTGGA
BoGUS A-121-200B	80	GGATTTCCTTGGTCACGCCAATGTCATTGTAACTGCTTGGGACG	58
		GCCATACTAATAGTGTCGGTCAGCTTGCTTTCGTAC
BoGUS A-161-240T	80	CCAAGCAGTTACAATGACATTGGCGTGACCAAGGAAATCCGCAA	59
		CCATATCGGATATGTCTGGTACGAACGTGAGTTCAC
BoGUS A-201-280B	80	GCGGAGCACGATACGCTGATCCTTCAGATAGGCCGGCACCGTGA	60
		ACTCACGTTCGTACCAGACATATCCGATATGGTTGC
BoGUS A-241-320T	80	GGTGCCGGCCTATCTGAAGGATCAGCGTATCGTGCTCCGCTTCG	61
		GCTCTGCAACTCACAAAGCAATTGTCTATGTCAATG
BoGUS A-281-360B	80	AATGGCAGGAATCCGCCCTTGTGCTCCACGACCAGCTCACCATT	62
		GACATAGACAATTGCTTTGTGAGTTGCAGAGCCGAA
BoGUS A-321-400T	80	GTGAGCTGGTCGTGGAGCACAAGGGCGGATTCCTGCCATTCGAA	63
		GCGGAAATCAACAACTCGCTGCGTGATGGCATGAAT
BoGUS A-361-460B	100	GTACAGCCCCACCGGTAGGGTGCTATCGTCGAGGATGTTGTCCA	64
		CGGCGACGGTGACGCGATTCATGCCATCACGCAGCGAGTTGTTG
		ATTTCCGCTTCG
BoGUS A-401-456T	56	CGCGTCACCGTCGCCGTGGACAACATCCTCGACGATAGCACCCT	65
		ACCGGTGGGGCT
BoGUS A-41-120B	80	CACTTCTCTTCCAGTCCTTTCCCGTAGTCCAGCTTGAAGTTCCA	66
		GACGCCATTGAGGTCGAAGACGCCACGGGTCTCGGT
BoGUS A-6-40B	35	TTGATCGGGTACAGACTAGTCAGATCTACCATGGG	67
BoGUS A-81-160T	80	ACTTCAAGCTGGACTACGGGAAAGGACTGGAAGAGAAGTGGTAC	68
		GAAAGCAAGCTGACCGACACTATTAGTATGGCCGTC
BoGUS B-1-80T	80	GTACAGCGAGCGCCACGAAGAGGGCCTCGGAAAAGTCATTCGTA	69
		ACAAGCCGAACTTCGACTTCTTCAACTATGCAGGCC
BoGUS B-121-200B	80	CTTTGCCTTGAAAGTCCACCGTATAGGTCACAGTCCCGGTTGGG	70
		CCATTGAAGTCGGTCACAACCGAGATGTCCTCGACG
BoGUS B-161-240T	80	ACCGGGACTGTGACCTATACGGTGGACTTTCAAGGCAAAGCCGA	71
		GACCGTGAAAGTGTCGGTCGTGGATGAGGAAGGCAA
BoGUS B-201-280B	80	CTCCACGTTACCGCTCAGGCCCTCGGTGCTTGCGACCACTTTGC	72
		CTTCCTCATCCACGACCGACACTTTCACGGTCTCGG
BoGUS B-241-320T	80	AGTGGTCGCAAGCACCGAGGGCCTGAGCGGTAACGTGGAGATTC	73
		CGAATGTCATCCTCTGGGAACCACTGAACACGTATC
BoGUS B-281-360B	80	GTCAGTCCGTCGTTCACCAGTTCCACTTTGATCTGGTAGAGATA	74
		CGTGTTCAGTGGTTCCCAGAGGATGACATTCGGAAT
BoGUS B-321-400T	80	TCTACCAGATCAAAGTGGAACTGGTGAACGACGGACTGACCATC	75
		GATGTCTATGAAGAGCCGTTCGGCGTGCGGACCGTG
BoGUS B-361-440B	80	ACGGTTTGTTGTTGATGAGGAACTTGCCGTCGTTGACTTCCACG	76
		GTCCGCACGCCGAACGGCTCTTCATAGACATCGATG
BoGUS B-401-480T	80	GAAGTCAACGACGGCAAGTTCCTCATCAACAACAAACCGTTCTA	77
		CTTCAAGGGCTTTGGCAAACATGAGGACACTCCTAT
BoGUS B-41-120B	80	TACGTAAACGGGGTCGTGTAGATTTTCACCGGACGGTGCAGGCC	78
		TGCATAGTTGAAGAAGTCGAAGTTCGGCTTGTTACG
BoGUS B-441-520B	80	ATCCATCACATTGCTCGCTTCGTTAAAGCCACGGCCGTTGATAG	79
		GAGTGTCCTCATGTTTGCCAAAGCCCTTGAAGTAGA
BoGUS B-481-555T	75	CAACGGCCGTGGCTTTAACGAAGCGAGCAATGTGATGGATTTCA	80
		ATATCCTCAAATGGATCGGCGCCAACAGCTT
BoGUS B-5-40B	36	AATGACTTTTCCGAGGCCCTCTTCGTGGCGCTCGCT	81
BoGUS B-521-559B	39	CCGGAAGCTGTTGGCGCCGATCCATTTGAGGATATTGAA	82
BoGUS B-81-160T	80	TGCACCGTCCGGTGAAAATCTACACGACCCCGTTTACGTACGTC	83
		GAGGACATCTCGGTTGTGACCGACTTCAATGGCCCA
BoGUS C-1-80T	80	CCGGACCGCACACTATCCGTACTCTGAAGAGTTGATGCGTCTTG	84
		CGGATCGCGAGGGTCTGGTCGTGATCGACGAGACTC
BoGUS C-121-200B	80	GTTCACGGAGAACGTCTTGATGGTGCTCAAACGTCCGAATCTTC	85
		TCCCAGGTACTGACGCGCTCGCTGCCTTCGCCGAGT
BoGUS C-161-240T	80	ATTCGGACGTTTGAGCACCATCAAGACGTTCTCCGTGAACTGGT	86
		GTCTCGTGACAAGAACCATCCAAGCGTCGTGATGTG
BoGUS C-201-280B	80	CGCGCCCTCTTCCTCAGTCGCCGCCTCGTTGGCGATGCTCCACA	87
		TCACGACGCTTGGATGGTTCTTGTCACGAGACACCA
BoGUS C-241-320T	80	GAGCATCGCCAACGAGGCGGCGACTGAGGAAGAGGGCGCGTACG	88
		AGTACTTCAAGCCGTTGGTGGAGCTGACCAAGGAAC
BoGUS C-281-360B	80	ACAAACAGCACGATCGTGACCGGACGCTTCTGTGGGTCGAGTTC	89
		CTTGGTCAGCTCCACCAACGGCTTGAAGTACTCGTA
BoGUS C-321-400T	80	TCGACCCACAGAAGCGTCCGGTCACGATCGTGCTGTTTGTGATG	90
		GCTACCCCGGAGACGGACAAAGTCGCCGAACTGATT
BoGUS C-361-440B	80	CGAAGTACCATCCGTTATAGCGATTGAGCGCGATGACGTCAATC	91
		AGTTCGGCGACTTTGTCCGTCTCCGGGGTAGCCATC
BoGUS C-401-489T	89	GACGTCATCGCGCTCAATCGCTATAACGGATGGTACTTCGATGG	92
		CGGTGATCTCGAAGCGGCCAAAGTCCATCTCCGCCAGGAATTTC
		A
BoGUS C-41-120B	80	CCCGTGGTGGCCATGAAGTTGAGGTGCACGCCAACTGCCGGAGT	93
		CTCGTCGATCACGACCAGACCCTCGCGATCCGCAAG
BoGUS C-441-493B	53	CGCGTGAAATTCCTGGCGGAGATGGACTTTGGCCGCTTCGAGAT	94
		CACCGCCAT
BoGUS C-5-40B	36	ACGCATCAACTCTTCAGAGTACGGATAGTGTGCGGT	95
BoGUS C-81-160T	80	CGGCAGTTGGCGTGCACCTCAACTTCATGGCCACCACGGGACTC	96
		GGCGAAGGCAGCGAGCGCGTCAGTACCTGGGAGAAG
BoGUS D-1-80T	80	CGCGTGGAACAAGCGTTGCCCAGGAAAGCCGATCATGATCACTG	97
		AGTACGGCGCAGACACCGTTGCGGGCTTTCACGACA
BoGUS D-121-200B	80	TCGCGAAGTCCGCGAAGTTCCACGCTTGCTCACCCACGAAGTTC	98
		TCAAACTCATCGAACACGACGTGGTTCGCCTGGTAG
BoGUS D-161-240T	80	TTCGTGGGTGAGCAAGCGTGGAACTTCGCGGACTTCGCGACCTC	99
		TCAGGGCGTGATGCGCGTCCAAGGAAACAAGAAGGG
BoGUS D-201-280B	80	GTGCGCGGCGAGCTTCGGCTTGCGGTCACGAGTGAACACGCCCT	100
		TCTTGTTTCCTTGGACGCGCATCACGCCCTGAGAGG
BoGUS D-241-320T	80	CGTGTTCACTCGTGACCGCAAGCCGAAGCTCGCCGCGCACGTCT	101
		TTCGCGAGCGCTGGACCAACATTCCAGATTTCGGCT
BoGUS D-281-369B	89	CGGTCACCAATTCACACGTGATGGTGATGGTGATGGCTAGCGTT	102
		CTTGTAGCCGAAATCTGGAATGTTGGTCCAGCGCTCGCGAAAGA
		C
BoGUS D-321-373T	53	ACAAGAACGCTAGCCATCACCATCACCATCACGTGTGAATTGGT	103
		GACCGGGCC
BoGUS D-41-120B	80	TACTCGACTTGATATTCCTCGGTGAACATCACTGGATCAATGTC	104
		GTGAAAGCCCGCAACGGTGTCTGCGCCGTACTCAGT
BoGUS D-5-40B	36	GATCATGATCGGCTTTCCTGGGCAACGCTTGTTCCA	105
BoGUS D-81-160T	80	TTGATCCAGTGATGTTCACCGAGGAATATCAAGTCGAGTACTAC	106
		CAGGCGAACCACGTCGTGTTCGATGAGTTTGAGAAC

The A1 form of microbial GUS in pLITMUS 39 is transfected into KW1 host E. coli cells. Bacterial cells are collected by centrifugation, washed with Mg salt solution and resuspended in IMAC buffer (50 mMNa 3 PO 4 , ph 7.0, 300 mM KC1, 0.1% Triton® X-100, 1 mMPMSF). For hexa-His fusion proteins, the lysate is clarified by centrifugation at 20,000 rpm for 30 min and batch absorbed on a Ni-IDA-Sepharose® column. The matrix is poured into a column and washed with IMAC buffer containing 75 mM imidazole. The β-glucuronidases protein bound to the matrix is eluted with IMAC buffer containing 10 mM EDTA.

If GUS is cloned without the hexa-His tail, the lysate is centrifuged at 50,000 rpm for 45 min, and diluted with 20 mM NaPO 4 , 1 mM EDTA, pH 7.0 (buffer A). The diluted supernatant is then loaded onto a SP-Sepharose® or equivalent column, and a linear gradient of 0 to 30% SP Buffer B (1 M NaCl, 20 mM Na PO 4 , 1 mM EDTA, pH 7.0) in Buffer A with a total of 6 column volumes is applied. Fractions containing GUS are combined. Further purifications can be performed.

Example 7

Muteins of Codon Optimized β-Glucuronidase

Muteins of the codon-optimized GUS genes are constructed. Each of the four GUS genes described above, A0, AI, AII, and AIII, contain none, one, or four amino acid alterations. The muteins that contain one alteration have a Leu 141 to His codon change. The muteins that contain four alterations have the Leu 141 to His change as well as Val 138 to Ala, Tyr 204 to Asp, and Thr 560 to Ala changes. pLITMUS 39 containing these 12 muteins are transfected into KW1. Colonies are tested for secretion of the introduced GUS gene by staining with X-GlcA. A white colony indicates undetectable GUS activity, a light blue colony indicates some detectable activity, and a dark blue colony indicates a higher level of detectable activity. As shown in Table 5 below, when GUS has the four mutations, no GUS activity is detectable. When GUS has a single Leu 141 to His mutation, three of the four constructs exhibit no GUS activity, while the AI construct exhibits a low level of GUS activity. All constructs exhibit GUS activity when no mutations are present. Thus, the Leu 141 to His mutation dramatically affects the activity of GUS.

TABLE 5
Number of	GUS construct
Mutations	A0	AI	AII	AIII
4	white	white	white	white
1	white	light blue	white	white
0	light blue	dark blue	light blue	light blue

Example 8

Expression of Microbial β-Glucuronidases in Yeast, Plants and E. coli

A series of expression vector constructs of three different GUS genes, E. coli GUS, Bacillus GUS, and the A0 version of codon-optimized Bacillus GUS, are prepared and tested for enzymatic activity in E. coli, yeast, and plants (rice, Millin variety). The GUS genes are cloned in vectors that either contain a signal peptide suitable for the host or do not contain a signal peptide. The E. coli vector contains a sequence encoding a pelB signal peptide, the yeast vectors contain a sequence encoding either an invertase or Mat alpha signal peptide, and the plant vectors contain a sequence encoding either a glycine-rich protein (GRP) or extensin signal peptide.

Intertase signal sequence:
ATGCTTTTGC AAGCCTTCCT TTTCCTTTTG GCTGGTTTTG CAGCCAAAAT ATCTGCAATG
(SEQ ID NO. 107)
Mat alpha signal sequence:
ATGAGATTTC CTTCAATTTT TACTGCAGTT TTATTCGCAG CATCCTCCGC ATTAGCTGCT
CCAGTCAACA CTACAACAGA AGATGAAACG GCACAAATTC CGGCTGAAGC TGTCATCGGT
TACTTAGATT TAGAAGGGGA TTTCGATGTT GCTGTTTTGC CATTTTCCAA CAGCACAAAT
AACGGGTTAT TGTTTATAAA TACTACTATT GCCAGCATTG CTGCTAAAGA AGAAGGGGTA
TCTTTGGATA AAAGAGAG  (SEQ ID NO. 108)
Extensin signal sequence
CATGGGAAAA ATGGCTTCTC TATTTGCCAC ATTTTTAGTG GTTTTAGTGT CACTTAGCTT
AGCTTCTGAA AGCTCAGCAA ATTATCAA (SEQ ID NO. 109)
GRP signal sequence
CATGGCTACT ACTAAGCATT TGGCTCTTGC CATCCTTGTC CTCCTTAGCA TTGGTATGAC
CACCAGTGCA AGAACCCTCC TA  (SEQ ID NO. 110)

The GUS genes are cloned into each of these vectors using standard recombinant techniques of isolation of a GUS-gene containing fragment and ligation into an appropriately restricted vector. The recombinant vectors are then transfected into the appropriate host and transfectants are tested for GUS activity.

As shown in the Table below, all tested transfectants exhibit GUS activity (indicated by a +). Moreover, similar results are obtained regardless of the presence or absence of a signal peptide.

	TABLE 6
	E. coli	Yeast	Plants
GUS	No SP*	pelB	No SP	Invertase	Mat α	No SP	GRP	Extensin
E. coli GUS	+	NT	+	+	+	+	+	+
Bacillus GUS	+	NT	+	+	+	+	+	+
*SP = signal peptide

Example 9

Eliminiation of the Potential N-Glycosylation Site of Bacillus β-Glucuronidase

The consensus N-glycosylation sequence Asn-X-Ser/Thr is present in Bacillus GUS at amino acids 118-120, Asn-Asn-Ser (FIGS. 3 A-B). Glycosylation could interfere with secretion or activity of β-glucuronidase upon entering the ER. To remove potential N-glycosylation, the Asn at residue 118 is changed to another amino acid in the plasmid pTANE95m (AI) is altered. The GUS in this plasmid is a synthetic GUS gene with a completely native 5′ end.

The oligonucleotides Asn-T, 5′-A TTC CTG CCA TTC GAG GCG GAA ATC NNG AAC TCG CTG CGT GAT-3′ (SEQ ID No. 111) and Asn-B, 5′-ATC ACG CAG CGA GTT CNN GAT TTC CGC CTC GAA TGG CAG GAA T-3′ (SEQ ID No. 112), are used in the “quikchange” mutagenesis method by Stratagene (La Jolla, Calif.) to randomize the first two nucleotides of the Asn 118 codon, AAC. The third base is changed to a G nucleotide, so that reversion to Asn is not possible. In theory a total of 13 different amino acids are created at position 118.

Because expression of GUS from the plasmid pTANE95m (AI) exhibits a range of colony phenotypes from white to dark blue, a restriction enzyme digestion assay is used to confirm presence of mutants. Therefore, an elimination of a BstB I restriction site which does not change any amino acid, is also introduced into the mutagenizing oligonucleotides to facilitate restriction digestion screening of mutants.

Sixty colonies were randomly picked and assayed by BstB I digestion. Twenty-one out of the 60 colonies have the BstB I site removed and are thus mutants. DNA sequence analysis of these candidate mutants show that a total of 8 different amino acids are obtained. Five of the N118 mutants are chosen as suitable for further experimentation. In these mutants, the N118 residue is changed to a Ser, Arg, Leu, Pro, or Met.

Example 10

Expression of β-Glucuronidase in Transgenic Rice Plants

Microbial GUS can be used as a non-destructible marker. In this example, transgenic rice expressing a GUS gene encoding a secreted form are assayed for GUS expression in planta.

