Genomic sequence of NGR234 symbiotic plasmid, its gene map, and its use in diagnostics and gene transfer in agriculture

Abstract

The sequencing and analysis of the complete nucleotide sequence of symbiotic plasmid pNGR234a isolated from Rhizobium sp. NGR234. The complete sequence of pNGR234a is presented. The analysis includes the identification of a number of novel ORFs and the proteins expressible therefrom which have been ascribed putative functions.

Description

TECHNICAL FIELD

[0001] This invention relates to a symbiotic plasmid of the broad host-range Rhizobium sp. NGR234 and its use. In particular, this invention relates to the isolation and analysis of the complete sequence of the NGR234 symbiotic plasmid pNGR234a, and the open reading frames (ORFs) identifiable therein as well as the proteins expressible from said ORFs.

BACKGROUND OF THE INVENTION

[0002] Together with carbon, hydrogen and oxygen, nitrogen is one of the essential components in organic chemistry. Although it is present in vast quantities in the atmosphere, nitrogen in its diatomic form N2 remains unassimilable by living organisms. The nitrogen cycle begins by the fixation of nitrogen into ammonia which is chemically more reactive and can be assimilated into the food chain. A large fraction of the total nitrogen fixed every year is produced by microorganisms. Among these, the soil bacteria of the genera Azorhizobium, Bradyrhizobium, Sinorhizobium and Rhizobium, generally referred to as rhizobia, fix nitrogen in symbiotic associations with many plants from the Leguminosae family. This highly specific interaction leads to the formation of specialized root-, and in the case of Azorhizobium, stem-structures called nodules. It is within these nodules that rhizobia differentiate into bacteroids capable of fixing atmospheric nitrogen into ammonia. In turn, ammonia diffuses into the vegetal cells and sustains plant growth even under limiting nitrogen conditions.

[0003] The Rhizobium-legume interaction presents many interesting features. Obviously, the possibility of using this symbiosis as an “environmentally friendly” way to provide some of the most important world crops (such as soybean, bean and many other legumes) with fixed nitrogen without using nitrate-rich fertilizers, has important economic consequences. It is also an ideal model to study a non-pathogenic interaction between bacteria and a highly developed, multicellular organism such as the host plant. Furthermore, the various steps involved in the establishment of a functional nitrogen symbiosis, which include some dramatic morphological changes as well as processes of cellular differentiation, require a complex exchange of molecular signals. Despite many decades of studies, it is only recently that the Rhizobium-legume interaction has been partially understood at the molecular level. The establishment of a functional symbiosis can be divided into two major steps as follows.

[0004] (A) Rhizosphere Ecology and Nodulation

[0005] Rhizobia are soil bacteria that proliferate in the rhizosphere of compatible plants, taking advantage of the many compounds released by plant roots. In return it has been shown that the presence of rhizobia in the rhizosphere reduces susceptibility of plants to many root diseases. In the case of low nitrogen levels in the soil, compatible rhizobia can interact with host plants and start the nodulation process (Long, 1989; Fellay et al., 1995; van Rhijn and Vanderleyden, 1995). Molecular signalling between the two partners begins with the release by the plant of phenolic compounds (mostly flavonoids) that induce the expression of nodulation genes (referred to as nod, nol and noe genes). The NodD1 gene product appears to be the central mediator between the plant signal and nodulation gene induction (Bender et al., 1988). It is modified by the binding of flavonoids and acts as a positive regulator on the expression of the remaining nodulation genes. Among them, the nodABC loci encode products responsible for the synthesis of the core structure of lipooligosaccharides called Nod factors (Relic et al., 1994). More nodulation genes are involved in strain-specific modifications of the Nod factors as well as in its secretion. It seems established now that variability in the structure of Nod factors may play a significant role in the determination of the host-range of a given Rhizobium strain, that is in its ability to efficiently nodulate different legumes. For example, the strain Rhizobium meliloti can only nodulate Medicago, Melilotus and Trigonella ssp., whereas Rhizobium sp. NGR234 can symbiotically interact with more than 105 different genera of plants, including the non-legume Parasponia andersonii.

[0006] The structure of many Nod factors, their isolation from Rhizobium strains and their commercial application in agriculture have been described (NodNGR-Faktoren: Relić et al., 1994; WO 94/00466; NodRm-Faktoren: WO 91/15496). Secreted Nod factors act in turn as signal molecules that allow rhizobia to enter young root hairs of a host plant, and induce root-cortical cell division that will produce the future nodule. Invaginated rhizobia progress towards the forming nodule within infection threads that are synthesized by the plant cells. Bacteria are then released into the cytoplasm of dividing nodule cells where they differentiate into bacteroids capable of fixing atmospheric nitrogen.

[0007] With respect to regulation of the nodulation genes, other regulatory genes with similarities to nodD1 (genes that belong to the lysR family) have been identified in various strains (Davis and Johnston, 1990). The function of these genes, called nodD2, nodD3 or syrM, is only partially understood. Some nodD genes have been described (WO 94/00466; CA 1314249; WO 87/07910; U.S. Pat. No. 5,023,180). Also, recombinant DNA molecules including the consensus sequence of the promoters of nodD1-regulated genes, called nod-boxes (Fisher and Long, 1993), have been disclosed (U.S. Pat. No. 5,484,718; U.S. Pat. No. 5,085,588). Finally, recombinant plasmids with the nodABC genes or, in one case (Bradyrhizobium japonicum), a sequence influencing host specificity have been disclosed (U.S. Pat. No. 5,045,461; U.S. Pat. No. 4966847).