Seeds of T0 plants, which are the primary transformed plants, from pTANG86.1/2/3/4/5/6 (see Table 7 below) transformed plants, seeds of pCAM1301 ( E. coli GUS with N358-Q change to remove N-glycosylation signal sequence) transformed plants, or untransformed Millin rice seeds are germinated in water containing 1 mM MUG or 50 μg/mL X-GlcA with or without hygromycin (for nontransformed plants). Resulting plants are observed for any reduced growth due to the presence of MUG, X-GlcA. No toxic effects of X-GlcA are detected, but roots of the plants grown in MUG are somewhat stunted.

For assaying GUS activity in planta, seeds are germinated in water with or without hygromycin (for nontransformed plants). Roots of the seedlings are submerged in water containing 1 mM MUG, or 50 μg/mL X-GlcA. Fluorescence (in the case of MUG staining) or indigo dye (in the case of X-GlcA staining) are assayed in the media and roots over time.

Secondary roots from seedlings of pTANG86.3 and pTANG86.5 (BoGUS fused with signal peptides) plants show indigo color after ½ hour incubation in water containing X-GlcA. Evidence that GUS is a non-destructive marker is obtained by plant growth after transferring the stained plant to water. Furthermore, stained roots also grow further.

Example 11

Expression of β-Glucuronidase in Yeast

All the yeast plasmids are based on the Ycp backbone, which contains a yeast centromere and is stable at low copy number. Yeast strain InvSc1 (mat α his3 Δ1 leu2 trp1-289 ura3-52) from Invitrogen (Carlsbad, Calif.) is transformed with the E. coli GUS and Bacillus GUS plasmids indicated in the table below. Transformants are plated on both selection media (minimal media supplemented with His, Leu, Trp, and 2% glucose as a carbon source to suppress the expression of the gene driven by the gal1 promoter) and expression media (media supplemented with His, Leu, Trp, 1% raffinose, 1% galactose as carbon source and with 50 μg/ml X-GlcA).

	TABLE 7
	Yeast	Plants
	No SP	Invertase	Mat alpha	No SP	GRP	Extensin
E. coli	pAKD80.3	pAKD80.6	pTANG87.4	pTANG86.2	pTANG86.4	pTANG86.6
Syn BGUS	pTANG87.1	pTANG87.2	pTANG87.3	pTANG86.1	pTANG86.3	pTANG86.5
Nat BGUS	pAKD102.1	pAKE2.1	pAKE11.4	pAKD40	pAKC30.1	pAKC30.3

With the exception of pAKD80.6, all other transformed yeast colonies are white on X-GlcA plates. The transformants do express GUS, however, which is evidenced by lysing the cells on the plates with hot agarose containing X-GlcA and observing the characteristic indigo color. The yeast transformants are white when GUS is not secreted, as X-GlcA cannot be taken by the yeast cell. All the yeast colonies transformed with pAKD80.6 are blue on X-GlcA plates and have a blue halo around each colony, clearly indicating that the enzyme is secreted into the medium.

Bacillus GUS enzyme has a potential N-glycosylation site, which may interfere with the secretion process or cause inactivation of the enzyme upon secretion. To determine whether the N-glycosylation site has a deleterious effect, on secretion, yeast colonies are streaked on expression plates containing X-GlcA and from 0.1 to 20 μg/ml of tunicamycin (to inhibit all N-glycosylation). At high concentrations of tunicamycin (5, 10, and 20 μg/ml), yeast colonies do not grow, likely due to toxicity of the drug. However, in yeast transformed with pTANG87.3, the cells that do survive at these tunicamycin concentrations are blue. This indicates that glycosylation may affect the secretion or activity of Bacillus GUS. Any effect should be overcome by mutating the glycosylation signal sequence as described.

Example 12

Expression of Low-Cysteine E. Coli β-Glucuronidase

The E. coli GUS protein has nine cysteine residues, whereas, human GUS has four and Bacillus GUS has one. Low-cysteine muteins of E. coli GUS are constructed to provide a form of EcGUS that is secretable.

Single and multiple Cys muteins are constructed by site-directed mutagenesis techniques. Eight of the nine cysteine residues in E. coli GUS are changed to the corresponding residue found in human GUS based on alignment of the two protein sequences. One of the E. coli GUS cysteine residues, amino acid 463, aligns with a cysteine residue in human GUS and was not altered. The corresponding amino acids between E. coli GUS and human GUS are shown below.

TABLE 8
		Human GUS
		corresponding amino
Identifier	EcGUS Cys residue no.	acid
A	28	Asn
B	133	Ala
C	197	Ser
D	253	Glu
E	262	Ser
F	442	Phe
G	448	Tyr
H	463	Cys
I	527	Lys

The mutein GUS genes are cloned into a pBS backbone. The mutations are confirmed by diagnostic restriction site changes and by DNA sequence analysis. Recombinant vectors are transfected into KW1 and GUS activity assayed by staining with X-GlcA (5-bromo-4-chloro-3-indolyl-β-D-glucuronide).

As shown in the Table below, when the Cys residues at 442 (F), 448 (G), and 527 (I) are altered, GUS activity is greatly or completely diminished. In contrast, when the N-terminal five Cys residues (A, B, C, D, and E) are altered, GUS activity remains detectable.

TABLE 9
Cys changes      GUS activity
A                Yes
B                Yes
C                Yes
I                No
D, E             Yes
F, G             No
C, D, E          Yes
B, C, D, E       Yes
A, B, C, D, E    Yes
A, B, C, D, E, I No