[0008] (B) Symbiotic Nitrogen Fixation

[0009] Inside the nodules, rhizobia differentiate into bacteroids that express the enzymatic complex (nitrogenase) required for the reduction of atmospheric nitrogen into ammonia. The nitrogenase is encoded by three genes nifH, nifD and nifK which are well conserved in nitrogen fixing organisms (Badenoch-Jones et al., 1989). Many additional loci are necessary for functional nitrogenase activity. Those originally identified in Klebsiella pneumoniae are known as nif genes, whereas those found only in Rhizobium strains are described as fix genes (Fischer, 1994). Some of these gene products are required for the biosynthesis of cofactors, the assembly of the enzymatic complex or play regulatory and different accessory roles (oxygen-limited respiration, etc.). Many of these genes are less conserved among the various rhizobial strains and in some cases their function is still not fully understood. The high sensitivity of the nitrogenase complex to free oxygen requires a very strict control of most nif and fix gene expression. In this respect, the FixL, FixJ, FixK, NifA and RpoN proteins have been identified in representative Rhizobium species as the major regulatory elements that, in microanaerobic conditions, activate the synthesis of the nitrogenase complex (Fischer, 1994). Recombinant DNA molecules containing nif genes/promoters have been disclosed: nifH promoters of B. japonicum (U.S. Pat. No. 5008194), nifH and nifD promoter of R. japonicum (EP 164245), nifA of B. japonicum and R. meliloti (EP 339830), nifHDK and hydrogen-uptake (hup) genes of R. japonicum (EP 205071).

[0010] Many more genetic determinants play a significant role in the Rhizobium-legume symbiosis. Genes (exo, lps and ndv genes) involved in the production of extracellular polysaccharides (EPS), lipopolysaccharides (LPS) and cyclic glucanes of rhizobia play an essential role in the symbiotic interaction (Long et al., 1988; Stanfield et al., 1988). Mutation in these genes negatively influences the development of functional nodules. In this respect, some exopolysaccharides of the NGR234 derivative strain ANU280, have been disclosed (WO 87/06796). Although Nod factors seem to play a key role in the nodulation process, experimental data indicate that other signal molecules produced by the bacterial symbionts are required for functional symbiosis and may play a role in coordinating various steps such as the controlled invasion process, the release of rhizobia from the infection thread into the plant cell cytoplasm, the bacteroid differentiation process, etc. Moreover, the need for rhizobia to survive in the rhizosphere and to compete adequately with other microorganisms requires many more unidentified genes that, although they may not be characterised as proper symbiotic loci, do affect the efficiency of the various strains to induce functional nitrogen fixing symbiosis in field conditions. Finally, in our view genetic engineering of improved rhizobial strains cannot be pursued without a more extended knowledge of the structure and complexity of the Rhizobium symbiotic genome.

[0011] In this respect we decided to determine the complete DNA sequence of a symbiotic plasmid of Rhizobium sp. NGR234. In contrast to Bradyrhizobium and Azorhizobium that carry symbiotic genes on large chromosomes (ca. 8 Mbp) and to R. meliloti that harbours two very large symbiotic plasmids of 1.4 and 1.6 Mbp, NGR234 carries a single plasmid of ca. 500 kbp, pNGR234a. Moreover, it has been shown by transfer of pNGR234a into heterologous rhizobia, and even into non-nodulating Agrobacterium tumefaciens, that most nodulation functions are encoded by this plasmid (Broughton et al., 1984). The fact that NGR234 is able to interact symbiotically with more plants than any other known strain, and that a complete ordered cosmid library of pNGR234a was available, reinforced NGR234 as the best choice for a large-scale sequencing effort on a symbiotic plasmid (Perret et al., 1991; Freiberg et al., 1997).

[0012] Automated fluorescent methods have been used to sequence cosmids from eukaryotic organisms, including Saccharomyces cerevisiae (Levy, 1994), Caenorhabditis elegans (Sulston et al., 1992), Drosophila melanogaster (Hartl and Palazzolo, 1993), and Homo sapiens (Bodmer, 1994), as well as chromosomes from the prokaryotes Haemophilus influenzae (Fleischmann et al., 1995) and Mycoplasma genitalium (Fraser et al., 1995). In most large-scale sequencing centres this technology is based mainly on the shotgun approach. After random fragmentation of DNA (e.g. cosmids, bacterial artificial chromosomes (BACs), entire chromosomes) using sonication or mechanical forces, size-selected fragments are subcloned into M13 phages, phagemids or plasmids and sequenced by cycle sequencing using dye primers (Craxton, 1993). A disadvantage of this method is that DNA regions with elevated GC contents produce large numbers of compressions (unresolvable foci in sequence gels) in the dye primer sequences leading to several hundred compressions per assembled cosmid sequence. It is known that the use of dye terminators—fluorescently labelled dideoxynucleoside triphosphates—instead of dye primers reduces the number of compressions (Rosenthal and Charnock-Jones, 1993). Therefore, dye terminators are frequently being used for gap closure and proofreading after assembly of the shotgun data.

[0013] To sequence GC-rich cosmids with the highest accuracy, the effectiveness of shotgun sequencing with dye terminators in comparison to dye primer sequencing was investigated. To improve the incorporation of dye terminators into DNA, a modified Tag DNA polymerase carrying a single mutation was used (Tabor and Richardson, 1995). This enzyme has properties similar to a thermostable “sequenase” and is commercially available as Thermo Sequenase (Amersham, Buckinghamshire, UK) or AmpliTaq FS (Perkin-Elmer, Foster City, Calif., USA). Concentrations of dye terminators needed in the cycle sequencing reactions can be reduced by 20-250 times. It was found that dye terminator shotgun sequencing leads to compression-free raw data that can be assembled much faster than shotgun data mainly obtained by dye primer sequencing. This strategy thus allows a several-fold increase in speed to sequence individual cosmids. This was demonstrated by comparing assembly of the sequence data of two cosmids from pNGR234a generated by different chemistries: Cosmid pXB296 was sequenced with dye terminators, whereas data for pXB110 were obtained using the common dye primer method. Also disclosed is the analysis of the entire pXB296 sequence.

[0014] Moreover, the dye terminator shotgun sequencing strategy used to generate the sequence data for pXB296 was also used to sequence all the other remaining overlapping cosmids of the plasmid pNGR234a. In summary, 20 cosmids have been sequenced together with two PCR products and a subcloned DNA fragment derived from a cosmid identified as pXB564 in order to generate the plasmid's complete nucleotide sequence.

[0015] After its assembly, the analysis of the entire nucleotide sequence of pNGR234a, especially the determination of putative coding regions and the prediction of their expressible proteins and putative functions, was performed. Initially, analysis of the region covered by cosmid pXB296 was extended to cosmids pXB368 and pXB110. Thus, in approximately 100 kb of the plasmid (position 417,796-517,279) most ORFs and their deduced proteins with different putative functions were predicted. Subsequently, the rest of pNGR234a was analyzed.

SUMMARY OF THE INVENTION

[0016] The present invention provides the complete nucleotide sequence of symbiotic plasmid pNGR234a or degenerate variants thereof of Rhizobium sp. NGR234.

[0017] The present invention also contemplates sequence variants of the plasmid pNGR234a altered by mutation, deletion or insertion.

[0018] Also encompassed by the present invention are each of the ORFs derivable from the nucleotide sequence of pNGR234a or variants thereof.

[0019] In a preferred embodiment, the ORFs derived from the nucleotide sequence of pNGR234a encode the functions of nitrogen fixation, nodulation, transportation, permeation, synthesis and modification of surface poly- or oligosaccharides, lipo-oligosaccharides or secreted oligosaccharide derivatives, secretion (of proteins or other biomolecules), transcriptional regulation or DNA-binding, peptidolysis or proteolysis, transposition or integration, plasmid stability, plasmid replication or conjugal plasmid transfer, stress response (such as heat shock, cold shock or osmotic shock), chemotaxis, electron transfer, synthesis of isoprenoid compounds, synthesis of cell wall components, rhizopine metabolism, synthesis and utilization of amino acids, rhizopines, amino acid derivatives or other biomolecules, degradation of xenobiotic compounds, or encode proteins exhibiting similarities to proteins of amino acid metabolism or related ORFs, or enzymes (such as oxidoreductase, transferase, hydrolase, lyase, isomerase or ligase).

[0020] In another preferred embodiment, the ORFs are under the control of their natural regulatory elements or under the control of analogues to such natural regulatory elements.

[0021] The present invention also provides the sequences of the intergenic regions of pNGR234a which, in a preferred embodiment, are regulatory DNA sequences or repeated elements. In a further preferred embodiment, the intergenic sequences are ORF-fragments.

[0022] Also provided by the present invention are mobile elements (insertion elements or mosaic elements) derivable from the nucleotide sequences of the present invention.

[0023] The present invention also contemplates the use of the disclosed nucleotide sequences or ORFs in the analysis of genome structure, organisation or dynamics.

[0024] Also provided by the present invention is the use of the nucleotide sequences or ORFs in the subcloning of new nucleotide sequences. In a preferred embodiment, the new nucleotide sequences are coding sequences or non-coding sequences.

[0025] In yet a further preferred embodiment, the nucleotide sequences or ORFs are used in genome analysis and subcloning methods as oligonucleotide primers or hybridization probes.

[0026] The present invention further provides proteins expressible from the disclosed nucleotide sequences or ORFs.

[0027] Also contemplated by the present invention is the use of the disclosed nucleotide sequences, individual ORFs or groups of ORFs or the proteins expressible therefrom in the identification and classification of organisms and their genetic information, the identification and characterisation of nucleotide sequences, the identification and characterisation of amino acid sequences or proteins, the transportation of compounds to and from an organism which is host to said nucleotide sequences, ORFs or proteins, the degradation and/or metabolism of organic, inorganic, natural or xenobiotic substances in a host organism, or the modification of the host-range, nitrogen fixation abilities, fitness or competitiveness of organisms.

[0028] The present invention also provides plasmid pNGR234a of Rhizobium sp. NGR234 comprising the disclosed nucleotide sequence or any degenerate variant thereof.

[0029] The present invention also provides a plasmid harbouring at least one of the disclosed ORFs or any degenerate variant thereof.

[0030] The plasmids of the invention may be produced recombinantly and/or by mutation, deletion, insertion or inactivation of an ORF, ORFs or groups of ORFs.

[0031] The present invention also provides the use of the disclosed plasmids or variants thereof in obtaining a synthetic minimal set of ORFs required for functional Rhizobium-legume symbiosis, the modification of the host-range of rhizobia, the augmentation of the fitness or competitiveness of Rhizobium sp. NGR234 in the soil and its nodulation efficiency on host plants, the introduction of desired phenotypes into host plants using the disclosed plasmids as stable shuttle systems for foreign DNA encoding said desired phenotypes, or the direct transfer of the disclosed plasmids into rhizobia or other microorganisms without using other vectors for mobilization.

[0032] The nucleotide sequences of the present invention were advantageously obtained using known cycle sequencing methods. The preferred dye terminator/thermostable sequenase shotgun sequencing method used to generate the nucleotide sequences of the present invention, when applied to cosmids and when compared to other sequencing methods, was shown to yield sequence reads of the highest fidelity. Consequently, the speed of assembly of particular cosmids was increased, and the resultant high-quality sequences required little editing or proofreading. Thus, the preferred sequencing method described herein was successfully used to generate the complete nucleotide sequence of all the overlapping cosmids of plasmid pNGR234a, thereby resulting in the assembly of the complete sequence of the plasmid.

[0033] The complete sequence of pNGR234a is disclosed for the first time in this application, as are the majority of the ORFs predicted within the sequence. Putative functions have been ascribed to the novel and inventive ORFs disclosed herein and the proteins for which they code.

BRIEF DESCRIPTION OF DRAWINGS

[0034] The present invention is described below and illustrated thereafter in the appended examples, with reference to the following figures:

[0035] FIG. 1 A comparative graph showing the comparison of sequences from pXB296 created by different cycle sequencing methods.

[0036] FIG. 2 A schematic diagram showing the organization of the predicted ORFs in pXB296 from Rhizobium sp. NGR234.

[0037] FIG. 3 The complete nucleotide sequence of plasmid pNGR234a (with the pages labelled sequentially from 19961 to 1996142).

[0038] FIG. 4 A schematic diagram showing the map of the 20 sequenced cosmids covering the 536 kb symbiotic plasmid pNGR234a of Rhizobium sp. NGR234.

[0039] FIG. 5 A diagram indicating multiple alignments of the nucleotide sequence of the replication origins of various plasmids.

[0040] FIG. 6 A diagram indicating multiple DNA sequence alignments of the regions containing the origin of transfer of various plasmids.