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

112 1 2100 DNA Bacillus sp. 1agcctttact tttctttcaa cttttcatcc cgatactttt ttgtaatagt ttttttcatt 60aataatacaa gtcctgattt tgcaagaata atccttttta gataaaaata tctatgctaa 120taataacatg taaccactta catttaaaaa ggagtgctat catgttatat ccaatcaata 180cagaaacccg aggagttttt gatttaaatg gggtctggaa ttttaaatta gattacggca 240aaggactgga agaaaagtgg tatgaatcaa aactgacaga taccatatca atggctgtac 300cttcctccta taatgatatc ggtgttacga aggaaattcg aaaccatatc ggctatgtat 360ggtacgagcg tgaatttacc gttcctgctt atttaaaaga tcagcgcatc gtcctgcgtt 420ttggttcagc aacacataag gctattgtat acgttaacgg agaactagta gttgaacaca 480aaggcggctt cttaccgttt gaggcagaaa taaacaacag cttaagagac ggaatgaatc 540gtgtaacagt agcggttgat aatattttag atgattctac gctcccagtt gggctatata 600gtgaaagaca tgaagaaggt ttgggaaaag tgattcgtaa taaacctaat tttgacttct 660ttaactatgc aggcttacat cgtcctgtaa aaatttatac aacccctttt acctatgttg 720aggatatatc ggttgtaacc gattttaacg gtccaacggg aacagttacg tatacagttg 780attttcaggg taaggcagaa accgtaaagg ttagtgtagt tgatgaagaa gggaaagttg 840ttgcttcaac tgaaggcctc tctggtaatg ttgagattcc taacgttatc ctttgggaac 900ctttaaatac ctatctctat caaattaaag ttgagttagt aaatgatggt ctaactattg 960atgtatacga agagccattt ggagttcgaa ccgttgaagt aaacgacggg aaattcctca 1020ttaataacaa accattttat tttaaagggt tcggaaaaca cgaggatact ccaataaatg 1080gaagaggctt taatgaagca tcaaatgtaa tggattttaa tattttgaaa tggatcggtg 1140cgaattcctt tcggacggcg cactatcctt attctgaaga actgatgcgg ctcgcagatc 1200gtgaagggtt agtcgtcata gatgaaaccc cagcagttgg tgttcatttg aactttatgg 1260caacgactgg tttgggcgaa ggttcagaga gagtgagtac ttgggaaaaa atccggacct 1320ttgaacatca tcaagatgta ctgagagagc tggtttctcg tgataaaaac cacccctctg 1380ttgtcatgtg gtcgattgca aatgaagcgg ctacggaaga agaaggcgct tatgaatact 1440ttaagccatt agttgaatta acgaaagaat tagatccaca aaaacgccca gttaccattg 1500ttttgttcgt aatggcgaca ccagaaacag ataaagtggc ggagttaatt gatgtgattg 1560cattgaatcg atacaacggc tggtattttg atgggggtga tcttgaagcc gcgaaagtcc 1620accttcgtca ggaatttcat gcgtggaata aacgctgtcc aggaaaacct ataatgataa 1680cagagtatgg ggctgatacc gtagctggtt ttcatgatat tgatccggtt atgtttacag 1740aagagtatca ggttgaatat taccaagcaa atcatgtagt atttgatgaa tttgagaact 1800ttgttggcga gcaggcctgg aattttgcag actttgctac aagccagggt gtcatgcgtg 1860ttcaaggtaa caaaaaaggt gttttcacac gcgaccgcaa accaaaatta gcagcacatg 1920ttttccgcga acgttggaca aacatcccgg atttcggtta taaaaattaa taaaaagctg 1980gttctccaat aggaggccag cttttttaca tggatacaat ggttgtaaat taaaaaccct 2040cttcattttt tatataaaaa tgaagagggt tttaattttt taaatgttat tacatttttt 2100 2 602 PRT Bacillus sp. 2Met Leu Tyr Pro Ile Asn Thr Glu Thr Arg Gly Val Phe Asp Leu Asn 1 5 10 15Gly Val Trp Asn Phe Lys Leu Asp Tyr Gly Lys Gly Leu Glu Glu Lys 20 25 30Trp Tyr Glu Ser Lys Leu Thr Asp Thr Ile Ser Met Ala Val Pro Ser 35 40 45Ser Tyr Asn Asp Ile Gly Val Thr Lys Glu Ile Arg Asn His Ile Gly 50 55 60Tyr Val Trp Tyr Glu Arg Glu Phe Thr Val Pro Ala Tyr Leu Lys Asp65 70 75 80Gln Arg Ile Val Leu Arg Phe Gly Ser Ala Thr His Lys Ala Ile Val 85 90 95Tyr Val Asn Gly Glu Leu Val Val Glu His Lys Gly Gly Phe Leu Pro 100 105 110Phe Glu Ala Glu Ile Asn Asn Ser Leu Arg Asp Gly Met Asn Arg Val 115 120 125Thr Val Ala Val Asp Asn Ile Leu Asp Asp Ser Thr Leu Pro Val Gly 130 135 140Leu Tyr Ser Glu Arg His Glu Glu Gly Leu Gly Lys Val Ile Arg Asn145 150 155 160Lys Pro Asn Phe Asp Phe Phe Asn Tyr Ala Gly Leu His Arg Pro Val 165 170 175Lys Ile Tyr Thr Thr Pro Phe Thr Tyr Val Glu Asp Ile Ser Val Val 180 185 190Thr Asp Phe Asn Gly Pro Thr Gly Thr Val Thr Tyr Thr Val Asp Phe 195 200 205Gln Gly Lys Ala Glu Thr Val Lys Val Ser Val Val Asp Glu Glu Gly 210 215 220Lys Val Val Ala Ser Thr Glu Gly Leu Ser Gly Asn Val Glu Ile Pro225 230 235 240Asn Val Ile Leu Trp Glu Pro Leu Asn Thr Tyr Leu Tyr Gln Ile Lys 245 250 255Val Glu Leu Val Asn Asp Gly Leu Thr Ile Asp Val Tyr Glu Glu Pro 260 265 270Phe Gly Val Arg Thr Val Glu Val Asn Asp Gly Lys Phe Leu Ile Asn 275 280 285Asn Lys Pro Phe Tyr Phe Lys Gly Phe Gly Lys His Glu Asp Thr Pro 290 295 300Ile Asn Gly Arg Gly Phe Asn Glu Ala Ser Asn Val Met Asp Phe Asn305 310 315 320Ile Leu Lys Trp Ile Gly Ala Asn Ser Phe Arg Thr Ala His Tyr Pro 325 330 335Tyr Ser Glu Glu Leu Met Arg Leu Ala Asp Arg Glu Gly Leu Val Val 340 345 350Ile Asp Glu Thr Pro Ala Val Gly Val His Leu Asn Phe Met Ala Thr 355 360 365Thr Gly Leu Gly Glu Gly Ser Glu Arg Val Ser Thr Trp Glu Lys Ile 370 375 380Arg Thr Phe Glu His His Gln Asp Val Leu Arg Glu Leu Val Ser Arg385 390 395 400Asp Lys Asn His Pro Ser Val Val Met Trp Ser Ile Ala Asn Glu Ala 405 410 415Ala Thr Glu Glu Glu Gly Ala Tyr Glu Tyr Phe Lys Pro Leu Val Glu 420 425 430Leu Thr Lys Glu Leu Asp Pro Gln Lys Arg Pro Val Thr Ile Val Leu 435 440 445Phe Val Met Ala Thr Pro Glu Thr Asp Lys Val Ala Glu Leu Ile Asp 450 455 460Val Ile Ala Leu Asn Arg Tyr Asn Gly Trp Tyr Phe Asp Gly Gly Asp465 470 475 480Leu Glu Ala Ala Lys Val His Leu Arg Gln Glu Phe His Ala Trp Asn 485 490 495Lys Arg Cys Pro Gly Lys Pro Ile Met Ile Thr Glu Tyr Gly Ala Asp 500 505 510Thr Val Ala Gly Phe His Asp Ile Asp Pro Val Met Phe Thr Glu Glu 515 520 525Tyr Gln Val Glu Tyr Tyr Gln Ala Asn His Val Val Phe Asp Glu Phe 530 535 540Glu Asn Phe Val Gly Glu Gln Ala Trp Asn Phe Ala Asp Phe Ala Thr545 550 555 560Ser Gln Gly Val Met Arg Val Gln Gly Asn Lys Lys Gly Val Phe Thr 565 570 575Arg Asp Arg Lys Pro Lys Leu Ala Ala His Val Phe Arg Glu Arg Trp 580 585 590Thr Asn Ile Pro Asp Phe Gly Tyr Lys Asn 595 600 3 372 PRT Enterobacter sp. / Salmonella sp. VARIANT (1)...(372) Xaa = Any Amino Acid 3Gly Lys Leu Ser Pro Thr Pro Thr Ala Tyr Ile Gln Asp Val Thr Val 1 5 10 15Xaa Thr Asp Val Leu Glu Asn Thr Glu Gln Ala Thr Val Leu Gly Asn 20 25 30Val Gly Ala Asp Gly Asp Ile Arg Val Glu Leu Arg Asp Gly Gln Gln 35 40 45Gln Ile Val Ala Gln Gly Leu Gly Ala Thr Gly Ile Phe Glu Leu Asp 50 55 60Asn Pro His Leu Trp Glu Pro Gly Glu Gly Tyr Leu Tyr Glu Leu Arg65 70 75 80Val Thr Cys Glu Ala Asn Gly Glu Cys Asp Glu Tyr Pro Val Arg Val 85 90 95Gly Ile Arg Ser Ile Thr Xaa Lys Gly Glu Gln Phe Leu Ile Asn His 100 105 110Lys Pro Phe Tyr Leu Thr Gly Phe Gly Arg His Glu Asp Ala Asp Phe 115 120 125Arg Gly Lys Gly Phe Asp Pro Val Leu Met Val His Asp His Ala Leu 130 135 140Met Asn Trp Ile Gly Ala Asn Ser Tyr Arg Thr Ser His Tyr Pro Tyr145 150 155 160Ala Glu Lys Met Leu Asp Trp Ala Asp Glu His Val Ile Val Val Ile 165 170 175Asn Glu Thr Ala Ala Gly Gly Phe Asn Thr Leu Ser Leu Gly Ile Thr 180 185 190Phe Asp Ala Gly Glu Arg Pro Lys Glu Leu Tyr Ser Glu Glu Ala Ile 195 200 205Asn Gly Glu Thr Ser Gln Gln Ala His Leu Gln Ala Ile Lys Glu Leu 210 215 220Ile Ala Arg Asp Lys Asn His Pro Ser Val Val Cys Trp Ser Ile Ala225 230 235 240Asn Glu Pro Asp Thr Arg Pro Asn Gly Ala Arg Glu Tyr Phe Ala Pro 245 250 255Leu Ala Lys Ala Thr Arg Glu Leu Asp Pro Thr Arg Pro Ile Thr Cys 260 265 270Val Asn Val Met Phe Cys Asp Ala Glu Ser Asp Thr Ile Thr Asp Leu 275 280 285Phe Asp Val Val Cys Leu Asn Arg Tyr Tyr Gly Trp Tyr Val Gln Ser 290 295 300Gly Asp Leu Glu Lys Ala Glu Gln Met Leu Glu Gln Glu Leu Leu Ala305 310 315 320Trp Gln Ser Lys Leu His Arg Pro Ile Ile Ile Thr Glu Tyr Gly Val 325 330 335Asp Thr Leu Ala Gly Met Pro Ser Val Tyr Pro Asp Met Trp Ser Glu 340 345 350Lys Tyr Gln Trp Lys Trp Leu Glu Met Tyr His Arg Val Phe Asp Arg 355 360 365Gly Ser Val Cys 370 4 376 PRT Staphylococcus homini VARIANT (1)...(376) Xaa = Any Amino Acid 4Gly Leu Ser Gly Asn Val Glu Ile Pro Asn Val Ile Leu Trp Glu Pro 1 5 10 15Leu Asn Thr Tyr Leu Tyr Gln Ile Lys Val Glu Leu Val Asn Asp Gly 20 25 30Leu Thr Ile Asp Val Tyr Glu Glu Pro Phe Gly Val Arg Thr Val Glu 35 40 45Val Asn Asp Gly Lys Phe Leu Ile Asn Asn Lys Pro Phe Tyr Phe Lys 50 55 60Gly Phe Gly Lys His Glu Asp Thr Pro Ile Asn Gly Arg Gly Phe Asn65 70 75 80Glu Ala Ser Asn Val Met Asp Phe Asn Ile Leu Lys Trp Ile Gly Ala 85 90 95Asn Ser Phe Arg Thr Ala His Tyr Pro Tyr Ser Glu Glu Leu Met Arg 100 105 110Leu Ala Asp Arg Glu Gly Leu Val Val Ile Asp Glu Thr Pro Ala Val 115 120 125Gly Val His Leu Asn Phe Met Ala Thr Thr Gly Leu Gly Glu Gly Ser 130 135 140Glu Arg Val Ser Thr Trp Glu Lys Ile Arg Thr Phe Glu His His Gln145 150 155 160Asp Val Leu Arg Glu Leu Val Ser Arg Asp Lys Asn His Pro Ser Val 165 170 175Val Met Trp Ser Ile Ala Asn Glu Ala Ala Thr Glu Glu Glu Gly Ala 180 185 190Tyr Glu Tyr Phe Lys Pro Leu Gly Gly Ala Ala Lys Glu Leu Asp Pro 195 200 205Xaa Lys Arg Pro Val Thr Ile Val Leu Phe Val Met Ala Thr Pro Glu 210 215 220Thr Asp Lys Val Ala Glu Leu Ile Asp Val Ile Ala Leu Asn Arg Tyr225 230 235 240Asn Gly Trp Tyr Phe Asp Gly Gly Asp Leu Glu Ala Ala Lys Val His 245 250 255Leu Arg Gln Glu Phe His Ala Trp Asn Lys Arg Cys Pro Gly Lys Pro 260 265 270Ile Met Ile Thr Glu Tyr Gly Ala Asp Thr Val Ala Gly Phe His Asp 275 280 285Ile Asp Pro Val Met Phe Thr Glu Glu Tyr Gln Val Glu Tyr Tyr Gln 290 295 300Ala Asn His Val Val Phe Asp Glu Phe Glu Asn Phe Val Gly Glu Gln305 310 315 320Ala Trp Asn Phe Ala Asp Phe Ala Thr Ser Gln Gly Val Met Arg Val 325 330 335Gln Gly Asn Lys Lys Gly Val Phe Thr Arg Asp Arg Lys Pro Xaa Leu 340 345 350Ala Ala His Val Phe Arg Glu Arg Arg Thr Asn Ile Pro Asp Phe Gly 355 360 365Tyr Lys Asn Ala Ser His His His 370 375 5 540 PRT Staphylococcus warneri VARIANT (1)...(540) Xaa = Any Amino Acid 5Leu Xaa Leu Leu His Pro Ile Thr Thr Gly Thr Arg Gly Gly Phe Ala 1 5 10 15Leu Tyr Gly Xaa Xaa Asn Leu Met Leu Asp Tyr Gly Xaa Gly Leu Thr 20 25 30Asp Thr Trp Thr Xaa Ser Leu Leu Thr Glu Leu Ser Arg Leu Val Val 35 40 45Leu Ser Trp Thr Thr His Xaa Leu Thr Gly Glu Xaa Pro Ala Ile Ser 50 55 60Ile Leu Trp Pro Asn Ser Glu Leu Thr Val Ser Xaa Leu Tyr Xaa Gly65 70 75 80Ser Leu Xaa Ser Ser Ser Xaa Leu Cys Ser Ser Leu Thr Xaa His Val 85 90 95Val Ile Cys Gln Xaa Val Thr Leu Xaa Val Asp His Thr Gly Leu Ile 100 105 110Xaa Xaa Phe Glu Phe Met Ser Thr Thr Cys Cys Xaa Xaa Asp Glu Leu 115 120 125Val Thr Gly Thr Leu Ala Xaa Ile Leu Tyr His Xaa Ile Leu Pro His 130 135 140Gly Leu Tyr Arg Lys Arg His Glu Xaa Gly Leu Gly Lys Xaa Asn Phe145 150 155 160Tyr Xaa Leu His Phe Ala Phe Phe Xaa Tyr Ala Xaa Leu Xaa Arg Thr 165 170 175Val Xaa Met Tyr Xaa Asn Leu Val Arg Xaa Gln Asp Ile Xaa Val Val 180 185 190Thr Xaa Xaa His Xaa Xaa Xaa Xaa Thr Val Glu Gln Cys Val Xaa Xaa 195 200 205Asn Xaa Lys Ile Xaa Ser Val Lys Ile Thr Ile Leu Asp Glu Asn Asp 210 215 220His Ala Ile Xaa Glu Ser Glu Gly Ala Lys Gly Asn Val Thr Ile Gln225 230 235 240Asn Pro Ile Leu Trp Gln Pro Leu His Ala Tyr Leu Tyr Asn Met Lys 245 250 255Val Glu Leu Leu Asn Asp Asn Glu Cys Val Asp Val Tyr Thr Glu Arg 260 265 270Phe Gly Ile Arg Ser Val Glu Val Lys Asp Gly Gln Phe Leu Ile Asn 275 280 285Asp Lys Pro Phe Tyr Phe Lys Gly Phe Gly Lys His Glu Asp Thr Tyr 290 295 300Xaa Asn Gly Arg Gly Leu Asn Glu Ser Ala Asn Val Met Asp Ile Asn305 310 315 320Leu Met Lys Trp Ile Gly Ala Asn Ser Phe Arg Thr Ser His Tyr Pro 325 330 335Tyr Ser Glu Glu Met Met Arg Leu Ala Asp Glu Gln Gly Ile Val Val 340 345 350Ile Asp Glu Thr Thr Xaa Val Gly Ile His Leu Asn Phe Met Xaa Thr 355 360 365Leu Gly Gly Ser Xaa Ala His Asp Thr Trp Xaa Glu Phe Asp Thr Leu 370 375 380Glu Phe His Lys Glu Val Ile Xaa Asp Leu Ile Xaa Arg Asp Lys Asn385 390 395 400His Ala Trp Val Val Met Trp Xaa Phe Gly Asn Glu Xaa Gly Xaa Asn 405 410 415Lys Gly Gly Ala Lys Ala Xaa Phe Glu Pro Phe Val Asn Leu Ala Gly 420 425 430Glu Lys Asp Xaa Xaa Xaa Xaa Pro Val Thr Ile Val Thr Ile Leu Xaa 435 440 445Ala Xaa Arg Asn Val Cys Glu Val Xaa Asp Leu Val Asp Val Val Cys 450 455 460Leu Xaa Xaa Xaa Xaa Gly Trp Tyr Xaa Gln Ser Gly Asp Leu Glu Gly465 470 475 480Ala Lys Xaa Ala Leu Asp Lys Glu Xaa Xaa Glu Trp Trp Lys Xaa Gln 485 490 495Xaa Asn Lys Pro Xaa Met Phe Thr Glu Tyr Gly Val Asp Xaa Val Val 500 505 510Gly Leu Xaa Xaa Xaa Pro Asp Lys Met Xaa Pro Glu Glu Tyr Lys Met 515 520 525Xaa Phe Tyr Lys Gly Tyr Xaa Lys Ile Met Asp Lys 530 535 540 6 563 PRT Thermotoga maritima VARIANT (1)...(563) Xaa = Any Amino Acid 6Met Val Arg Pro Gln Arg Asn Lys Lys Arg Phe Ile Leu Ile Leu Asn 1 5 10 15Gly Val Trp Asn Leu Glu Val Thr Ser Lys Asp Arg Pro Ile Ala Val 20 25 30Pro Gly Ser Trp Asn Glu Gln Tyr Gln Asp Leu Cys Tyr Glu Glu Gly 35 40 45Pro Phe Thr Tyr Lys Thr Thr Phe Tyr Val Pro Lys Xaa Leu Ser Gln 50 55 60Lys His Ile Arg Leu Tyr Phe Ala Ala Val Asn Thr Asp Cys Glu Val65 70 75 80Phe Leu Asn Gly Glu Lys Val Gly Glu Asn His Ile Glu Tyr Leu Pro 85 90 95Phe Glu Val Asp Val Thr Gly Lys Val Lys Ser Gly Glu Asn Glu Leu 100 105 110Arg Val Val Val Glu Asn Arg Leu Lys Val Gly Gly Phe Pro Ser Lys 115 120 125Val Pro Asp Ser Gly Thr His Thr Val Gly Phe Phe Gly Ser Phe Pro 130 135 140Pro Ala Asn Phe Asp Phe Phe Pro Tyr Gly Gly Ile Ile Arg Pro Val145 150 155 160Leu Ile Glu Phe Thr Asp His Ala Arg Ile Leu Asp Ile Trp Val Asp 165 170 175Thr Ser Glu Ser Glu Pro Glu Lys Lys Leu Gly Lys Val Lys Val Lys 180 185 190Ile Glu Val Ser Glu Glu Ala Val Gly Gln Glu Met Thr Ile Lys Leu 195 200 205Gly Glu Glu Glu Lys Lys Ile Arg Thr Ser Asn Arg Phe Val Glu Gly 210 215 220Glu Phe Ile Leu Glu Asn Ala Arg Phe Trp Ser Leu Glu Asp Pro Tyr225 230 235 240Leu Tyr Pro Leu Lys Val Glu Leu Glu Lys Asp Glu Tyr Thr Leu Asp 245 250 255Ile Gly Ile Arg Thr Ile Ser Trp Asp Glu Lys Arg Leu Tyr Leu Asn 260 265 270Gly Lys Pro Val Phe Leu Lys Gly Phe Gly Lys His Glu Glu Phe Pro 275 280 285Val Leu Gly Gln Gly Thr Phe Tyr Pro Leu Met Ile Lys Asp Phe Asn 290 295 300Leu Leu Lys Trp Ile Asn Ala Asn Ser Phe Arg Thr Ser His Tyr Pro305 310 315 320Tyr Ser Glu Glu Trp Leu Asp Leu Ala Asp Arg Leu Gly Ile Leu Val 325 330 335Ile Asp Glu Ala Pro His Val Gly Ile Thr Arg Tyr His Tyr Asn Pro 340 345 350Glu Thr Gln Lys Ile Ala Glu Asp Asn Ile Arg Arg Met Ile Asp Arg 355 360 365His Lys Asn His Pro Ser Val Ile Met Trp Ser Val Ala Asn Glu Pro 370 375 380Glu Ser Asn His Pro Asp Ala Glu Gly Phe Phe Lys Ala Leu Tyr Glu385 390 395 400Thr Ala Asn Glu Met Asp Arg Thr Arg Pro Val Val Met Val Ser Met 405 410 415Met Asp Ala Pro Asp Glu Arg Thr Arg Asp Val Ala Leu Lys Tyr Phe 420 425 430Asp Ile Val Cys Val Asn Arg Tyr Tyr Gly Trp Tyr Ile Tyr Gln Gly 435 440 445Arg Ile Glu Glu Gly Leu Gln Ala Leu Glu Lys Asp Ile Glu Glu Leu 450 455 460Tyr Ala Arg His Arg Lys Pro Ile Phe Val Thr Glu Phe Gly Ala Asp465 470 475 480Ala Ile Ala Gly Ile His Tyr Asp Pro Pro Gln Met Phe Ser Glu Glu 485 490 495Tyr Gln Ala Glu Leu Val Glu Lys Thr Ile Arg Leu Leu Leu Lys Lys 500 505 510Asp Tyr Ile Ile Gly Thr His Val Trp Ala Phe Ala Asp Phe Lys Thr 515 520 525Pro Gln Asn Val Arg Arg Pro Ile Leu Asn His Lys Gly Val Phe Thr 530 535 540Arg Asp Arg Gln Pro Lys Leu Val Ala His Val Leu Arg Arg Leu Trp545 550 555 560Ser Glu Val 7 1806 DNA Bacillus sp. CDS (1)...