[0041] FIG. 7 A schematic diagram showing a circular representation of the symbiotic plasmid pNGR234a of NGR234.

DETAILED DESCRIPTION OF THE INVENTION AND BEST MODE

[0042] Comparison of Different Shotgun Sequencing Strategies

[0043] The following is a more detailed description of certain key aspects of the present invention.

[0044] GC-rich cosmids were examined to investigate whether they could be sequenced much more efficiently using dye terminators throughout the shotgun phase instead of dye primers. As a test case, cosmid pXB296 with a GC content of 58 mol % from pNGR234a, the symbiotic plasmid of Rhizobium sp. NGR234, was exclusively sequenced using dye terminators in combination with a thermostable sequenase [Thermo Sequenase (Amersham)]. Another rhizobial cosmid with identical GC content, pXB110, was sequenced using traditional dye primer chemistry and Taq DNA polymerase.

[0045] Using the dye terminator/thermostable sequenase shotgun strategy, it was shown that most, if not all, compressions could be resolved and reads were produced with the highest fidelity among all sequencing chemistries tested. As a result, a much faster assembly of cosmid pXB296 in comparison to pXB110 was obtained. The shotgun data could be assembled into a high-quality sequence without extensive editing and proofreading. By measuring the error rate in overlapping regions between individual cosmids from pNGR234a, as well as the cosmid vector sequence itself (data not shown), it was estimated that the accuracy of the pXB296 sequence is higher than 99.98%. Using other thermostable sequenases such as AmpliTaq FS (Perkin-Elmer), similar results were expected because thermostable sequenases have similar properties.

[0046] Dye primer chemistry in combination with Thermo Sequenase was also examined. Although the peak uniformity of signals was much improved over dye primer/Taq DNA polymerase data, the number of compressions in GC-rich shotgun reads was not reduced significantly. Compressions in shotgun raw data enormously increase the overall effort of editing, proofreading, and finishing a cosmid as shown for pXB110 (Table 1).

[0047] Because of their longer reading potential, dye primer reads are helpful for gap closure. However, using ABI 373A sequencers (Applied Biosystems, Inc. (ABI), Perkin-Elmer, Foster City, Calif., USA), dye primer reads are, on average, only ˜50 bases longer than dye terminator reads.

[0048] Using the experimental conditions of the present invention, shotgun sequencing with dye terminators and a thermostable sequenase is superior because for GC-rich cosmid templates it removes most of the compressions and this leads to a several-fold improvement in assembling and finishing of cosmid-sized projects. Although dye terminators are slightly more expensive than dye primers, the overall saving in time for finishing projects has, in our experience, a much greater effect on general costs.

[0049] It has been shown that the strategy of the present invention is effective for high-throughput shotgun sequencing of GC-rich templates. This strategy was therefore used to sequence the remaining 19 overlapping cosmids of the symbiotic plasmid pNGR234a of Rhizobium sp. NGR234. In total, 20 cosmids, two PCR products (1.5 and

TABLE 1
Comparison of the assembly of the sequence data from cosmids
pXB296 (dye terminator shotgun reads) and pXB110 (dye primer
shotgun reads)
Data assembly	pXB296	pXB110
Average length of the shotgun reads (bases)	332	378
No. of shotgun reads used for assembly	786	899
No. of shotgun reads assembled with 4% mismatcha	736	308
No. of shotgun reads assembled with 25% mismatcha	775	879
No. of contigsb longer than 1 kbp	3	25
No. of contigs left after editingc	2	4
No. of additional reads (gap closure and proofreading)d	32	191
Total length of cosmid insert (bp)	34,010	34,573
Sequencing redundancy (per bp)	8.0	10.5
aAssembling program: XGAP; principal autoassembling conditions: normal shotgun assembly, joins
# permitted, minimum initial match = 15, maximum no. of pads per reading during the alignment procedure = 8,
# maximum no. of pads per reading in contig to align any new reading = 8, alignment mismatches 4% and 25%,
# respectively.
bContiguous parts of sequence created by overlapping reads.
cLengths of contigs: 6-10 kbp (pXB296); 2-12 kbp (pXB110).
dReads necessary for closing gaps and making single-stranded regions double-stranded by primer walking
# on selected templates and, in case of pXB110, for solving ambiguities (compressions) by the resequencing of clones
# with universal primer and dye terminators.

[0050] 2.0 kb in length) and a 1.5 kb restriction fragment were sequenced in order to generate the complete pNGR234a sequence (FIG. 4).

[0051] Genetic organization of pXB296

[0052] All 28 predicted open reading frames (ORFS) in pXB296 (FIG. 2) show significant homologies to database entries (Table 2). The first putative gene cluster (cluster I) containing ORF1 to ORF5 corresponds to various oligopeptide permease operons (Hiles et al., 1987; Perego et al., 1990). Only ORF5 shows homology to a gene from a different bacterium, Bacillus anthracis (Makino et al., 1989). Each homologue encodes membrane-bound or membrane-associated proteins suggesting that all five ORFs are involved in oligopeptide permeation.

[0053] Organization of the predicted gene cluster IV, including the nifA homologue ORF16 (fixABCX, nifA, nifB, fdxN, ORF, fixU homologues, position 16,746-24,731), the predicted locations of the σ54-dependent promoters and the nifA upstream activator sequences (FIG. 2), correspond to the organization found in Rhizobium meliloti and Rhizobium leguminosarum bv. trifolli. (Iismaa et al., 1989; Fischer, 1994). NifA is a positive transcriptional activator (Buikema et al., 1985), whereas nif and fix genes are essential for symbiotic nitrogen fixation. Identification of σ54-dependent promoter sequences, together with the upstream activator motifs upstream of ORF21, ORF22, and ORF23, suggests that these ORFs may play an important, but still undefined, role in symbiosis.

[0054] Inevitably, large-scale sequencing uncovers differences with already published sequences. van Slooten et al. (1992) cloned a 5.8 kb EcoRI fragment from Rhizobium sp. NGR234 and sequenced 2067 bp by manual radioactive methods (EMBL accession no. S38912). This sequence exhibits 2.4% mismatches with the corresponding sequence in pXB296.