(1806) 7atg tta tat cca atc aat aca gaa acc cga gga gtt ttt gat tta aat 48Met Leu Tyr Pro Ile Asn Thr Glu Thr Arg Gly Val Phe Asp Leu Asn 1 5 10 15ggg gtc tgg aat ttt aaa tta gat tac ggc aaa gga ctg gaa gaa aag 96Gly Val Trp Asn Phe Lys Leu Asp Tyr Gly Lys Gly Leu Glu Glu Lys 20 25 30tgg tat gaa tca aaa ctg aca gat acc ata tca atg gct gta cct tcc 144Trp Tyr Glu Ser Lys Leu Thr Asp Thr Ile Ser Met Ala Val Pro Ser 35 40 45tcc tat aat gat atc ggt gtt acg aag gaa att cga aac cat atc ggc 192Ser Tyr Asn Asp Ile Gly Val Thr Lys Glu Ile Arg Asn His Ile Gly 50 55 60tat gta tgg tac gag cgt gaa ttt acc gtt cct gct tat tta aaa gat 240Tyr Val Trp Tyr Glu Arg Glu Phe Thr Val Pro Ala Tyr Leu Lys Asp 65 70 75 80cag cgc atc gtc ctg cgt ttt ggt tca gca aca cat aag gct att gta 288Gln Arg Ile Val Leu Arg Phe Gly Ser Ala Thr His Lys Ala Ile Val 85 90 95tac gtt aac gga gaa cta gta gtt gaa cac aaa ggc ggc ttc tta ccg 336Tyr Val Asn Gly Glu Leu Val Val Glu His Lys Gly Gly Phe Leu Pro 100 105 110ttt gag gca gaa ata aac aac agc tta aga gac gga atg aat cgt gta 384Phe Glu Ala Glu Ile Asn Asn Ser Leu Arg Asp Gly Met Asn Arg Val 115 120 125aca gta gcg gtt gat aat att tta gat gat tct acg ctc cca gtt ggg 432Thr Val Ala Val Asp Asn Ile Leu Asp Asp Ser Thr Leu Pro Val Gly 130 135 140cta tat agt gaa aga cat gaa gaa ggt ttg gga aaa gtg att cgt aat 480Leu Tyr Ser Glu Arg His Glu Glu Gly Leu Gly Lys Val Ile Arg Asn145 150 155 160aaa cct aat ttt gac ttc ttt aac tat gca ggc tta cat cgt cct gta 528Lys Pro Asn Phe Asp Phe Phe Asn Tyr Ala Gly Leu His Arg Pro Val 165 170 175aaa att tat aca acc cct ttt acc tat gtt gag gat ata tcg gtt gta 576Lys Ile Tyr Thr Thr Pro Phe Thr Tyr Val Glu Asp Ile Ser Val Val 180 185 190acc gat ttt aac ggt cca acg gga aca gtt acg tat aca gtt gat ttt 624Thr Asp Phe Asn Gly Pro Thr Gly Thr Val Thr Tyr Thr Val Asp Phe 195 200 205cag ggt aag gca gaa acc gta aag gtt agt gta gtt gat gaa gaa ggg 672Gln Gly Lys Ala Glu Thr Val Lys Val Ser Val Val Asp Glu Glu Gly 210 215 220aaa gtt gtt gct tca act gaa ggc ctc tct ggt aat gtt gag att cct 720Lys Val Val Ala Ser Thr Glu Gly Leu Ser Gly Asn Val Glu Ile Pro225 230 235 240aac gtt atc ctt tgg gaa cct tta aat acc tat ctc tat caa att aaa 768Asn Val Ile Leu Trp Glu Pro Leu Asn Thr Tyr Leu Tyr Gln Ile Lys 245 250 255gtt gag tta gta aat gat ggt cta act att gat gta tac gaa gag cca 816Val Glu Leu Val Asn Asp Gly Leu Thr Ile Asp Val Tyr Glu Glu Pro 260 265 270ttt gga gtt cga acc gtt gaa gta aac gac ggg aaa ttc ctc att aat 864Phe Gly Val Arg Thr Val Glu Val Asn Asp Gly Lys Phe Leu Ile Asn 275 280 285aac aaa cca ttt tat ttt aaa ggg ttc gga aaa cac gag gat act cca 912Asn Lys Pro Phe Tyr Phe Lys Gly Phe Gly Lys His Glu Asp Thr Pro 290 295 300ata aat gga aga ggc ttt aat gaa gca tca aat gta atg gat ttt aat 960Ile Asn Gly Arg Gly Phe Asn Glu Ala Ser Asn Val Met Asp Phe Asn305 310 315 320att ttg aaa tgg atc ggt gcg aat tcc ttt cgg acg gcg cac tat cct 1008Ile Leu Lys Trp Ile Gly Ala Asn Ser Phe Arg Thr Ala His Tyr Pro 325 330 335tat tct gaa gaa ctg atg cgg ctc gca gat cgt gaa ggg tta gtc gtc 1056Tyr Ser Glu Glu Leu Met Arg Leu Ala Asp Arg Glu Gly Leu Val Val 340 345 350ata gat gaa acc cca gca gtt ggt gtt cat ttg aac ttt atg gca acg 1104Ile Asp Glu Thr Pro Ala Val Gly Val His Leu Asn Phe Met Ala Thr 355 360 365act ggt ttg ggc gaa ggt tca gag aga gtg agt act tgg gaa aaa atc 1152Thr Gly Leu Gly Glu Gly Ser Glu Arg Val Ser Thr Trp Glu Lys Ile 370 375 380cgg acc ttt gaa cat cat caa gat gta ctg aga gag ctg gtt tct cgt 1200Arg Thr Phe Glu His His Gln Asp Val Leu Arg Glu Leu Val Ser Arg385 390 395 400gat aaa aac cac ccc tct gtt gtc atg tgg tcg att gca aat gaa gcg 1248Asp Lys Asn His Pro Ser Val Val Met Trp Ser Ile Ala Asn Glu Ala 405 410 415gct acg gaa gaa gaa ggc gct tat gaa tac ttt aag cca tta gtt gaa 1296Ala Thr Glu Glu Glu Gly Ala Tyr Glu Tyr Phe Lys Pro Leu Val Glu 420 425 430tta acg aaa gaa tta gat cca caa aaa cgc cca gtt acc att gtt ttg 1344Leu Thr Lys Glu Leu Asp Pro Gln Lys Arg Pro Val Thr Ile Val Leu 435 440 445ttc gta atg gcg aca cca gaa aca gat aaa gtg gcg gag tta att gat 1392Phe Val Met Ala Thr Pro Glu Thr Asp Lys Val Ala Glu Leu Ile Asp 450 455 460gtg att gca ttg aat cga tac aac ggc tgg tat ttt gat ggg ggt gat 1440Val Ile Ala Leu Asn Arg Tyr Asn Gly Trp Tyr Phe Asp Gly Gly Asp465 470 475 480ctt gaa gcc gcg aaa gtc cac ctt cgt cag gaa ttt cat gcg tgg aat 1488Leu Glu Ala Ala Lys Val His Leu Arg Gln Glu Phe His Ala Trp Asn 485 490 495aaa cgc tgt cca gga aaa cct ata atg ata aca gag tat ggg gct gat 1536Lys Arg Cys Pro Gly Lys Pro Ile Met Ile Thr Glu Tyr Gly Ala Asp 500 505 510acc gta gct ggt ttt cat gat att gat ccg gtt atg ttt aca gaa gag 1584Thr Val Ala Gly Phe His Asp Ile Asp Pro Val Met Phe Thr Glu Glu 515 520 525tat cag gtt gaa tat tac caa gca aat cat gta gta ttt gat gaa ttt 1632Tyr Gln Val Glu Tyr Tyr Gln Ala Asn His Val Val Phe Asp Glu Phe 530 535 540gag aac ttt gtt ggc gag cag gcc tgg aat ttt gca gac ttt gct aca 1680Glu Asn Phe Val Gly Glu Gln Ala Trp Asn Phe Ala Asp Phe Ala Thr545 550 555 560agc cag ggt gtc atg cgt gtt caa ggt aac aaa aaa ggt gtt ttc aca 1728Ser Gln Gly Val Met Arg Val Gln Gly Asn Lys Lys Gly Val Phe Thr 565 570 575cgc gac cgc aaa cca aaa tta gca gca cat gtt ttc cgc gaa cgt tgg 1776Arg Asp Arg Lys Pro Lys Leu Ala Ala His Val Phe Arg Glu Arg Trp 580 585 590aca aac atc ccg gat ttc ggt tat aaa aat 1806Thr Asn Ile Pro Asp Phe Gly Tyr Lys Asn 595 600 8 602 PRT Bacillus sp. 8Met Leu Tyr Pro Ile Asn Thr Glu Thr Arg Gly Val Phe Asp Leu Asn Gly1 5 10 15Val Trp Asn Phe Lys Leu Asp Tyr Gly Lys Gly Leu Glu Glu Lys Trp 20 25 30Tyr Glu Ser Lys Leu Thr Asp Thr Ile Ser Met Ala Val Pro Ser Ser 35 40 45Tyr Asn Asp Ile Gly Val Thr Lys Glu Ile Arg Asn His Ile Gly Tyr 50 55 60Val Trp Tyr Glu Arg Glu Phe Thr Val Pro Ala Tyr Leu Lys Asp Gln65 70 75 80Arg Ile Val Leu Arg Phe Gly Ser Ala Thr His Lys Ala Ile Val Tyr 85 90 95Val Asn Gly Glu Leu Val Val Glu His Lys Gly Gly Phe Leu Pro Phe 100 105 110Glu Ala Glu Ile Asn Asn Ser Leu Arg Asp Gly Met Asn Arg Val Thr 115 120 125Val Ala Val Asp Asn Ile Leu Asp Asp Ser Thr Leu Pro Val Gly Leu 130 135 140Tyr Ser Glu Arg His Glu Glu Gly Leu Gly Lys Val Ile Arg Asn Lys145 150 155 160Pro Asn Phe Asp Phe Phe Asn Tyr Ala Gly Leu His Arg Pro Val Lys 165 170 175Ile Tyr Thr Thr Pro Phe Thr Tyr Val Glu Asp Ile Ser Val Val Thr 180 185 190Asp Phe Asn Gly Pro Thr Gly Thr Val Thr Tyr Thr Val Asp Phe Gln 195 200 205Gly Lys Ala Glu Thr Val Lys Val Ser Val Val Asp Glu Glu Gly Lys 210 215 220Val Val Ala Ser Thr Glu Gly Leu Ser Gly Asn Val Glu Ile Pro Asn225 230 235 240Val Ile Leu Trp Glu Pro Leu Asn Thr Tyr Leu Tyr Gln Ile Lys Val 245 250 255Glu Leu Val Asn Asp Gly Leu Thr Ile Asp Val Tyr Glu Glu Pro Phe 260 265 270Gly Val Arg Thr Val Glu Val Asn Asp Gly Lys Phe Leu Ile Asn Asn 275 280 285Lys Pro Phe Tyr Phe Lys Gly Phe Gly Lys His Glu Asp Thr Pro Ile 290 295 300Asn Gly Arg Gly Phe Asn Glu Ala Ser Asn Val Met Asp Phe Asn Ile305 310 315 320Leu Lys Trp Ile Gly Ala Asn Ser Phe Arg Thr Ala His Tyr Pro Tyr 325 330 335Ser Glu Glu Leu Met Arg Leu Ala Asp Arg Glu Gly Leu Val Val Ile 340 345 350Asp Glu Thr Pro Ala Val Gly Val His Leu Asn Phe Met Ala Thr Thr 355 360 365Gly Leu Gly Glu Gly Ser Glu Arg Val Ser Thr Trp Glu Lys Ile Arg 370 375 380Thr Phe Glu His His Gln Asp Val Leu Arg Glu Leu Val Ser Arg Asp385 390 395 400Lys Asn His Pro Ser Val Val Met Trp Ser Ile Ala Asn Glu Ala Ala 405 410 415Thr Glu Glu Glu Gly Ala Tyr Glu Tyr Phe Lys Pro Leu Val Glu Leu 420 425 430Thr Lys Glu Leu Asp Pro Gln Lys Arg Pro Val Thr Ile Val Leu Phe 435 440 445Val Met Ala Thr Pro Glu Thr Asp Lys Val Ala Glu Leu Ile Asp Val 450 455 460Ile Ala Leu Asn Arg Tyr Asn Gly Trp Tyr Phe Asp Gly Gly Asp Leu465 470 475 480Glu Ala Ala Lys Val His Leu Arg Gln Glu Phe His Ala Trp Asn Lys 485 490 495Arg Cys Pro Gly Lys Pro Ile Met Ile Thr Glu Tyr Gly Ala Asp Thr 500 505 510Val Ala Gly Phe His Asp Ile Asp Pro Val Met Phe Thr Glu Glu Tyr 515 520 525Gln Val Glu Tyr Tyr Gln Ala Asn His Val Val Phe Asp Glu Phe Glu 530 535 540Asn Phe Val Gly Glu Gln Ala Trp Asn Phe Ala Asp Phe Ala Thr Ser545 550 555 560Gln Gly Val Met Arg Val Gln Gly Asn Lys Lys Gly Val Phe Thr Arg 565 570 575Asp Arg Lys Pro Lys Leu Ala Ala His Val Phe Arg Glu Arg Trp Thr 580 585 590Asn Ile Pro Asp Phe Gly Tyr Lys Asn 595 600 9 1327 DNA Enterobacter sp. / Salmonella sp. misc_feature (1)...(1327) n = A,T,C or G 9cattggggaa actttccccc acacctactg cgtatattca ggatgttacg gttnttactg 60atgttttgga aaatactgaa caggcgaccg taactgggga atgtgggggc tgatggtgat 120attcgggttg agcttcgcga tgggcagcaa caaatagtgg cacaagggct gggggccaca 180ggtatatttg aactggataa tcctcatctt tgggaaccag gtgaagggta tttgtacgag 240ctgcgggtta cctgcgaagc caatggtgag tgtgacgaat atccagtacg tgtcggtatc 300cgttccatta cggntaaggg tgagcagttt ttgattaacc acaaaccgtt ttatttaacc 360cggttttggt cgacatgaag atgcagattt tcgcggcaaa ggtttcgacc cgggtgttga 420tggttcacga ccacgcgttg atgaactgga ttgggctaac tcctatcgca cgtcccacta 480cccttacgcg gaaaagatgc tcgattgggc tgatgagcac gtatcgtagt gattaatgaa 540accgcggcgg gtggctttaa cactttatcg ttgggaatca cttttgacgc aggcgaaaga 600cctaaagaac ttctacagcg aagaggcgat taatggcgag acttcagcag gctcacttgc 660aggctataaa agagcttatt gcccgggata aaaaccatcc aagtgtagtg tgtggagtat 720tgccaatgag cccgacaccc gtccaaatgg agccagagag tactttgcgc ctttagctaa 780ggccactcgt gaactggatc cgacacgtcc gattacctgc gtaaacgtga tgttctgcga 840tgccgaaagc gacaccatca ccgacctgtt cgacgtggtt tgtctgaatc gctattacgg 900ctggtatgtg caatcaggtg atttggaaaa agcagaacag atgctggagc aagaactgct 960ggcctggcag tcaaaactac atcgcccaat tattattacg gaatacggtg tcgatacgct 1020ggcaggaatg ccctcggttt atcccgacat gtggagtgaa aagtaccagt gaaatggctt 1080gaaatgtatc accgtgtctt tgaccggggg agcgtttgca agcgcnaagc ttagttaaca 1140ccggnggtac cgatcacgcg tnaggcgccn cccatggnca tatgngctag cntgcggccg 1200cnatgcattc tgcagcgatc gcagctgagt acacgagctc acccgcggag tcgacaagat 1260ccaagtacta cccgggnata cgtaactagt gcatgctcgc gaaatattta ggccttatcg 1320aattaat 1327 10 729 DNA Pseudomonas sp. misc_feature (1)...(729) n = A,T,C or G 10cttgctggac nacngttnag gatttttaga cacgnggagc taaagcttgc tgaccnaact 60atcacgccgg ncgtgcangc ttggaccgcg acattncctg acangngaaa nactccgcca 120tatccatctt tgctggccca acagtgagtt nacngtnncg nacnntnnga nggatcagtg 180natcgagctc cnttnanntt ctncgctaac ataacatgtn gcatatgtca atnaatnacg 240ctggncgtgg ancncaccgg gctnattcgn tgnnattcga attgnatgnc aacaactntg 300ntgcacgntg gnaaanaatt gcgtnacagg gactttggcc ncttcctaaa ccatngcatc 360ctcccnatgg gctgtacacg aatgngcccc caaaanggcn ttcagaaagg caatttntaa 420caaggcngan ntttgacttt ttcaactatg cagnnctgca ccggacgctg aaaatgtaca 480ngaccctggg tacgtncnac caagacatnn aagtngtgac cgactccatt gtnctaaccg 540ggactgtacc tataatgcgg actatcangg caatgcatga cgtngaancg acacaccagg 600atnaggaaaa caantggtgg nancncacca ngccatgatt gtcacgtttt gttagcntng 660anacnaattc nattgctttn ttagcttntt anatnagcct ntttanatta ganttctnan 720tgagactgt 729 11 1062 DNA Salmonella sp. misc_feature (1)...(1062) n = A,T,C or G 11nctcatgacc cncccntttt ngtancntnt ttgnnanctg ctgcannnga tcacnacnng 60ganncggggn gggttcgnnc tctatggcnc gnggaacnnn atgntggncn acngttnang 120actgacagac acgtggagct aaagcttgct gccgaactat cactcagntc ntgnaagttg 180gacaacacat tncctgacan gngaaaagcc cgccatatcc atactgtgct ggcccaacan 240tgagttcacn gtcgtcgnac tntatgangg atcacctgta tcganctccn ttnatnttct 300ncagctaaca taactgtgng catatgtcaa tgnatgacct ggtcggtgna ncacaccggg 360cgtnattgnt gnnattcgaa tttnatgtca acaactttgn tgcangntgg aatgaatctg 420ggggccaggg actttggcca ncttcctnaa ccattcgcan cctcccccag tgggcttgta 480cacnattgng ccccaaaaag gcntcagata ggcattttga caagctccan nttaactttt 540tcaactatgc ngncctgcac cggacgctga aaaangtaca nganccttgt acgttccacc 600aaganattta aggtgtgacc cacntccatt ttcctaacng gactgtgact nataaaggnt 660gaccnttcan ggacacattg caatgaccct ttnaaacgga anaacccccg gnttaaagga 720aaaacaaatt tggttgggna gtccanccaa gggccaatta nttgttncnc gggggantaa 780ancccccncc aatcgatctt cgaaatttaa acagcgctcc ggccgccacg tgcgaattcc 840gatatcggat gaggccagcg cnaagcttag ttaacaccgg nggtaccgat cacgcgtnag 900gcgccnccca tggncatatg ngctagcntg cggccgcnat gcattctgca gcgatcgcag 960ctgagtacac gagctcaccc gcggagtcga caagatccaa gtactacccg ggnatacgta 1020actagtgcat gctcgcgaaa tatttaggcc ttatcgaatt aa 1062 12 1738 DNA Staphylococcus warneri misc_feature (1)...(1738) n = A,T,C or G 12tanancttgt ntctgctgca cccnatcacg acagggaccc ggggngggtt cgcgctctat 60ggcncgngga acttaatgct ggactacggt tnaggactga cagacacgtg gactnaaagc 120ttgctgaccg aactatcacg actggtcgtg ctaagttgga ccacacattn cctgacaggg 180gaaanacccg ccatatccat cttgtggccc aacagtgagt taaccgtgtc gancttatat 240ganggatcac tgnattcgag ctccntctta tgttcttcgc taacatanca tgtngtcata 300tgtcaatang tgacnctggn cgtggatcac accgggctna ttgntgnatt cgaatttatg 360tcaacaactt gttgcangnt ggatgaattg gtnacaggga ctttggccan catcctatac 420catngcatcc ttccccatgg gctttaccga aagcgccacg aaaanggcct cggaaaagnc 480aatttttacn ggctccactt tgcntttttc aantatgcng anctgnaccg gacggtnana 540atgtacanga accttgtacg tcnncaagac atttaggttg tgaccgntta gcatnagcng 600tnntaaacag tagaacaatg tgtganccnt aactaaaaaa tanacagcgt taaaatcacg 660attctggatg aaaatgatca tgcaatancc gaaagcgaag gcgctaaagg caatgtaact 720attcaaaatc ctatattgtg gcaaccttta catgcctatt tatacaatat gaaagtagaa 780ttactcaacg ataatgagtg tgtagatgtt tatacagaac gtttcggtat tcgatctgtn 840gaagtgaagg atggacagtt tttaattaat gacaaaccat tttatttcaa aggtttcggt 900aaacatgaag atacctatta aaatggtcga ggcttaaacg aatcagccaa cgtcatggac 960atcaacttaa tgaaatggat aggtgctaat tcatttagaa cctctcatta cccatattca 1020gaagaaatga tgcgtttagc agatgaacaa ggtattgtag tgatagatga gacaacangt 1080gtcggtatac atcttaattt tatggnnacc ttaggtggct ccnttgcaca tgatacatgg 1140aangaatttg acactctcga gtttcataaa gaagtcatan aagacttgat tgngagagac 1200aagaatcatg catgggtagt catgtggtna tttggcaatg agcnagggtn aaataaaggg 1260ggtgctaaag catnctttga gccatttgtt aatttagcag gtgaaaaaga tnntcngnnt 1320ngcccagtga ctatcgttac tatattanct gcnnancgaa atgtatgtga agttnnagat 1380ttagtcgatg tggtttgtct nnnnagnnnn tanggttggt atncacaatc aggtgattta 1440gaaggtgcta aacnagcatt agataaggag ntagncgaat ggtggaaang acaacnaaat 1500aagccaatna tgtttacaga gtatggtgtg gatanngttg taggtttaca nncgatncct 1560gataaaatgc nnccagaaga gtataaaatg agnttttata aaggntatna taaaattatg 1620gataaacgat cgcagctgag tacacgagct cacccgcgga gtcgacaaga tccaagtact 1680acccgggnat acgtaactag tgcatgctcg cgaaatattt aggccttatc gaattaat 1738 13 628 DNA Staphylococcus homini misc_feature (1)...(628) n = A,T,C or G 13tgtgggnctt tgttccttgn tcagctcccc aacggcttga agtactcgta cgcgccctct 60tcctcagtcg ccgcctcgtt ggcgatgctc cacatcacga cgcttggatg gttcttgtca 120cgagacacca gttcacggag aacgtcttga tggtgctcaa acgtccgaat cttctcccag 180gtactgacgc gctcgctgcc ttcgccgagt cccgtggtgg ccatgaagtt gaggtgcacg 240ccaactgccg gagtctcgtc gatcacgacc agaccctcgc gatccgcaag acgcatcaac 300tcttcagagt acggatagtg tgcggtccgg aagctgttgg cgccgatcca tttgaggata 360ttgaaatcca tcacattgct cgcttcgtta aagccacggc cgttgatagg agtgtcctca 420tgtttgccaa agcccttgaa gtagaacggt ttgttgttga tgaggaactt gccgtcgttg 480acttcacggt ccgcacgccg aacggctctt catagacatc gatggtcaag tcccgtcgtt 540caccagttcc actttgatct ggtagagata cgtgttcaag tggttcccag aggatgacat 600tcggaatctt cacgttaccg ctcaagcc 628 14 1689 DNA Thermotoga maritima misc_feature (1)...(1689) n = A,T,C or G 14atggtaagac cgcaacgaaa caagaagaga tttattctta tcttgaatgg agtttggaat 60cttgaagtaa ccagcaaaga cagaccaatc gccgttcctg gaagctggaa tgagcagtac 120caggatctgt gctacgaaga aggacccttc acctacaaaa ccaccttcta cgttccgaag 180naactttcac aaaaacacat cagactttac tttgctgcgg tgaacacgga ctgcgaggtc 240ttcctcaacg gagagaaagt gggagagaat cacattgaat accttccctt cgaagtagat 300gtgacgggga aagtgaaatc cggagagaac gaactcaggg tggttgttga gaacagattg 360aaagtgggag gatttccctc gaaggttcca gacagcggca ctcacaccgt gggatttttt 420ggaagttttc cacctgcaaa cttcgacttc ttcccctacg gtggaatcat aaggcctgtt 480ctgatagagt tcacagacca cgcgaggata ctcgacatct gggtggacac gagtgagtct 540gaaccggaga agaaacttgg aaaagtgaaa gtgaagatag aagtctcaga agaagcggtg 600ggacaggaga tgacgatcaa acttggagag gaagagaaaa agattagaac atccaacaga 660ttcgtcgaag gggagttcat cctcgaaaac gccaggttct ggagcctcga agatccatat 720ctttatcctc tcaaggtgga acttgaaaaa gacgagtaca ctctggacat cggaatcaga 780acgatcagct gggacgagaa gaggctctat ctgaacggga aacctgtctt tttgaagggc 840tttggaaagc acgaggaatt ccccgttctg gggcagggca ccttttatcc attgatgata 900aaagacttca accttctgaa gtggatcaac gcgaattctt tcaggacctc tcactatcct 960tacagtgaag agtggctgga tcttgccgac agactcggaa tccttgtgat agacgaagcc 1020ccgcacgttg gtatcacaag gtaccactac aatcccgaga ctcagaagat agcagaagac 1080aacataagaa gaatgatcga cagacacaag aaccatccca gtgtgatcat gtggagtgtg 1140gcgaacgaac cagagtccaa ccatccagac gcggagggtt tcttcaaagc cctttatgag 1200actgccaatg aaatggatcg aacacgcccc gttgtcatgg tgagcatgat ggacgcacca 1260gacgagagaa caagagacgt ggcgctgaag tacttcgaca tcgtctgtgt gaacaggtac 1320tacggctggt acatctatca gggaaggata gaagaaggac ttcaagctct ggaaaaagac 1380atagaagagc tctatgcaag gcacagaaag cccatctttg tcacagaatt cggtgcggac 1440gcgatagctg gcatccacta cgatccacct caaatgttct ccgaagagta ccaagcagag 1500ctcgttgaaa agacgatcag gctccttttg aaaaaagact acatcatcgg aacacacgtg 1560tgggcctttg cagattttaa gactcctcag aatgtgagaa gacccattct caaccacaag 1620ggtgttttca caagagacag acaacccaaa ctcgttgctc atgtactgag aagactgtgg 1680agtgaggtt 1689 15 602 PRT Bacillus sp. 15Met Leu Tyr Pro Ile Asn Thr Glu Thr Arg Gly Val Phe Asp Leu Asn 1 5 10 15Gly Val Trp Asn Phe Lys Leu Asp Tyr Gly Lys Gly Leu Glu Glu Lys 20 25 30Trp Tyr Glu Ser Lys Leu Thr Asp Thr Ile Ser Met Ala Val Pro Ser 35 40 45Ser Tyr Asn Asp Ile Gly Val Thr Lys Glu Ile Arg Asn His Ile Gly 50 55 60Tyr Val Trp Tyr Glu Arg Glu Phe Thr Val Pro Ala Tyr Leu Lys Asp65 70 75 80Gln Arg Ile Val Leu Arg Phe Gly Ser Ala Thr His Lys Ala Ile Val 85 90 95Tyr Val Asn Gly Glu Leu Val Val Glu His Lys Gly Gly Phe Leu Pro 100 105 110Phe Glu Ala Glu Ile Asn Asn Ser Leu Arg Asp Gly Met Asn Arg Val 115 120 125Thr Val Ala Val Asp Asn Ile Leu Asp Asp Ser Thr Leu Pro Val Gly 130 135 140Leu Tyr Ser Glu Arg His Glu Glu Gly Leu Gly Lys Val Ile Arg Asn145 150 155 160Lys Pro Asn Phe Asp Phe Phe Asn Tyr Ala Gly Leu His Arg Pro Val 165 170 175Lys Ile Tyr Thr Thr Pro Phe Thr Tyr Val Glu Asp Ile Ser Val Val 180 185 190Thr Asp Phe Asn Gly Pro Thr Gly Thr Val Thr Tyr Thr Val Asp Phe 195 200 205Gln Gly Lys Ala Glu Thr Val Lys Val Ser Val Val Asp Glu Glu Gly 210 215 220Lys Val Val Ala Ser Thr Glu Gly Leu Ser Gly Asn Val Glu Ile Pro225 230 235 240Asn Val Ile Leu Trp Glu Pro Leu Asn Thr Tyr Leu Tyr Gln Ile Lys 245 250 255Val Glu Leu Val Asn Asp Gly Leu Thr Ile Asp Val Tyr Glu Glu Pro 260 265 270Phe Gly Val Arg Thr Val Glu Val Asn Asp Gly Lys Phe Leu Ile Asn 275 280 285Asn Lys Pro Phe Tyr Phe Lys Gly Phe Gly Lys His Glu Asp Thr Pro 290 295 300Ile Asn Gly Arg Gly Phe Asn Glu Ala Ser Asn Val Met Asp Phe Asn305 310 315 320Ile Leu Lys Trp Ile Gly Ala Asn Ser Phe Arg Thr Ala His Tyr Pro 325 330 335Tyr Ser Glu Glu Leu Met Arg Leu Ala Asp Arg Glu Gly Leu Val Val 340 345 350Ile Asp Glu Thr Pro Ala Val Gly Val His Leu Asn Phe Met Ala Thr 355 360 365Thr Gly Leu Gly Glu Gly Ser Glu Arg Val Ser Thr Trp Glu Lys Ile 370 375 380Arg Thr Phe Glu His His Gln Asp Val Leu Arg Glu Leu Val Ser Arg385 390 395 400Asp Lys Asn His Pro Ser Val Val Met Trp Ser Ile Ala Asn Glu Ala 405 410 415Ala Thr Glu Glu Glu Gly Ala Tyr Glu Tyr Phe Lys Pro Leu Val Glu 420 425 430Leu Thr Lys Glu Leu Asp Pro Gln Lys Arg Pro Val Thr Ile Val Leu 435 440 445Phe Val Met Ala Thr Pro Glu Thr Asp Lys Val Ala Glu Leu Ile Asp 450 455 460Val Ile Ala Leu Asn Arg Tyr Asn Gly Trp Tyr Phe Asp Gly Gly Asp465 470 475 480Leu Glu Ala Ala Lys Val His Leu Arg Gln Glu Phe His Ala Trp Asn 485 490 495Lys Arg Cys Pro Gly Lys Pro Ile Met Ile Thr Glu Tyr Gly Ala Asp 500 505 510Thr Val Ala Gly Phe His Asp Ile Asp Pro Val Met Phe Thr Glu Glu 515 520 525Tyr Gln Val Glu Tyr Tyr Gln Ala Asn His Val Val Phe Asp Glu Phe 530 535 540Glu Asn Phe Val Gly Glu Gln Ala Trp Asn Phe Ala Asp Phe Ala Thr545 550 555 560Ser Gln Gly Val Met Arg Val Gln Gly Asn Lys Lys Gly Val Phe Thr 565 570 575Arg Asp Arg Lys Pro Lys Leu Ala Ala His Val Phe Arg Glu Arg Trp 580 585 590Thr Asn Ile Pro Asp Phe Gly Tyr Lys Asn 595 600 16 613 PRT Homo sapien 16Leu Gly Leu Gln Gly Gly Met Leu Tyr Pro Gln Glu Ser Pro Ser Arg 1 5 10 15Glu Cys Lys Glu Leu Asp Gly Leu Trp Ser Phe Arg Ala Asp Phe Ser 20 25 30Asp Asn Arg Arg Arg Gly Phe Glu Glu Gln Trp Tyr Arg Arg Pro Leu 35 40 45Trp Glu Ser Gly Pro Thr Val Asp Met Pro Val Pro Ser Ser Phe Asn 50 55 60Asp Ile Ser Gln Asp Trp Arg Leu Arg His Phe Val Gly Trp Val Trp65 70 75 80Tyr Glu Arg Glu Val Ile Leu Pro Glu Arg Trp Thr Gln Asp Leu Arg 85 90 95Thr Arg Val Val Leu Arg Ile Gly Ser Ala His Ser Tyr Ala Ile Val 100 105 110Trp Val Asn Gly Val Asp Thr Leu Glu His Glu Gly Gly Tyr Leu Pro 115 120 125Phe Glu Ala Asp Ile Ser Asn Leu Val Gln Val Gly Pro Leu Pro Ser 130 135 140Arg Leu Arg Ile Thr Ile Ala Ile Asn Asn Thr Leu Thr Pro Thr Thr145 150 155 160Leu Pro Pro Gly Thr Ile Gln Tyr Leu Thr Asp Thr Ser Lys Tyr Pro 165 170 175Lys Gly Tyr Phe Val Gln Asn Thr Tyr Phe Asp Phe Phe Asn Tyr Ala 180 185 190Gly Leu Gln Arg Ser Val Leu Leu Tyr Thr Thr Pro Thr Thr Tyr Ile 195 200 205Asp Asp Ile Thr Val Thr Thr Ser Val Glu Gln Asp Ser Gly Leu Val 210 215 220Asn Tyr Gln Ile Ser Val Lys Gly Ser Asn Leu Phe Lys Leu Glu Val225 230 235 240Arg Leu Leu Asp Ala Glu Asn Lys Val Val Ala Asn Gly Thr Gly Thr 245 250 255Gln Gly Gln Leu Lys Val Pro Gly Val Ser Leu Trp Trp Pro Tyr Leu 260 265 270Met His Glu Arg Pro Ala Tyr Leu Tyr Ser Leu Glu Val Gln Leu Thr 275 280 285Ala Gln Thr Ser Leu Gly Pro Val Ser Asp Phe Tyr Thr Leu Pro Val 290 295 300Gly Ile Arg Thr Val Ala Val Thr Lys Ser Gln Phe Leu Ile Asn Gly305 310 315 320Lys Pro Phe Tyr Phe His Gly Val Asn Lys His Glu Asp Ala Asp Ile 325 330 335Arg Gly Lys Gly Phe Asp Trp Pro Leu Leu Val Lys Asp Phe Asn Leu 340 345 350Leu Arg Trp Leu Gly Ala Asn Ala Phe Arg Thr Ser His Tyr Pro Tyr 355 360 365Ala Glu Glu Val Met Gln Met Cys Asp Arg Tyr Gly Ile Val Val Ile 370 375 380Asp Glu Cys Pro Gly Val Gly Leu Ala Leu Pro Gln Phe Phe Asn Asn385 390 395 400Val Ser Leu His His His Met Gln Val Met Glu Glu Val Val Arg Arg 405 410 415Asp Lys Asn His Pro Ala Val Val Met Trp Ser Val Ala Asn Glu Pro 420 425 430Ala Ser His Leu Glu Ser Ala Gly Tyr Tyr Leu Lys Met Val Ile Ala 435 440 445His Thr Lys Ser Leu Asp Pro Ser Arg Pro Val Thr Phe Val Ser Asn 450 455 460Ser Asn Tyr Ala Ala Asp Lys Gly Ala Pro Tyr Val Asp Val Ile Cys465 470 475 480Leu Asn Ser Tyr Tyr Ser Trp Tyr His Asp Tyr Gly His Leu Glu Leu 485 490 495Ile Gln Leu Gln Leu Ala Thr Gln Phe Glu Asn Trp Tyr Lys Lys Tyr 500 505 510Gln Lys Pro Ile Ile Gln Ser Glu Tyr Gly Ala Glu Thr Ile Ala Gly 515 520 525Phe His Gln Asp Pro Pro Leu Met Phe Thr Glu Glu Tyr Gln Lys Ser 530 535 540Leu Leu Glu Gln Tyr His Leu Gly Leu Asp Gln Lys Arg Arg Lys Tyr545 550 555 560Val Val Gly Glu Leu Ile Trp Asn Phe Ala Asp Phe Met Thr Glu Gln 565 570 575Ser Pro Thr Arg Val Leu Gly Asn Lys Lys Gly Ile Phe Thr Arg Gln 580 585 590Arg Gln Pro Lys Ser Ala Ala Phe Leu Leu Arg Glu Arg Tyr Trp Lys 595 600 605Ile Ala Asn Glu Thr 610 17 603 PRT Escherichia coli 17Met Leu Arg Pro Val Glu Thr Pro Thr Arg Glu Ile Lys Lys Leu Asp 1 5 10 15Gly Leu Trp Ala Phe Ser Leu Asp Arg Glu Asn Cys Gly Ile Asp Gln 20 25 30Arg Trp Trp Glu Ser Ala Leu Gln Glu Ser Arg Ala Ile Ala Val Pro 35 40 45Gly Ser Phe Asn Asp Gln Phe Ala Asp Ala Asp Ile Arg Asn Tyr Ala 50 55 60Gly Asn Val Trp Tyr Gln Arg Glu Val Phe Ile Pro Lys Gly Trp Ala65 70 75 80Gly Gln Arg Ile Val Leu Arg Phe Asp Ala Val Thr His Tyr Gly Lys 85 90 95Val Trp Val Asn Asn Gln Glu Val Met Glu His Gln Gly Gly Tyr Thr 100 105 110Pro Phe Glu Ala Asp Val Thr Pro Tyr Val Ile Ala Gly Lys Ser Val 115 120 125Arg Ile Thr Val Cys Val Asn Asn Glu Leu Asn Trp Gln Thr Ile Pro 130 135 140Pro Gly Met Val Ile Thr Asp Glu Asn Gly Lys Lys Lys Gln Ser Tyr145 150 155 160Phe His Asp Phe Phe Asn Tyr Ala Gly Ile His Arg Ser Val Met Leu 165 170 175Tyr Thr Thr Pro Asn Thr Trp Val Asp Asp Ile Thr Val Val Thr His 180 185 190Val Ala Gln Asp Cys Asn His Ala Ser Val Asp Trp Gln Val Val Ala 195 200 205Asn Gly Asp Val Ser Val Glu Leu Arg Asp Ala Asp Gln Gln Val Val 210 215 220Ala Thr Gly Gln Gly Thr Ser Gly Thr Leu Gln Val Val Asn Pro His225 230 235 240Leu Trp Gln Pro Gly Glu Gly Tyr Leu Tyr Glu Leu Cys Val Thr Ala 245 250 255Lys Ser Gln Thr Glu Cys Asp Ile Tyr Pro Leu Arg Val Gly Ile Arg 260 265 270Ser Val Ala Val Lys Gly Glu Gln Phe Leu Ile Asn His Lys Pro Phe 275 280 285Tyr Phe Thr Gly Phe Gly Arg His Glu Asp Ala Asp Leu Arg Gly Lys 290 295 300Gly Phe Asp Asn Val Leu Met Val His Asp His Ala Leu Met Asp Trp305 310 315 320Ile Gly Ala Asn Ser Tyr Arg Thr Ser His Tyr Pro Tyr Ala Glu Glu 325 330 335Met Leu Asp Trp Ala Asp Glu His Gly Ile Val Val Ile Asp Glu Thr 340 345 350Ala Ala Val Gly Phe Asn Leu Ser Leu Gly Ile Gly Phe Glu Ala Gly 355 360 365Asn Lys Pro Lys Glu Leu Tyr Ser Glu Glu Ala Val Asn Gly Glu Thr 370 375 380Gln Gln Ala His Leu Gln Ala Ile Lys Glu Leu Ile Ala Arg Asp Lys385 390 395 400Asn His Pro Ser Val Val Met Trp Ser Ile Ala Asn Glu Pro Asp Thr 405 410 415Arg Pro Gln Gly Ala Arg Glu Tyr Phe Ala Pro Leu Ala Glu Ala Thr 420 425 430Arg Lys Leu Asp Pro Thr Arg Pro Ile Thr Cys Val Asn Val Met Phe 435 440 445Cys Asp Ala His Thr Asp Thr Ile Ser Asp Leu Phe Asp Val Leu Cys 450 455 460Leu Asn Arg Tyr Tyr Gly Trp Tyr Val Gln Ser Gly Asp Leu Glu Thr465 470 475 480Ala Glu Lys Val Leu Glu Lys Glu Leu Leu Ala Trp Gln Glu Lys Leu 485 490 495His Gln Pro Ile Ile Ile Thr Glu Tyr Gly Val Asp Thr Leu Ala Gly 500 505 510Leu His Ser Met Tyr Thr Asp Met Trp Ser Glu Glu Tyr Gln Cys Ala 515 520 525Trp Leu Asp Met Tyr His Arg Val Phe Asp Arg Val Ser Ala Val Val 530 535 540Gly Glu Gln Val Trp Asn Phe Ala Asp Phe Ala Thr Ser Gln Gly Ile545 550 555 560Leu Arg Val Gly Gly Asn Lys Lys Gly Ile Phe Thr Arg Asp Arg Lys 565 570 575Pro Lys Ser Ala Ala Phe Leu Leu Gln Lys Arg Trp Thr Gly Met Asn 580 585 590Phe Gly Glu Lys Pro Gln Gln Gly Gly Lys Gln 595 600 18 607 PRT Bacillus sp. 18Met Val Asp Leu Thr Ser Leu Tyr Pro Ile Asn Thr Glu Thr Arg Gly 1 5 10 15Val Phe Asp Leu Asn Gly Val Trp Asn Phe Lys Leu Asp Tyr Gly Lys 20 25 30Gly Leu Glu Glu Lys Trp Tyr Glu Ser Lys Leu Thr Asp Thr Ile Ser 35 40 45Met Ala Val Pro Ser Ser Tyr Asn Asp Ile Gly Val Thr Lys Glu Ile 50 55 60Arg Asn His Ile Gly Tyr Val Trp Tyr Glu Arg Glu Phe Thr Val Pro65 70 75 80Ala Tyr Leu Lys Asp Gln Arg Ile Val Leu Arg Phe Gly Ser Ala Thr 85 90 95His Lys Ala Ile Val Tyr Val Asn Gly Glu Leu Val Val Glu His Lys 100 105 110Gly Gly Phe Leu Pro Phe Glu Ala Glu Ile Asn Asn Ser Leu Arg Asp 115 120 125Gly Met Asn Arg Val Thr Val Ala Val Asp Asn Ile Leu Asp Asp Ser 130 135 140Thr Leu Pro Val Gly Leu Tyr Ser Glu Arg His Glu Glu Gly Leu Gly145 150 155 160Lys Val Ile Arg Asn Lys Pro Asn Phe Asp Phe Phe Asn Tyr Ala Gly 165 170 175Leu His Arg Pro Val Lys Ile Tyr Thr Thr Pro Phe Thr Tyr Val Glu 180 185 190Asp Ile Ser Val Val Thr Asp Phe Asn Gly Pro Thr Gly Thr Val Thr 195 200 205Tyr Thr Val Asp Phe Gln Gly Lys Ala Glu Thr Val Lys Val Ser Val 210 215 220Val Asp Glu Glu Gly Lys Val Val Ala Ser Thr Glu Gly Leu Ser Gly225 230 235 240Asn Val Glu Ile Pro Asn Val Ile Leu Trp Glu Pro Leu Asn Thr Tyr 245 250 255Leu Tyr Gln Ile Lys Val Glu Leu Val Asn Asp Gly Leu Thr Ile Asp 260 265 270Val Tyr Glu Glu Pro Phe Gly Val Arg Thr Val Glu Val Asn Asp Gly 275 280 285Lys Phe Leu Ile Asn Asn Lys Pro Phe Tyr Phe Lys Gly Phe Gly Lys 290 295 300His Glu Asp Thr Pro Ile Asn Gly Arg Gly Phe Asn Glu Ala Ser Asn305 310 315 320Val Met Asp Phe Asn Ile Leu Lys Trp Ile Gly Ala Asn Ser Phe Arg 325 330 335Thr Ala His Tyr Pro Tyr Ser Glu Glu Leu Met Arg Leu Ala Asp Arg 340 345 350Glu Gly Leu Val Val Ile Asp Glu Thr Pro Ala Val Gly Val His Leu 355 360 365Asn Phe Met Ala Thr Thr Gly Leu Gly Glu Gly Ser Glu Arg Val Ser 370 375 380Thr Trp Glu Lys Ile Arg Thr Phe Glu His His Gln Asp Val Leu Arg385 390 395 400Glu Leu Val Ser Arg Asp Lys Asn His Pro Ser Val Val Met Trp Ser 405 410 415Ile Ala Asn Glu Ala Ala Thr Glu Glu Glu Gly Ala Tyr Glu Tyr Phe 420 425 430Lys Pro Leu Val Glu Leu Thr Lys Glu Leu Asp Pro Gln Lys Arg Pro 435 440 445Val Thr Ile Val Leu Phe Val Met Ala Thr Pro Glu Thr Asp Lys Val 450 455 460Ala Glu Leu Ile Asp Val Ile Ala Leu Asn Arg Tyr Asn Gly Trp Tyr465 470 475 480Phe Asp Gly Gly Asp Leu Glu Ala Ala Lys Val His Leu Arg Gln Glu 485 490 495Phe His Ala Trp Asn Lys Arg Cys Pro Gly Lys Pro Ile Met Ile Thr 500 505 510Glu Tyr Gly Ala Asp Thr Val Ala Gly Phe His Asp Ile Asp Pro Val 515 520 525Met Phe Thr Glu Glu Tyr Gln Val Glu Tyr Tyr Gln Ala Asn His Val 530 535 540Val Phe Asp Glu Phe Glu Asn Phe Val Gly Glu Gln Ala Trp Asn Phe545 550 555 560Ala Asp Phe Ala Thr Ser Gln Gly Val Met Arg Val Gln Gly Asn Lys 565 570 575Lys Gly Val Phe Thr Arg Asp Arg Lys Pro Lys Leu Ala Ala His Val 580 585 590Phe Arg Glu Arg Trp Thr Asn Ile Pro Asp Phe Gly Tyr Lys Asn 595 600 605 19 376 PRT Staphylococcus homini VARIANT (1)...(376) Xaa = Any Amino Acid 19Gly Leu Ser Gly Asn Val Glu Ile Pro Asn Val Ile Leu Trp Glu Pro 1 5 10 15Leu Asn Thr Tyr Leu Tyr Gln Ile Lys Val Glu Leu Val Asn Asp Gly 20 25 30Leu Thr Ile Asp Val Tyr Glu Glu Pro Phe Gly Val Arg Thr Val Glu 35 40 45Val Asn Asp Gly Lys Phe Leu Ile Asn Asn Lys Pro Phe Tyr Phe Lys 50 55 60Gly Phe Gly Lys His Glu Asp Thr Pro Ile Asn Gly Arg Gly Phe Asn65 70 75 80Glu Ala Ser Asn Val Met Asp Phe Asn Ile Leu Lys Trp Ile Gly Ala 85 90 95Asn Ser Phe Arg Thr Ala His Tyr Pro Tyr Ser Glu Glu Leu Met Arg 100 105 110Leu Ala Asp Arg Glu Gly Leu Val Val Ile Asp Glu Thr Pro Ala Val 115 120 125Gly Val His Leu Asn Phe Met Ala Thr Thr Gly Leu Gly Glu Gly Ser 130 135 140Glu Arg Val Ser Thr Trp Glu Lys Ile Arg Thr Phe Glu His His Gln145 150 155 160Asp Val Leu Arg Glu Leu Val Ser Arg Asp Lys Asn His Pro Ser Val 165 170 175Val Met Trp Ser Ile Ala Asn Glu Ala Ala Thr Glu Glu Glu Gly Ala 180 185 190Tyr Glu Tyr Phe Lys Pro Leu Gly Gly Ala Ala Lys Glu Leu Asp Pro 195 200 205Xaa Lys Arg Pro Val Thr Ile Val Leu Phe Val Met Ala Thr Pro Glu 210 215 220Thr Asp Lys Val Ala Glu Leu Ile Asp Val Ile Ala Leu Asn Arg Tyr225 230 235 240Asn Gly Trp Tyr Phe Asp Gly Gly Asp Leu Glu Ala Ala Lys Val His 245 250 255Leu Arg Gln Glu Phe His Ala Trp Asn Lys Arg Cys Pro Gly Lys Pro 260 265 270Ile Met Ile Thr Glu Tyr Gly Ala Asp Thr Val Ala Gly Phe His Asp 275 280 285Ile Asp Pro Val Met Phe Thr Glu Glu Tyr Gln Val Glu Tyr Tyr Gln 290 295 300Ala Asn His Val Val Phe Asp Glu Phe Glu Asn Phe Val Gly Glu Gln305 310 315 320Ala Trp Asn Phe Ala Asp Phe Ala Thr Ser Gln Gly Val Met Arg Val 325 330 335Gln Gly Asn Lys Lys Gly Val Phe Thr Arg Asp Arg Lys Pro Xaa Leu 340 345 350Ala Ala His Val Phe Arg Glu Arg Arg Thr Asn Ile Pro Asp Phe Gly 355 360 365Tyr Lys Asn Ala Ser His His His 370 375 20 535 PRT Staphylococcus warneri VARIANT (1)...