TABLE 2
Putative ORFs of pXB296 and homologies of the deduced amino acid sequences to
known proteins
		riboso-
		mal binding site:		homo-
		SD-sequence-dis-		logous
		tance from start	no. of	amino	homologous
	position on	codon (bases)-	deduced	acids	protein		iden-	simi-
		cosmid	start codond	amino	(po-		length		acces-	tity	larity
ORFa	st.b	(based no.)c	SD-sequence:	acids	sition)	name	(aa)c	functionf	sion no	(%)g	(%)g
ORF1h	+	00001-00625	5′-TAAGGAGGTGA-3′	>207	1-207	OppB	306	oligopeptide	X05491	45	68
ORF2	+	00628-01503	GTATCCGGT-7-ATG	291	2-289	OppC	305	permease	X56347	37	63
ORF3	+	01505-02512	AGCGGAGG-7-ATG	335	8-327	OppD	336	proteins	X56347	49	69
ORF4	+	02509-03570	TGAAGTGGT-6-ATG	353	2-323	OppF	334		X05491	51	69
ORF5	+	03606-04991	CAAGGA-6-ATG	461	1-458	CapA	411	encapsulation	M24150	25	48
								protein
ORF6	+	05460-06863	CCGAGAGG-8-ATG	467	1-464	BioA	455	aminotrans-	M29292	29	55
								ferase
ORF7	+	06888-08426	GCCTTCGG-5-GTG	512	97-509	ORFi	417	unknown	D37877	36	58
					34-510	GapD	482	succinic	M38417	33	57
								semialdehyde
								dehydrogenase
ORF8	−	09781-10860	GAACGTGG-8-ATG	359	72-299	ORFi	414	transposase	X15942	30	48
								homologue
								minicircle DNA
ORF9	+	11124-12455	?-7-ATG	443	2-443	GLUDI	558	glutamate	M37154	41	60
								dehydrogenase
ORF10	−	13370-14116	AAAGGA-6-ATG	248	1-245	ORF21	231	transposase	X79443	45	64
ORF11	−	14128-15672	CATGGAG-7-TTG	514	1-513	ORF11	558	homologues,	X79443	41	62
								IS1162
ORF12	−	16712-16942	GAAGGA-8-ATG	76	1-70	FixU	70	unknown	P42710	63	80
ORF13	−	16939-17265	ACAAGAGG-7-ATG	109	1-79	ORF2i	>78	unknown	X07567	53	81
					15-107	NifZ	159	involved in	M20568	39	56
								FeMocofactor
								synthesis
ORF14	−	17349-17543	CCAGGAG-9-ATG	64	1-64	FdxN	64	ferredoxin-	M21841	80	87
								like
ORF15	−	17585-19066	AGTGGAG-7-ATG	493	1-493	NifB	490	involved in	M15544	73	84
								FeMocofactor
								synthesis
ORF16	−	19292-20962	ATTGG-12-ATG	556	9-556	NifA	541	transcrip-	X02615	59	72
								tional
								regulator
ORF17	−	21129-21422	AGGGGAG-7-ATG	97	1-97	FixX	98	required for	M15546	84	87
ORF18	−	21437-22744	AACTGAGGT-7-ATG	435	1-435	FixC	435	nitrogen	M15546	83	90
ORF19	−	22755-23864	ATAGGAG-6-ATG	369	18-369	FixB	353	fixation	M15546	79	89
ORF20	−	23874-24731	TAAAGAG-5-ATG	285	1-285	FixA	292		M15546	74	85
ORF21	−	25148-25468	CCAGGAG-10-ATG	106	1-106	ORF118i	108	unknown	X13691	55	71
ORF22	−	26145-26711	GAAGGAG-9-ATG	188	9-199	−	241	hypothetical	U32739	47	64
								protein
					1-173	−	166	peroxisomal	U11244	32	57
								protein
ORF23	+	27169-27861	GAAGGA-7-ATG	230	1-167	NifQ	167	probably in-	X13303	37	57
								volved in
								Mo-processing
ORF24	+	27920-29434	CTGGGAGG-18-ATG	504	1-454	DctA1	456	C4-dicarboxy-	S38912	97	98
								late
					8-454	DctA2	449	transporter	S38912	97	98
ORF25	+	29431-30675	TTCGGCGG-12-ATG	414	2-414	CamC	415	cytP450-like	M12546	34	53
ORF26	+	30676-31332	TTGGG-5-TTG	218	30-190	LinA	155	γ-hexachloro-	D90355	27	51
								cyclohexan-
								dechlorinase
ORP27	+	31329-33035	AGTGGAG-10-ATG	568	28-270	FabG	244	reductase	M84991	38	57
					294-534					30	57
ORF28k	+	33173-34010	CAAGGAG-5-ATG	>279	1-279	LuxA	355	luciferase	M10961	23	49
								α-subunit
a(ORF) Open reading frame.
b(st.) Plus or minus strand.
cPosition on cosmid: from the first base of the start codon to the last base of the stop codon; alternative start points are 6912/6927/7017 (ORF7), 10665/10656 (ORF8), 11220 (ORF9), 15699/15651 (ORF 11), 17322/17271 (ORF13), 20995/21076 (ORF16), 26744 (ORF22), 27229/27304 (ORF23), 27941 (ORF24), and 30751/30754 (ORF26).
d(SD sequence) Shine-Dalgarno sequence (Shine and Dalgarno 1974). Bases underlined are identical with the Shine-Dalgarno sequence. The following possible start codons were considered: ATG, GTG, or TTG.
e(aa) Amino acids.
fOrganisms: Salmonella typhimurium, Bacillus subtilis (OppBCDF), Bacillus anthracis (CapA), Bacillus sphaericus (BioA), Streptomyces hygroscopius (ORF7 homolog), Escherichia coli (GapD). Streptomyces coelicolor (ORF8 homolog), Homo sapiens (GLUD1), Pseudomonas fluorescens (ORF10, ORF11 homologs). Rhizobium leguminosarum (FixU), Rhodobacter capsulatus (ORF13 homolog), Azo
#tobacter vinelandii (NifZ), Rhizobium meliloti (FdxN, NifBA, FIxXCBA), Bradyrhizobium japonicum (ORF118), Haemophilus influenzae (hypothetical protein), Lipomyces kononenkoae (peroxisomal protein), Klebsiella pneumonia (NifQ), Rhizobium sp. NGR234 (DctA), Pseudomonas putida (CamC), Pseudomonas paucimobilis (LinA), Escherichia coli (FabG), Vibrio harveyi (LuxA).
gidentity and similarity were calculated using the program BESTFIT (Smith and Waterman 1981).
h(ORF1) 3′ end.
iTranslated ORF.
k(ORF28) 5′ end.