(535) Xaa = Any Amino Acid 20Leu Xaa Leu Leu His Pro Ile Thr Thr Gly Thr Arg Gly Gly Phe Ala 1 5 10 15Leu Tyr Gly Xaa Xaa Asn Leu Met Leu Asp Tyr Gly Xaa Gly Leu Thr 20 25 30Asp Thr Trp Thr Xaa Ser Leu Leu Thr Glu Leu Ser Arg Leu Val Val 35 40 45Leu Ser Trp Thr Thr His Xaa Leu Thr Gly Glu Xaa Pro Ala Ile Ser 50 55 60Ile Leu Trp Pro Asn Ser Glu Leu Thr Val Ser Xaa Leu Tyr Xaa Gly65 70 75 80Ser Leu Xaa Ser Ser Ser Xaa Leu Cys Ser Ser Leu Thr Xaa His Val 85 90 95Val Ile Cys Gln Xaa Val Thr Leu Xaa Val Asp His Thr Gly Leu Ile 100 105 110Xaa Xaa Phe Glu Phe Met Ser Thr Thr Cys Cys Xaa Xaa Asp Glu Leu 115 120 125Val Thr Gly Thr Leu Ala Xaa Ile Leu Tyr His Xaa Ile Leu Pro His 130 135 140Gly Leu Tyr Arg Lys Arg His Glu Xaa Gly Leu Gly Lys Xaa Asn Phe145 150 155 160Tyr Xaa Leu His Phe Ala Phe Phe Xaa Tyr Ala Xaa Leu Xaa Arg Thr 165 170 175Val Xaa Met Tyr Xaa Asn Leu Val Arg Xaa Gln Asp Ile Val Val Thr 180 185 190Xaa His Xaa Xaa Xaa Thr Val Glu Gln Cys Val Xaa Xaa Asn Lys Ile 195 200 205Xaa Ser Val Lys Ile Thr Ile Leu Asp Glu Asn Asp His Ala Ile Xaa 210 215 220Glu Ser Glu Gly Ala Lys Gly Asn Val Thr Ile Gln Asn Pro Ile Leu225 230 235 240Trp Gln Pro Leu His Ala Tyr Leu Tyr Asn Met Lys Val Glu Leu Leu 245 250 255Asn Asp Asn Glu Cys Val Asp Val Tyr Thr Glu Arg Phe Gly Ile Arg 260 265 270Ser Val Glu Val Lys Asp Gly Gln Phe Leu Ile Asn Asp Lys Pro Phe 275 280 285Tyr Phe Lys Gly Phe Gly Lys His Glu Asp Thr Tyr Asn Gly Arg Gly 290 295 300Leu Asn Glu Ser Ala Asn Val Met Asp Ile Asn Leu Met Lys Trp Ile305 310 315 320Gly Ala Asn Ser Phe Arg Thr Ser His Tyr Pro Tyr Ser Glu Glu Met 325 330 335Met Arg Leu Ala Asp Glu Gln Gly Ile Val Val Ile Asp Glu Thr Thr 340 345 350Xaa Val Gly Ile His Leu Asn Phe Met Xaa Thr Leu Gly Gly Ser Xaa 355 360 365Ala His Asp Thr Trp Xaa Glu Phe Asp Thr Leu Glu Phe His Lys Glu 370 375 380Val Ile Xaa Asp Leu Ile Xaa Arg Asp Lys Asn His Ala Trp Val Val385 390 395 400Met Trp Xaa Phe Gly Asn Glu Xaa Gly Xaa Asn Lys Gly Gly Ala Lys 405 410 415Ala Xaa Phe Glu Pro Phe Val Asn Leu Ala Gly Glu Lys Asp Xaa Xaa 420 425 430Xaa Xaa Pro Val Thr Ile Val Thr Ile Leu Xaa Ala Xaa Arg Asn Val 435 440 445Cys Glu Val Xaa Asp Leu Val Asp Val Val Cys Leu Xaa Xaa Xaa Xaa 450 455 460Gly Trp Tyr Xaa Gln Ser Gly Asp Leu Glu Gly Ala Lys Xaa Ala Leu465 470 475 480Asp Lys Glu Xaa Xaa Glu Trp Trp Lys Xaa Gln Xaa Asn Lys Pro Xaa 485 490 495Met Phe Thr Glu Tyr Gly Val Asp Xaa Val Val Gly Leu Xaa Xaa Xaa 500 505 510Pro Asp Lys Met Xaa Pro Glu Glu Tyr Lys Met Xaa Phe Tyr Lys Gly 515 520 525Tyr Xaa Lys Ile Met Asp Lys 530 535 21 563 PRT Thermotoga maritima VARIANT (1)...(563) Xaa = Any Amino Acid 21Met Val Arg Pro Gln Arg Asn Lys Lys Arg Phe Ile Leu Ile Leu Asn 1 5 10 15Gly Val Trp Asn Leu Glu Val Thr Ser Lys Asp Arg Pro Ile Ala Val 20 25 30Pro Gly Ser Trp Asn Glu Gln Tyr Gln Asp Leu Cys Tyr Glu Glu Gly 35 40 45Pro Phe Thr Tyr Lys Thr Thr Phe Tyr Val Pro Lys Xaa Leu Ser Gln 50 55 60Lys His Ile Arg Leu Tyr Phe Ala Ala Val Asn Thr Asp Cys Glu Val65 70 75 80Phe Leu Asn Gly Glu Lys Val Gly Glu Asn His Ile Glu Tyr Leu Pro 85 90 95Phe Glu Val Asp Val Thr Gly Lys Val Lys Ser Gly Glu Asn Glu Leu 100 105 110Arg Val Val Val Glu Asn Arg Leu Lys Val Gly Gly Phe Pro Ser Lys 115 120 125Val Pro Asp Ser Gly Thr His Thr Val Gly Phe Phe Gly Ser Phe Pro 130 135 140Pro Ala Asn Phe Asp Phe Phe Pro Tyr Gly Gly Ile Ile Arg Pro Val145 150 155 160Leu Ile Glu Phe Thr Asp His Ala Arg Ile Leu Asp Ile Trp Val Asp 165 170 175Thr Ser Glu Ser Glu Pro Glu Lys Lys Leu Gly Lys Val Lys Val Lys 180 185 190Ile Glu Val Ser Glu Glu Ala Val Gly Gln Glu Met Thr Ile Lys Leu 195 200 205Gly Glu Glu Glu Lys Lys Ile Arg Thr Ser Asn Arg Phe Val Glu Gly 210 215 220Glu Phe Ile Leu Glu Asn Ala Arg Phe Trp Ser Leu Glu Asp Pro Tyr225 230 235 240Leu Tyr Pro Leu Lys Val Glu Leu Glu Lys Asp Glu Tyr Thr Leu Asp 245 250 255Ile Gly Ile Arg Thr Ile Ser Trp Asp Glu Lys Arg Leu Tyr Leu Asn 260 265 270Gly Lys Pro Val Phe Leu Lys Gly Phe Gly Lys His Glu Glu Phe Pro 275 280 285Val Leu Gly Gln Gly Thr Phe Tyr Pro Leu Met Ile Lys Asp Phe Asn 290 295 300Leu Leu Lys Trp Ile Asn Ala Asn Ser Phe Arg Thr Ser His Tyr Pro305 310 315 320Tyr Ser Glu Glu Trp Leu Asp Leu Ala Asp Arg Leu Gly Ile Leu Val 325 330 335Ile Asp Glu Ala Pro His Val Gly Ile Thr Arg Tyr His Tyr Asn Pro 340 345 350Glu Thr Gln Lys Ile Ala Glu Asp Asn Ile Arg Arg Met Ile Asp Arg 355 360 365His Lys Asn His Pro Ser Val Ile Met Trp Ser Val Ala Asn Glu Pro 370 375 380Glu Ser Asn His Pro Asp Ala Glu Gly Phe Phe Lys Ala Leu Tyr Glu385 390 395 400Thr Ala Asn Glu Met Asp Arg Thr Arg Pro Val Val Met Val Ser Met 405 410 415Met Asp Ala Pro Asp Glu Arg Thr Arg Asp Val Ala Leu Lys Tyr Phe 420 425 430Asp Ile Val Cys Val Asn Arg Tyr Tyr Gly Trp Tyr Ile Tyr Gln Gly 435 440 445Arg Ile Glu Glu Gly Leu Gln Ala Leu Glu Lys Asp Ile Glu Glu Leu 450 455 460Tyr Ala Arg His Arg Lys Pro Ile Phe Val Thr Glu Phe Gly Ala Asp465 470 475 480Ala Ile Ala Gly Ile His Tyr Asp Pro Pro Gln Met Phe Ser Glu Glu 485 490 495Tyr Gln Ala Glu Leu Val Glu Lys Thr Ile Arg Leu Leu Leu Lys Lys 500 505 510Asp Tyr Ile Ile Gly Thr His Val Trp Ala Phe Ala Asp Phe Lys Thr 515 520 525Pro Gln Asn Val Arg Arg Pro Ile Leu Asn His Lys Gly Val Phe Thr 530 535 540Arg Asp Arg Gln Pro Lys Leu Val Ala His Val Leu Arg Arg Leu Trp545 550 555 560Ser Glu Val 22 372 PRT Enterobacter sp. / Salmonella sp. VARIANT (1)...(372) Xaa = Any Amino Acid 22Gly Lys Leu Ser Pro Thr Pro Thr Ala Tyr Ile Gln Asp Val Thr Val 1 5 10 15Xaa Thr Asp Val Leu Glu Asn Thr Glu Gln Ala Thr Val Leu Gly Asn 20 25 30Val Gly Ala Asp Gly Asp Ile Arg Val Glu Leu Arg Asp Gly Gln Gln 35 40 45Gln Ile Val Ala Gln Gly Leu Gly Ala Thr Gly Ile Phe Glu Leu Asp 50 55 60Asn Pro His Leu Trp Glu Pro Gly Glu Gly Tyr Leu Tyr Glu Leu Arg65 70 75 80Val Thr Cys Glu Ala Asn Gly Glu Cys Asp Glu Tyr Pro Val Arg Val 85 90 95Gly Ile Arg Ser Ile Thr Xaa Lys Gly Glu Gln Phe Leu Ile Asn His 100 105 110Lys Pro Phe Tyr Leu Thr Gly Phe Gly Arg His Glu Asp Ala Asp Phe 115 120 125Arg Gly Lys Gly Phe Asp Pro Val Leu Met Val His Asp His Ala Leu 130 135 140Met Asn Trp Ile Gly Ala Asn Ser Tyr Arg Thr Ser His Tyr Pro Tyr145 150 155 160Ala Glu Lys Met Leu Asp Trp Ala Asp Glu His Val Ile Val Val Ile 165 170 175Asn Glu Thr Ala Ala Gly Gly Phe Asn Thr Leu Ser Leu Gly Ile Thr 180 185 190Phe Asp Ala Gly Glu Arg Pro Lys Glu Leu Tyr Ser Glu Glu Ala Ile 195 200 205Asn Gly Glu Thr Ser Gln Gln Ala His Leu Gln Ala Ile Lys Glu Leu 210 215 220Ile Ala Arg Asp Lys Asn His Pro Ser Val Val Cys Trp Ser Ile Ala225 230 235 240Asn Glu Pro Asp Thr Arg Pro Asn Gly Ala Arg Glu Tyr Phe Ala Pro 245 250 255Leu Ala Lys Ala Thr Arg Glu Leu Asp Pro Thr Arg Pro Ile Thr Cys 260 265 270Val Asn Val Met Phe Cys Asp Ala Glu Ser Asp Thr Ile Thr Asp Leu 275 280 285Phe Asp Val Val Cys Leu Asn Arg Tyr Tyr Gly Trp Tyr Val Gln Ser 290 295 300Gly Asp Leu Glu Lys Ala Glu Gln Met Leu Glu Gln Glu Leu Leu Ala305 310 315 320Trp Gln Ser Lys Leu His Arg Pro Ile Ile Ile Thr Glu Tyr Gly Val 325 330 335Asp Thr Leu Ala Gly Met Pro Ser Val Tyr Pro Asp Met Trp Ser Glu 340 345 350Lys Tyr Gln Trp Lys Trp Leu Glu Met Tyr His Arg Val Phe Asp Arg 355 360 365Gly Ser Val Cys 370 23 603 PRT Escherichia coli 23Met Leu Arg Pro Val Glu Thr Pro Thr Arg Glu Ile Lys Lys Leu Asp 1 5 10 15Gly Leu Trp Ala Phe Ser Leu Asp Arg Glu Asn Cys Gly Ile Asp Gln 20 25 30Arg Trp Trp Glu Ser Ala Leu Gln Glu Ser Arg Ala Ile Ala Val Pro 35 40 45Gly Ser Phe Asn Asp Gln Phe Ala Asp Ala Asp Ile Arg Asn Tyr Ala 50 55 60Gly Asn Val Trp Tyr Gln Arg Glu Val Phe Ile Pro Lys Gly Trp Ala65 70 75 80Gly Gln Arg Ile Val Leu Arg Phe Asp Ala Val Thr His Tyr Gly Lys 85 90 95Val Trp Val Asn Asn Gln Glu Val Met Glu His Gln Gly Gly Tyr Thr 100 105 110Pro Phe Glu Ala Asp Val Thr Pro Tyr Val Ile Ala Gly Lys Ser Val 115 120 125Arg Ile Thr Val Cys Val Asn Asn Glu Leu Asn Trp Gln Thr Ile Pro 130 135 140Pro Gly Met Val Ile Thr Asp Glu Asn Gly Lys Lys Lys Gln Ser Tyr145 150 155 160Phe His Asp Phe Phe Asn Tyr Ala Gly Ile His Arg Ser Val Met Leu 165 170 175Tyr Thr Thr Pro Asn Thr Trp Val Asp Asp Ile Thr Val Val Thr His 180 185 190Val Ala Gln Asp Cys Asn His Ala Ser Val Asp Trp Gln Val Val Ala 195 200 205Asn Gly Asp Val Ser Val Glu Leu Arg Asp Ala Asp Gln Gln Val Val 210 215 220Ala Thr Gly Gln Gly Thr Ser Gly Thr Leu Gln Val Val Asn Pro His225 230 235 240Leu Trp Gln Pro Gly Glu Gly Tyr Leu Tyr Glu Leu Cys Val Thr Ala 245 250 255Lys Ser Gln Thr Glu Cys Asp Ile Tyr Pro Leu Arg Val Gly Ile Arg 260 265 270Ser Val Ala Val Lys Gly Glu Gln Phe Leu Ile Asn His Lys Pro Phe 275 280 285Tyr Phe Thr Gly Phe Gly Arg His Glu Asp Ala Asp Leu Arg Gly Lys 290 295 300Gly Phe Asp Asn Val Leu Met Val His Asp His Ala Leu Met Asp Trp305 310 315 320Ile Gly Ala Asn Ser Tyr Arg Thr Ser His Tyr Pro Tyr Ala Glu Glu 325 330 335Met Leu Asp Trp Ala Asp Glu His Gly Ile Val Val Ile Asp Glu Thr 340 345 350Ala Ala Val Gly Phe Asn Leu Ser Leu Gly Ile Gly Phe Glu Ala Gly 355 360 365Asn Lys Pro Lys Glu Leu Tyr Ser Glu Glu Ala Val Asn Gly Glu Thr 370 375 380Gln Gln Ala His Leu Gln Ala Ile Lys Glu Leu Ile Ala Arg Asp Lys385 390 395 400Asn His Pro Ser Val Val Met Trp Ser Ile Ala Asn Glu Pro Asp Thr 405 410 415Arg Pro Gln Gly Ala Arg Glu Tyr Phe Ala Pro Leu Ala Glu Ala Thr 420 425 430Arg Lys Leu Asp Pro Thr Arg Pro Ile Thr Cys Val Asn Val Met Phe 435 440 445Cys Asp Ala His Thr Asp Thr Ile Ser Asp Leu Phe Asp Val Leu Cys 450 455 460Leu Asn Arg Tyr Tyr Gly Trp Tyr Val Gln Ser Gly Asp Leu Glu Thr465 470 475 480Ala Glu Lys Val Leu Glu Lys Glu Leu Leu Ala Trp Gln Glu Lys Leu 485 490 495His Gln Pro Ile Ile Ile Thr Glu Tyr Gly Val Asp Thr Leu Ala Gly 500 505 510Leu His Ser Met Tyr Thr Asp Met Trp Ser Glu Glu Tyr Gln Cys Ala 515 520 525Trp Leu Asp Met Tyr His Arg Val Phe Asp Arg Val Ser Ala Val Val 530 535 540Gly Glu Gln Val Trp Asn Phe Ala Asp Phe Ala Thr Ser Gln Gly Ile545 550 555 560Leu Arg Val Gly Gly Asn Lys Lys Gly Ile Phe Thr Arg Asp Arg Lys 565 570 575Pro Lys Ser Ala Ala Phe Leu Leu Gln Lys Arg Trp Thr Gly Met Asn 580 585 590Phe Gly Glu Lys Pro Gln Gln Gly Gly Lys Gln 595 600 24 807 DNA Bacillus sp. 24atggtagatc tgactagtct gtacccgatc aacaccgaga cccgtggcgt cttcgacctc 60aatggcgtct ggaacttcaa gctggactac gggaaaggac tggaagagaa gtggtacgaa 120agcaagctga ccgacactat tagtatggcc gtcccaagca gttacaatga cattggcgtg 180accaaggaaa tccgcaacca tatcggatat gtctggtacg aacgtgagtt cacggtgccg 240gcctatctga aggatcagcg tatcgtgctc cgcttcggct ctgcaactca caaagcaatt 300gtctatgtca atggtgagct ggtcgtggag cacaagggcg gattcctgcc attcgaagcg 360gaaatcaaca actcgctgcg tgatggcatg aatcgcgtca ccgtcgccgt ggacaacatc 420ctcgacgata gcaccctccc ggtggggctg tacagcgagc gccacgaaga gggcctcgga 480aaagtcattc gtaacaagcc gaacttcgac ttcttcaact atgcaggcct gcaccgtccg 540gtgaaaatct acacgacccc gtttacgtac gtcgaggaca tctcggttgt gaccgacttc 600aatggcccaa ccgggactgt gacctatacg gtggactttc aaggcaaagc cgagaccgtg 660aaagtgtcgg tcgtggatga ggaaggcaaa gtggtcgcaa gcaccgaggg cctgagcggt 720aacgtggaga ttccgaatgt catcctctgg gaaccactga acacgtatct ctaccagatc 780aaagtggaac tggtgaacga cggactg 807 25 779 DNA Salmonella sp. misc_feature (1)...(779) n = A,T,C or G 25ccncccnttt tngtancntn tttgnnanct gctgcannng atcacnacnn gganncgggg 60ngggttcgnn ctctatggcn cgnggaacnn natgntggnc nacngttnan gactgacaga 120cacgtggagc taaagcttgc tgccgaacta tcactcagnt cntgnaagtt ggacaacaca 180ttncctgaca ngngaaaagc ccgccatatc catactgtgc tggcccaaca ntgagttcac 240ngtcgtcgna ctntatgang gatcacctgt atcganctcc nttnatnttc tncagctaac 300ataactgtgn gcatatgtca atgnatgacc tggtcggtgn ancacaccgg gcgtnattgn 360tgnnattcga atttnatgtc aacaactttg ntgcangntg gaatgaatct gggggccagg 420gactttggcc ancttcctna accattcgca ncctccccca gtgggcttgt acacnattgn 480gccccaaaaa ggcntcagat aggcattttg acaagctcca nnttaacttt ttcaactatg 540cngncctgca ccggacgctg aaaaangtac anganccttg tacgttccac caaganattt 600aaggtgtgac ccacntccat tttcctaacn ggactgtgac tnataaaggn tgaccnttca 660nggacacatt gcaatgaccc tttnaaacgg aanaaccccc ggnttaaagg aaaaacaaat 720ttggttgggn agtccancca agggccaatt anttgttncn cggggganta aancccccn 779 26 644 DNA Pseudomonas sp. misc_feature (1)...(644) n = A,T,C or G 26tgctggacna cngttnagga tttttagaca cgnggagcta aagcttgctg accnaactat 60cacgccggnc gtgcangctt ggaccgcgac attncctgac angngaaana ctccgccata 120tccatctttg ctggcccaac agtgagttna cngtnncgna cnntnngang gatcagtgna 180tcgagctccn ttnannttct ncgctaacat aacatgtngc atatgtcaat naatnacgct 240ggncgtggan cncaccgggc tnattcgntg nnattcgaat tgnatgncaa caactntgnt 300gcacgntggn aaanaattgc gtnacaggga ctttggccnc ttcctaaacc atngcatcct 360cccnatgggc tgtacacgaa tgngccccca aaanggcntt cagaaaggca atttntaaca 420aggcngannt ttgacttttt caactatgca gnnctgcacc ggacgctgaa aatgtacang 480accctgggta cgtncnacca agacatnnaa gtngtgaccg actccattgt nctaaccggg 540actgtaccta taatgcggac tatcanggca atgcatgacg tngaancgac acaccaggat 600naggaaaaca antggtggna ncncaccang ccatgattgt cacg 644 27 1888 DNA Bacillus sp. 27atacgactca ctagtgggtc gacccatggt agatctgact agtctgtacc cgatcaacac 60cgagacccgt ggcgtcttcg acctcaatgg cgtctggaac ttcaagctgg actacgggaa 120aggactggaa gagaagtggt acgaaagcaa gctgaccgac actattagta tggccgtccc 180aagcagttac aatgacattg gcgtgaccaa ggaaatccgc aaccatatcg gatatgtctg 240gtacgaacgt gagttcacgg tgccggccta tctgaaggat cagcgtatcg tgctccgctt 300cggctctgca actcacaaag caattgtcta tgtcaatggt gagctggtcg tggagcacaa 360gggcggattc ctgccattcg aagcggaaat caacaactcg ctgcgtgatg gcatgaatcg 420cgtcaccgtc gccgtggaca acatcctcga cgatagcacc ctcccggtgg ggctgtacag 480cgagcgccac gaagagggcc tcggaaaagt cattcgtaac aagccgaact tcgacttctt 540caactatgca ggcctgcacc gtccggtgaa aatctacacg accccgttta cgtacgtcga 600ggacatctcg gttgtgaccg acttcaatgg cccaaccggg actgtgacct atacggtgga 660ctttcaaggc aaagccgaga ccgtgaaagt gtcggtcgtg gatgaggaag gcaaagtggt 720cgcaagcacc gagggcctga gcggtaacgt ggagattccg aatgtcatcc tctgggaacc 780actgaacacg tatctctacc cagatcaaag tggaactggt gaacgacgga ctgaccatcg 840atgtctatga agagccgttc ggcgtgcgga ccgtggaagt caacgacggc aagttcctca 900tcaacaacaa accgttctac ttcaagggct ttggcaaaca tgaggacact cctatcaacg 960gccgtggctt taacgaagcg agcaatgtga tggatttcaa tatcctcaaa tggatcggcg 1020ccaacagctt ccggaccgca cactatccgt actctgaaga gttgatgcgt cttgcggatc 1080gcgagggtct ggtcgtgatc gacgagactc cggcagttgg cgtgcacctc aacttcatgg 1140ccaccacggg actcggcgaa ggcagcgagc gcgtcagtac ctgggagaag attcggacgt 1200ttgagcacca tcaagacgtt ctccgtgaac tggtgtctcg tgacaagaac catccaagcg 1260tcgtgatgtg gagcatcgcc aacgaggcgg cgactgagga agagggcgcg tacgagtact 1320tcaagccgtt ggtggagctg accaaggaac tcgacccaca gaagcgtccg gtcacgatcg 1380tgctgtttgt gatggctacc ccggagacgg acaaagtcgc cgaactgatt gacgtcatcg 1440cgctcaatcg ctataacgga tggtacttcg atggcggtga tctcgaagcg gccaaagtcc 1500atctccgcca ggaatttcac gcgtggaaca agcgttgccc aggaaagccg atcatgatca 1560ctgagtacgg cgcagacacc gttgcgggct ttcacgacat tgatccagtg atgttcaccg 1620aggaatatca agtcgagtac taccaggcga accacgtcgt gttcgatgag tttgagaact 1680tcgtgggtga gcaagcgtgg aacttcgcgg acttcgcgac ctctcagggc gtgatgcgcg 1740tccaaggaaa caagaagggc gtgttcactc gtgaccgcaa gccgaagctc gccgcgcacg 1800tctttcgcga gcgctggacc aacattccag atttcggcta caagaacgct agccatcacc 1860atcaccatca cgtgtgaatt ggtgaccg 1888 28 615 PRT Bacillus sp. 28Met Val Asp Leu Thr Ser Leu Tyr Pro Ile Asn Thr Glu Thr Arg Gly 1 5 10 15Val Phe Asp Leu Asn Gly Val Trp Asn Phe Lys Leu Asp Tyr Gly Lys 20 25 30Gly Leu Glu Glu Lys Trp Tyr Glu Ser Lys Leu Thr Asp Thr Ile Ser 35 40 45Met Ala Val Pro Ser Ser Tyr Asn Asp Ile Gly Val Thr Lys Glu Ile 50 55 60Arg Asn His Ile Gly Tyr Val Trp Tyr Glu Arg Glu Phe Thr Val Pro65 70 75 80Ala Tyr Leu Lys Asp Gln Arg Ile Val Leu Arg Phe Gly Ser Ala Thr 85 90 95His Lys Ala Ile Val Tyr Val Asn Gly Glu Leu Val Val Glu His Lys 100 105 110Gly Gly Phe Leu Pro Phe Glu Ala Glu Ile Asn Asn Ser Leu Arg Asp 115 120 125Gly Met Asn Arg Val Thr Val Ala Val Asp Asn Ile Leu Asp Asp Ser 130 135 140Thr Leu Pro Val Gly Leu Tyr Ser Glu Arg His Glu Glu Gly Leu Gly145 150 155 160Lys Val Ile Arg Asn Lys Pro Asn Phe Asp Phe Phe Asn Tyr Ala Gly 165 170 175Leu His Arg Pro Val Lys Ile Tyr Thr Thr Pro Phe Thr Tyr Val Glu 180 185 190Asp Ile Ser Val Val Thr Asp Phe Asn Gly Pro Thr Gly Thr Val Thr 195 200 205Tyr Thr Val Asp Phe Gln Gly Lys Ala Glu Thr Val Lys Val Ser Val 210 215 220Val Asp Glu Glu Gly Lys Val Val Ala Ser Thr Glu Gly Leu Ser Gly225 230 235 240Asn Val Glu Ile Pro Asn Val Ile Leu Trp Glu Pro Leu Asn Thr Tyr 245 250 255Leu Tyr Gln Ile Lys Val Glu Leu Val Asn Asp Gly Leu Thr Ile Asp 260 265 270Val Tyr Glu Glu Pro Phe Gly Val Arg Thr Val Glu Val Asn Asp Gly 275 280 285Lys Phe Leu Ile Asn Asn Lys Pro Phe Tyr Phe Lys Gly Phe Gly Lys 290 295 300His Glu Asp Thr Pro Ile Asn Gly Arg Gly Phe Asn Glu Ala Ser Asn305 310 315 320Val Met Asp Phe Asn Ile Leu Lys Trp Ile Gly Ala Asn Ser Phe Arg 325 330 335Thr Ala His Tyr Pro Tyr Ser Glu Glu Leu Met Arg Leu Ala Asp Arg 340 345 350Glu Gly Leu Val Val Ile Asp Glu Thr Pro Ala Val Gly Val His Leu 355 360 365Asn Phe Met Ala Thr Thr Gly Leu Gly Glu Gly Ser Glu Arg Val Ser 370 375 380Thr Trp Glu Lys Ile Arg Thr Phe Glu His His Gln Asp Val Leu Arg385 390 395 400Glu Leu Val Ser Arg Asp Lys Asn His Pro Ser Val Val Met Trp Ser 405 410 415Ile Ala Asn Glu Ala Ala Thr Glu Glu Glu Gly Ala Tyr Glu Tyr Phe 420 425 430Lys Pro Leu Val Glu Leu Thr Lys Glu Leu Asp Pro Gln Lys Arg Pro 435 440 445Val Thr Ile Val Leu Phe Val Met Ala Thr Pro Glu Thr Asp Lys Val 450 455 460Ala Glu Leu Ile Asp Val Ile Ala Leu Asn Arg Tyr Asn Gly Trp Tyr465 470 475 480Phe Asp Gly Gly Asp Leu Glu Ala Ala Lys Val His Leu Arg Gln Glu 485 490 495Phe His Ala Trp Asn Lys Arg Cys Pro Gly Lys Pro Ile Met Ile Thr 500 505 510Glu Tyr Gly Ala Asp Thr Val Ala Gly Phe His Asp Ile Asp Pro Val 515 520 525Met Phe Thr Glu Glu Tyr Gln Val Glu Tyr Tyr Gln Ala Asn His Val 530 535 540Val Phe Asp Glu Phe Glu Asn Phe Val Gly Glu Gln Ala Trp Asn Phe545 550 555 560Ala Asp Phe Ala Thr Ser Gln Gly Val Met Arg Val Gln Gly Asn Lys 565 570 575Lys Gly Val Phe Thr Arg Asp Arg Lys Pro Lys Leu Ala Ala His Val 580 585 590Phe Arg Glu Arg Trp Thr Asn Ile Pro Asp Phe Gly Tyr Lys Asn Ser 595 600 605His His His His His His Val 610 615 29 16 PRT Bacillus sp. 29Met Leu Ile Ile Thr Cys Asn His Leu His Leu Lys Arg Ser Ala Ile 1 5 10 15 30 4 PRT Unknown Sequence that directs proteins to cytoplasm that may be added to the reference GUS 30Lys Asp Glu Leu 1 31 6 PRT Escherichia coli 31Asp Phe Phe Asn Tyr Ala 1 5 32 5 PRT Escherichia coli 32Trp Asn Phe Ala Asp 1 5 33 36 DNA Artificial Sequence PCR primer 33aattaaccct cactaaacgg ayttyttyaa ytaygc 36 34 39 DNA Artificial Sequence PCR primer 34gtaatacgac tcactatagg ggaartcngc raarttcca 39 35 17 DNA Artificial Sequence PCR primer 35atcgcacgtc ccactac 17 36 18 DNA Artificial Sequence PCR primer 36cgtgcgatag gagttagc 18 37 22 DNA Artificial Sequence PCR primer 37atttagaaca tctcattatc cc 22 38 23 DNA Artificial Sequence PCR primer 38tgagatgttc taaatgaatt agc 23 39 17 DNA Artificial Sequence PCR primer 39atcgtgaccg gacgctt 17 40 17 DNA Artificial Sequence PCR primer 40gcgcgtaatc ttcctgg 17 41 19 DNA Artificial Sequence PCR primer 41tagcgacctt cgctttcgg 19 42 20 DNA Artificial Sequence PCR primer 42atcatgttta cagagtatgg 20 43 21 DNA Artificial Sequence PCR primer 43ggaatattgc acaatgggcg c 21 44 23 DNA Artificial Sequence PCR primer 44gatctctacg catttcaccg cta 23 45 17 DNA Artificial Sequence PCR primer 45atggtaagac cgcaacg 17 46 25 DNA Artificial Sequence PCR primer 46taaaaaccat ggtaagaccg caacg 25 47 20 DNA Artificial Sequence PCR primer 47cctcactcca cagtcttctc 20 48 30 DNA Artificial Sequence PCR primer 48agaccgctag cctcactcca cagtcttctc 30 49 22 DNA Artificial Sequence PCR primer 49tttgactttt tcaactatgc ag 22 50 23 DNA Artificial Sequence PCR primer 50aattctgcat agttgaaaaa gtc 23 51 35 DNA Artificial Sequence Product of sythesis to facilitate protein purification 51gtcgacccat ggtagatctg actagtctgt acccg 35 52 30 DNA Artificial Sequence Product of synthesis to facilitate construction and cloning 52gtcgacagga gtgctatcat gctgtacccg 30 53 30 DNA Artificial Sequence Product of synthesis to facilitate protein purification 53gtcgacagga gtgctaccat ggtgtacccg 30 54 36 DNA Artificial Sequence Product of synthesis to facilitate protein purification 54gtcgacagga gtgctaccat ggtagatctg tacccg 36 55 41 DNA Artificial Sequence Product of synthesis to facilitate protein purification 55gctagccatc accatcacca tcacgtgtga attggtgacc g 41 56 9 PRT Artificial Sequence Product of synthesis to facilitate protein purification 56Ser Ser His His His His His His Val 1 5 57 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 57tcgacccatg gtagatctga ctagtctgta cccgatcaac accgagaccc gtggcgtctt 60cgacctcaat ggcgtctgga 80 58 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 58ggatttcctt ggtcacgcca atgtcattgt aactgcttgg gacggccata ctaatagtgt 60cggtcagctt gctttcgtac 80 59 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 59ccaagcagtt acaatgacat tggcgtgacc aaggaaatcc gcaaccatat cggatatgtc 60tggtacgaac gtgagttcac 80 60 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 60gcggagcacg atacgctgat ccttcagata ggccggcacc gtgaactcac gttcgtacca 60gacatatccg atatggttgc 80 61 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 61ggtgccggcc tatctgaagg atcagcgtat cgtgctccgc ttcggctctg caactcacaa 60agcaattgtc tatgtcaatg 80 62 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 62aatggcagga atccgccctt gtgctccacg accagctcac cattgacata gacaattgct 60ttgtgagttg cagagccgaa 80 63 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 63gtgagctggt cgtggagcac aagggcggat tcctgccatt cgaagcggaa atcaacaact 60cgctgcgtga tggcatgaat 80 64 100 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 64gtacagcccc accggtaggg tgctatcgtc gaggatgttg tccacggcga cggtgacgcg 60attcatgcca tcacgcagcg agttgttgat ttccgcttcg 100 65 56 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 65cgcgtcaccg tcgccgtgga caacatcctc gacgatagca ccctaccggt ggggct 56 66 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 66cacttctctt ccagtccttt cccgtagtcc agcttgaagt tccagacgcc attgaggtcg 60aagacgccac gggtctcggt 80 67 35 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 67ttgatcgggt acagactagt cagatctacc atggg 35 68 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 68acttcaagct ggactacggg aaaggactgg aagagaagtg gtacgaaagc aagctgaccg 60acactattag tatggccgtc 80 69 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 69gtacagcgag cgccacgaag agggcctcgg aaaagtcatt cgtaacaagc cgaacttcga 60cttcttcaac tatgcaggcc 80 70 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 70ctttgccttg aaagtccacc gtataggtca cagtcccggt tgggccattg aagtcggtca 60caaccgagat gtcctcgacg 80 71 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 71accgggactg tgacctatac ggtggacttt caaggcaaag ccgagaccgt gaaagtgtcg 60gtcgtggatg aggaaggcaa 80 72 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 72ctccacgtta ccgctcaggc cctcggtgct tgcgaccact ttgccttcct catccacgac 60cgacactttc acggtctcgg 80 73 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 73agtggtcgca agcaccgagg gcctgagcgg taacgtggag attccgaatg tcatcctctg 60ggaaccactg aacacgtatc 80 74 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 74gtcagtccgt cgttcaccag ttccactttg atctggtaga gatacgtgtt cagtggttcc 60cagaggatga cattcggaat 80 75 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 75tctaccagat caaagtggaa ctggtgaacg acggactgac catcgatgtc tatgaagagc 60cgttcggcgt gcggaccgtg 80 76 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 76acggtttgtt gttgatgagg aacttgccgt cgttgacttc cacggtccgc acgccgaacg 60gctcttcata gacatcgatg 80 77 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 77gaagtcaacg acggcaagtt cctcatcaac aacaaaccgt tctacttcaa gggctttggc 60aaacatgagg acactcctat 80 78 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 78tacgtaaacg gggtcgtgta gattttcacc ggacggtgca ggcctgcata gttgaagaag 60tcgaagttcg gcttgttacg 80 79 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 79atccatcaca ttgctcgctt cgttaaagcc acggccgttg ataggagtgt cctcatgttt 60gccaaagccc ttgaagtaga 80 80 75 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 80caacggccgt ggctttaacg aagcgagcaa tgtgatggat ttcaatatcc tcaaatggat 60cggcgccaac agctt 75 81 36 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 81aatgactttt ccgaggccct cttcgtggcg ctcgct 36 82 39 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 82ccggaagctg ttggcgccga tccatttgag gatattgaa 39 83 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 83tgcaccgtcc ggtgaaaatc tacacgaccc cgtttacgta cgtcgaggac atctcggttg 60tgaccgactt caatggccca 80 84 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 84ccggaccgca cactatccgt actctgaaga gttgatgcgt cttgcggatc gcgagggtct 60ggtcgtgatc gacgagactc 80 85 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 85gttcacggag aacgtcttga tggtgctcaa acgtccgaat cttctcccag gtactgacgc 60gctcgctgcc ttcgccgagt 80 86 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 86attcggacgt ttgagcacca tcaagacgtt ctccgtgaac tggtgtctcg tgacaagaac 60catccaagcg tcgtgatgtg 80 87 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 87cgcgccctct tcctcagtcg ccgcctcgtt ggcgatgctc cacatcacga cgcttggatg 60gttcttgtca cgagacacca 80 88 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 88gagcatcgcc aacgaggcgg cgactgagga agagggcgcg tacgagtact tcaagccgtt 60ggtggagctg accaaggaac 80 89 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 89acaaacagca cgatcgtgac cggacgcttc tgtgggtcga gttccttggt cagctccacc 60aacggcttga agtactcgta 80 90 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 90tcgacccaca gaagcgtccg gtcacgatcg tgctgtttgt gatggctacc ccggagacgg 60acaaagtcgc cgaactgatt 80 91 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 91cgaagtacca tccgttatag cgattgagcg cgatgacgtc aatcagttcg gcgactttgt 60ccgtctccgg ggtagccatc 80 92 89 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 92gacgtcatcg cgctcaatcg ctataacgga tggtacttcg atggcggtga tctcgaagcg 60gccaaagtcc atctccgcca ggaatttca 89 93 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 93cccgtggtgg ccatgaagtt gaggtgcacg ccaactgccg gagtctcgtc gatcacgacc 60agaccctcgc gatccgcaag 80 94 53 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 94cgcgtgaaat tcctggcgga gatggacttt ggccgcttcg agatcaccgc cat 53 95 36 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 95acgcatcaac tcttcagagt acggatagtg tgcggt 36 96 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 96cggcagttgg cgtgcacctc aacttcatgg ccaccacggg actcggcgaa ggcagcgagc 60gcgtcagtac ctgggagaag 80 97 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 97cgcgtggaac aagcgttgcc caggaaagcc gatcatgatc actgagtacg gcgcagacac 60cgttgcgggc tttcacgaca 80 98 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 98tcgcgaagtc cgcgaagttc cacgcttgct cacccacgaa gttctcaaac tcatcgaaca 60cgacgtggtt cgcctggtag 80 99 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 99ttcgtgggtg agcaagcgtg gaacttcgcg gacttcgcga cctctcaggg cgtgatgcgc 60gtccaaggaa acaagaaggg 80 100 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable micorbial GUS 100gtgcgcggcg agcttcggct tgcggtcacg agtgaacacg cccttcttgt ttccttggac 60gcgcatcacg ccctgagagg 80 101 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable microbial GUS. 101cgtgttcact cgtgaccgca agccgaagct cgccgcgcac gtctttcgcg agcgctggac 60caacattcca gatttcggct 80 102 89 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable microbial GUS. 102cggtcaccaa ttcacacgtg atggtgatgg tgatggctag cgttcttgta gccgaaatct 60ggaatgttgg tccagcgctc gcgaaagac 89 103 53 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable microbial GUS. 103acaagaacgc tagccatcac catcaccatc acgtgtgaat tggtgaccgg gcc 53 104 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable microbial GUS. 104tactcgactt gatattcctc ggtgaacatc actggatcaa tgtcgtgaaa gcccgcaacg 60gtgtctgcgc cgtactcagt 80 105 36 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable microbial GUS. 105gatcatgatc ggctttcctg ggcaacgctt gttcca 36 106 80 DNA Artificial Sequence Oligonucleotide. Product of synthesis to overlap and create fragments of an engineered secreatable microbial GUS. 106ttgatccagt gatgttcacc gaggaatatc aagtcgagta ctaccaggcg aaccacgtcg 60tgttcgatga gtttgagaac 80 107 60 DNA Unknown Invertase signal sequence used in yeast vector. 107atgcttttgc aagccttcct tttccttttg gctggttttg cagccaaaat atctgcaatg 60 108 258 DNA Unknown Mat alpha signal sequence used in yeast vector. 108atgagatttc cttcaatttt tactgcagtt ttattcgcag catcctccgc attagctgct 60ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120tacttagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240tctttggata aaagagag 258 109 88 DNA Unknown Extensin signal sequence used in plant vector. 109catgggaaaa atggcttctc tatttgccac atttttagtg gttttagtgt cacttagctt 60agcttctgaa agctcagcaa attatcaa 88 110 82 DNA Unknown GRP signal sequence used in plant vector. 110catggctact actaagcatt tggctcttgc catccttgtc ctccttagca ttggtatgac 60caccagtgca agaaccctcc ta 82 111 42 DNA Artificial Sequence Oligonucleotide used in “quickchange” mutagenesis. 111ttcctgccat tcgaggcgga aatcnngaac tcgctgcgtg at 42 112 43 DNA Artificial Sequence Oligonucleotide used in “quickchange” mutagenesis. 112atcacgcagc gagttcnnga tttccgcctc gaatggcagg aat 43