[0055] It contains the gene dctA (encoding a C4-dicarboxylate permease), which is 144 bases shorter than in pXB296. In this respect, a single nucleotide deletion in position 29,248 of the cosmid sequence close to the 3′ end of the gene causes a frameshift leading to a DctA product extended by 48 residues. van Slooten et al. (1992) also failed to identify the nifQ homologue, ORF23 (position 27,169-27,861), presumably because they overlooked a small XhoI fragment located between positions 27,349 and 27,536 on pXB296. Expression studies allowed these investigators to define a putative a σ54-dependent promoter in a 1.7 kb SmaI fragment (position 27,094-28,818 in pXB296). This fragment stretches from the upstream region of ORF23 to the 5′ part of dctA. The 58 bp intergenic region between ORF23 and dctA contains a stem-loop structure but no obvious promoter sequence. Possibly the promoter that controls dctA is located upstream of ORF23 (e.g. the minimal consensus sequence included in GGGGGCACAATTGC at position 27,098-27,111). Although clones containing dctA complemented mutants of R. meliloti and R. leguminosarum for growth on dicarboxylates, the growth of the NGR234 dctA deletion mutant was not affected (van Slooten et al., 1992). Nevertheless, this mutant was unable to fix nitrogen in nodules. Because dctA is now possibly part of a larger transcription unit, the symbiotic phenotype may also result from the inactivation of downstream genes.

[0056] Interestingly, the GC content of the predicted pXB296 ORFs ranges from 53.3 mol % to 64.6 mol %, with an overall cosmid GC content of 58.5 mol %. Genomes of Azorhizobium, Bradyrhizobium, and Rhizobium species have GC contents of 59 mol % to 65 mol % (Padmanabhan et al., 1990), with 62 mol % reported for Rhizobium sp. NGR234 (Broughton et al., 1972). Although pXB296 covers <7% of the complete symbiotic plasmid sequence, its lower overall GC value suggests that symbiotic genes might have evolved by lateral transfer from other organisms. In this case, methods of the type applied in the present invention will become even more relevant in sequencing the whole genome.

[0057] Genetic Organization of the 100 kb Region Covered by Cosmids pXB296, pXB368 and pXB110

[0058] Extending the analysis of pXB296 to a 100 kb region stretching from position 417,796 to 517,279 on the symbiotic plasmid pNGR234a led initially to the assignation of only 76 ORFs listed within Table 3 (excluding the first incomplete ORF noted in the analysis of pXB296 (“ORF1” of Table 2)). The ORFs y4tQ to y4vJ (excluding ORFs y4uD and y4uG and excluding ORF-fragments fu1, fu2, fu3, fu4 and fv1; see Table 3) are identical to the ORFs 2 to 28 of the analysis of pXB296 in Table 2 apart from minor revisions (N.B. the analysis recited in Table 3 should be taken as the definitive analysis—Table 2 merely represents preliminary findings). The cosmid pXB110, which was sequenced with the dye primer shotgun sequencing strategy in order to compare it with the dye terminator shotgun sequencing strategy used to sequence cosmid pXB296, in combination with pXB296 and pXB368 cover nearly this entire region. A PCR product and a restriction fragment of cosmid pXB564 also had to be sequenced in order to fill in the gap from position 480,607 to 483,991 between cosmids pXB368 and pXB110 (FIG. 4). Among the 76 predicted ORFs, 7 ORFs and their deduced proteins show no homologies to database entries. The other predicted ORFs and their deduced proteins do exhibit such homologies and therefore play putative roles in nitrogen fixation (ORFs y4uJ to y4vB, y4vE, y4vN to y4vR, y4wK and y4wL), nodulation (ORFs y4yC and y4yH), transportation (ORFs y4tQ to y4uA, y4vF and y4wM), secretion of proteins or other biomolecules (ORFs y4yI and y4yO), transcriptional regulation/DNA binding (ORFs y4wC and y4xI), in amino acid metabolism or metabolism of amino acid derivatives (ORFs y4uB, y4uC, y4uF, y4wD, y4wE and y4xN to y4yA), degradation of xenobiotic compounds (ORFs y4vG to y4vI), in peptidolysis/proteolysis (ORFs y4wA and y4wB) or transposition (ORFs y4uE, y4uH and y4uI) (see Table 3). The

TABLE 3
List of the predicted functional ORFs and of fragments representing putative remnants of functional ORFs
				no. of	hom.
	func-		position in	deduced	amino	hom. protein
	tional		plasmid	amino	acids		length	accession	I/	S/
ORFa	name	st.b	(base no.)c	acids	(position)	name	(aa)d	no.e	%f	%f	noteg
y4aA		−2/3	534696-000474	647	16-646	Shc	658	X86552	78	88	prob. squalene-hopene-cyclase; put. operon y4aABCD:
											inv. in synthesis of an isoprenoid compound
y4aB		−3	000523-001776	417	6-415	ORF1	414	X80766	43	63	put. flavoprotein oxidoreductase
y4aC		−2	001776-002615	279	3-247	Psy1	419	X68017	34	50	put. phytoene synthase
y4aD		−1	002612-003490	292	10-195	CrtI	342	L37405	33	51	hyp. protein hom. to squalene and phytoene synthetases
fa1		−3	003487-004011								fragmentous character
y4aF	nolK	−3	005173-006117	314	9-310	ORF14.8	321	U46859	51	70	put. NAD-dep. nucleotide sugar epimerase/dehydrogenase;
											NoeJKL/NodZ/NolK inv. in biosynthesis of fucose
											moiety of Nod factors
y4aG	noeH	−2	006126-007181	351	4-339	RfbD	348	U24571	65	80	put. GDP-D-mannose dehydratase
y4aH	nodZ	−1	007426-008394	322	3-254	NodZ	324	L22756	69	83	put. fucosyltransferase
y4aI	noeK	−3	008623-010047	474	5-471	ORF5	483	U47057	42	59	put. phosphomannomutase
y4aJ	noeJ	+3	010110-011648	512	33-498	XanB	466	M83231	50	65	put. mannose-1-phosphate guanylyltransferase
y4aK		+2	012125-012277	50							hyp. 5.5 kd protein
y4aL	nodD1	+2	012380-013348	322	1-322	NodD1	322	Y00059	98	99	transcriptional regulator (LysR family); high similarity to
					1-310	NodD2	312	this work	68	84	Y4xH(NodD2)
y4aM		+3	013911-014342	143	7-132	ORF3	127	L13845	50	66	put. DNA-binding protein; high similarity to Y4wC
					1-143	Y4wC	143	this work	69	77
y4aN		+1	01014488-014934	148	1-129	ORF3	128	X04833	41	56	homologue located nearby the replicator region of pRiA4b
y4aO		+3	015065-015643	192							hyp. 21.8 kd protein; low similarity to Y4nF(<30% id.)
y4aP	mucR	+3	016161-016592	143	1-143	MucR	143	L37353	89	95	put. transcriptional regulator (Ros/MucR family);
											similarity to Y4pD; possibly inv. in regulation of
											exopolysaccharide synthesis
y4aQ		−2	017016-017582	188	15-167	No1265	266	X74068	33	50	hyp. 20.4 kd protein; similar to Y4hP, Y4jD, Y4qI
y4aR		+2	017798-018121	107							hyp. 12.1 kd protein
y4aS		+1	018121-018666	181							hyp. 20 kd protein
fa2		+3	018912-019664	250	126-250	Tnp	465	U04047	38	51	hyp. protein fragment
					78-150	Y4iG	90	this work	93	97
					3-266	Y4bF	457	this work	53	73
y4bA		−2	019674-021758	694	1-393	fo6	430	this work	89	95	hyp. 78.7 kd protein; identical to Y4pH
					406-532	fo5	136	this work	83	94
					532-694	fo4	143	this work	77	83
y4bB		−3	021748-022014	88	2-88	Y4oL	88	this work	63	69	hyp. 9.7 kd protein precursoer; identical to Y4pI
y4bC		−1	022034-022483	149	1-149	Y4oM	149	this work	79	88	hyp. 16.8 kd protein; identical to Y4pJ
y4bD		−2	022674-022943	89	20-89	Y4oN	70	this work	73	84	hyp. 10.2 kd protein; identical to Y4pK
fb1		+2	022985-023659	224	36-224	Y4bF	457	this work	42	63	hyp. protein fragment
y4bF		+1	023953-025326	457	130-436	Tnp	465	U04047	31	46	put. transposase;
					2-265	Fa2	266	this work	53	73	upstream of this ORF (23875-23987) 89% nt-id. to part
					77-169	Y4iG	90	this work	51	72	of origin of replication-region (R. meliloti, S66221)
					285-457	Fb1	188	this work	42	63
					410-457	Y4jM	70	this work	75	79
y4bG		+1	025870-026685	271							hyp. 30 kd protein precurser
y4bH		+1	028513-028788	91							hyp. 9.6 kd integral membrane protein
y4bI		+3	028860-029276	138	3-108	HI1631	190	U00085	41	61	hyp. 15.3 kd protein precurser
y4bJ		+1	029392-031284	630	429-564	HtrA	503	L20127	40	53	hyp. 67.9 kd integral membrane protein, distantly related
											to peptidase family S2C
y4bK		+2	031625-032293	222	83-212	ORF1	215	D84146	25	45	hyp. 24.3 kd protein
y4bL		+1	032641-034191	516	7-515	ORF1	558	X79443	44	63	identical to Y4kJ and Y4tB; similar to Fo3 and Fo7; put.
					6-516	Y4uI	515	this work	48	66	transpposase
y4bM		+3	034188-034979	263	1-203	ORF2	231	X79443	45	62	identical to Y4kI and Y4tA; put. insertion sequence ATP-
					6-248	Y4pL	245	this work	55	73	binding protein; similarity to Y4pL, Y4uH, also to
					6-254	Y4uH	248	this work	48	68	Y4sD/Y4nD/Y4iQ
					1-263	Y4iQ	298	this work	31	56
y4bN		+1	035278-036573	431							hyp. 47.6 kd protein
y4bO		+1	036646-038466	606							hyp. 66.8 kd protein
y4cA		−1	038576-042169	1197							hyp. 137.7 kd protein; largest protein in pNGR234a
y4cB		−3	042226-042522	98							hyp. 10.2 kd integral membrane protein
y4cC		−3	042556-044109	517							hyp. 57.8 kd protein
y4cD		−2	044106-046028	640							hyp. 71.6 kd protein
y4cE		−3	046486-047661	391							hyp. 43.4 kd protein
y4cF		−1	047687-048829	380							hyp. 41.8 kd protein
y4cG		+2	049361-050278	305	16-173	Pin	184	K00676	50	68	prob. DNA invertase “resolvase-type”
					17-222	Y41S	183	this work	40	60
y4cH		−2	050427-050636	69	4-65	CspS	70	L23115	56	70	prob. cold shock regulator
y4cI		−2	053202-054416	404	1-397	RepC	405	X04833	60	73	put. replication protein C
y4cJ		−3	054571-055551	326	1-317	RepB	319	X89447	39	55	put. replication protein B