Claims

1. An isolated nucleic acid molecule comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO:14) or a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO:14) and which encodes a functional β-glucuronidase.

2. An isolated nucleic acid molecule that encodes one of the amino acid sequences of SEQ ID NOs.: 19-21, or a variant thereof wherein the variant has at least 75% amino acid identity to one of SEQ ID NOs.: 19-21 and which encodes a functional β-glucuronidase.

3. An expression vector comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, wherein the β-glucuronidase is encoded by a nucleic acid molecule comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO: 14) or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO:14) and which encodes a functional β-glucuronidase.

4. The expression vector of claim 3, wherein the heterologous promoter is a promoter selected from the group consisting of a developmental type-specific promoter, a tissue type-specific promoter, a cell type-specific promoter and an inducible promoter.

5. The expression vector of claim 3, wherein the promoter is functional in a cell selected from the group consisting of a plant cell, a bacterial cell, an animal cell and a fungal cell.

6. The expression vector of claim 3, wherein the vector is a binary Agrobacterium tumefaciens plasmid vector.

7. The expression vector of claim 3, further comprising a nucleic acid sequence encoding a product of a gene of interest.

8. The expression vector of claim 7, wherein the product is a protein.

9. A host cell containing the vector according to claim 3.

10. The host cell of claim 9, wherein the host cell is selected form the group consisting of a plant cell, an nsect cell, a fungal cell, an animal cell and a bacterial cell.

11. An expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, wherein the microbial β-glucuronidase comprises one of the amino acid sequences of SEQ ID NOs.: 19-21, or variant thereof, wherein the variant has at least 75% amino acid identity to one of SEQ ID NOs.: 19-21, and which encodes a functional β-glucuronidase.

12. A host cell containing the vector according to claim 11.

13. A method for monitoring expression of a gene of interest or a portion thereof in a host cell, comprising:(a) introducing into the host cell a vector construct, the vector construct comprising a nucleic acid molecule comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO: 14) or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO: 14) and which encodes functional β-glucuronidase and a nucleic acid molecule encoding a product of the gene of interest; wherein the β-glucuronidase and the gene of interest are co-expressed; (b) detecting the presence of the microbial β-glucuronidase, thereby monitoring expression of the gene of interest.

14. A method for transforming a host cell with a gene of interest or portion thereof, comprising:(a) introducing into the host cell a vector construct, the vector construct comprising a nucleic acid sequence comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO: 14) or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO:14) and which encodes a functional β-glucuronidase, and a nucleic acid sequence encoding a product of the gene of interest, such that the vector construct integrates into the genome of the host cell; wherein the β-glucuronidase and the gene of interest are co-expressed; (b) detecting the presence of the microbial β-glucuronidase, thereby establishing that the host cell is transformed.

15. A method for positive selection for a transformed cell, comprising:(a) introducing into a host cell a vector construct, the vector construct comprising a nucleic acid sequence comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO: 14) or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO:14) and which encodes a functional β-glucuronidase; (b) exposing the host cell to a sample comprising a glucuronide, wherein the glucuronide is cleaved by the β-glucuronidase, such that an aglycone is released, wherein the aglycone is required for growth of the host cell; wherein a host cell that expresses the β-glucuronidase grows, thereby positively selecting a transformed cell.

16. The method of any of claims 13-15 wherein the host cell is selected from the group consisting of a plant cell, an animal cell, an insect cell, a fungal cell and a bacterial cell.

17. An isolated nucleic acid molecule that encodes the amino acid sequence of SEQ ID NO: 22, or a variant thereof wherein the variant has at least 90% amino acid identity to SEQ ID NO: 22 and which encodes a functional β-glucuronidase.

18. An expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, wherein the microbial β-glucuronidase comprises the amino acid sequence of SEQ ID NO: 22, or variant thereof, wherein the variant has at least 90% amino acid identity to SEQ ID NO: 22, and which encodes a functional β-glucuronidase.

19. A host cell containing the vector according to claim 18.