y4cK		−2	055608-056831	407	10-404	RepA	398	X89447	58	73	put. replication protein A
y4cL	traI	+2	057635-058261	208	1-206	TraI	212	U43675	55	66	prob. autoinducer synthetase (inv. in control of conjugal
											transfer)
y4cM	trbB	+3	058272-059249	325	3-325	TrbB	323	U43675	80	88	prob. conjugal transfer protein (PulE family)
					1-115	Y4oG	125	this work	25	51
y4cN	trbC	+1	059239-059622	127	7-127	TrbC	134	U43675	69	78	prob. conjugal transfer protein (integral membrane prot.)
y4cO	trbD	+2	059615-059914	99	1-99	TrbD	99	U43675	70	89	prob. conjugal transfer protein (integral membrane prot.)
y4cP	trbEa	+3	059925-060374	149	1-136	TrbE	820	U43675	80	91	prob. conjugal transfer protein (hom. to 5′ part of trbE)
y4cQ	trbEb	+1	060394-062382	662	5-659	TrbE	820	U43675	83	90	prob. conjugal transfer protein (hom. to 3′ part of trbE)
y4dA	trbJ	+2	062354-063157	267	1-107	TrbJ	175	U43675	60	69	prob. conjugal transfer protein
					194-267				71	79
y4dB	trbK	+1	063154-063351	65	5-65	TrbK	75	U43675	40	56	prob. conjugal transfer protein precurser
y4dC	trbL	+3	063345-064520	391	3-387	TrbL	395	U43675	74	85	prob. conjugal transfer protein (integral membrane prot.)
y4dD	trbF	+2	064544-065206	220	1-220	TrbF	220	U43675	80	90	prob. conjugal transfer protein
y4dE	trbG	+1	065224-066036	270	6-270	TrbG	284	U43675	74	84	prob. conjugal transfer protein precursor
y4dF	trbH	+1	066040-066486	148	1-147	TrbH	159	U43675	55	68	prob. conjugal transfer protein precurser (with lipid anchor)
y4dG	trbI	+3	066498-067793	431	1-430	TrbI	433	U43675	66	79	prob. conjugal transfer protein (integral membrane prot.)
y4dH	traR	+2	068096-68806	236	7-236	TraR	234	Z15003	28	45	prob. transcriptional activator of conjugal transfer genes
											(LuxR family)
y4dI	traM	−1	068810-069133	107	8-101	TraM	102	U43674	30	51	prob. modulator of TraR/autoinducer-mediated activation
											of tra genes
y4dJ		+3	069351-069584	77	1-67	ORF	84	X16458	37	59 hyp. transcriptional regulator (PbsX family); low
											similarity to N-terminus of Y4dL
y4dK		−1	069629-069949	106							hyp. 11.8 kd protein
fd1		−2	069936-070250	(105)	(2-85)	ORFA	400	X67861	39	58	put. transposase fragment
y4dL		+1	070603-071193	196	—	—	—	—	—	—	hyp. 21.8 kd protein; low similarity to Y4dJ
y4dM		+2	071186-072415	409	1-357	HipA	440	M61242	31	46	hyp. 45.3 kd protein; homolog affects frequency of
					3-405	Y4mE	420	this work	34	56	persistence after inhibition of cell wall or DNA synthesis
y4dN		+1	072787-072975	62	—	—	—	—	—	—	hyp. 7 kd protein
y4dO		−1	073550-073951	133	12-121	ORF	381	D83536	43	57	hyp. 14.9 kd (fragmentous?) protein; homology to intron
											protein of P. anserina continues in fr.-2 (73541-73467)
y4dP		−1	074423-075025	200	1-48	ORFR2	57	U43674	72	89	hyp. 21 kd protein; hom. to conjugal transfer region 1
					56-198	ORFR3	154		47	71
y4dQ	traB	−2	075042-076205	387	1-387	TraB	421	U40389	61	72	prob. conjugal transfer protein
y4dR	traF	−3	076195-076761	188	20-188	TraF	176	U40389	55	73	prob. conjugal transfer protein
y4dS	traA	−2	076758-080066	1102	1-1102	TraA	1100	U43674	67	79	prob. conjugal transfer protein (relaxase)
y4dT	traC	+3	080319-080627	102	1-102	TraC	98	U40389	64	80	prob. conjugal transfer protein
y4dU	traD	+1	080632-080847	71	1-71	TraD	71	U43674	77	84	prob. conjugal transfer protein
y4dV	traG	+2	080834-082756	640	1-631	TraG	658	U40389	71	83	prob. conjugal transfer protein
fd2		+	083002-083293			ORFL1	152	U43674			fragments hom. to ORFL1 (conjugal transfer region1);
											frameshifts: 83072 (1 > 3), 83161 (3 > 2)
y4dW		+1	083305-083919	204							hypothetical 22.9 kd protein
y4dX		+1	083944-84522	192							hypothetical 20.6 kd protein
y4eA		−2	084570-084836	88							hypothetical 9.9 kd protein
y4eB		−3	084976-085290	104							hypothetical 11.6 kd protein
fe1		−	085829-088007			MerA	474	X65467			put. fragments; homology to mercuric reductase, put.
											frameshifts: 86592 (−1 < −3), 87288 (−3 < −2)
y4eC		−2	088305-089228	307	14-306	TraC-1	1061	X59793	38	55	hyp. 34.2 kd protein; hom. to 5′end. of traC-1 from
											plasmid RP4
y4eD		+1	091051-092178	375	51-136	ORF145	145	X52594	29	55	put. phosphodiesterase; low homology to
											glycerophosphoryl-diester-phosphodiesterase
y4eE		+1	092212-093288	358							hyp. 38.5 kd protein
fe2		−	093572-093969			TnpA		U14952			fragments of put. transposase; put. frameshift: 93798
											(2 < 3)
y4eF		−1	093980-094735	251	2-236	Int	259	U14952	37	53	put. integrase/recombinase (“phage-type”); similar to
					1-251	Y4qK	308	this work	92	94	Y4rF (35% aa-id.); low similarity to Y4rABCDE
fe5		−1	094988-095188	66	1-66	Fq6	66	this work	79	94	put. defective integrase/recombinase
					1-66	Y4rC	332	this work	41	55
fe3		−	095343-096025			Int	259	U14952			fragments hom. to integrase; put. frameshift: 95559-
											95671 (−2 < −1)
y4eH	nolL	−2	096093-097193	366	11-359	NolL	373	U22899	63	77	nodulation protein; hyp. acetyl transferase
y4eI		−2	097914-098225	103							hyp. 11.1 kd protein with transmembrane domain
fe6		+3	098358-098657	99	3-98	AatB	410	L12149	40	55	hyp. 10.3 kd protein fragment, hom. to C-terminal part of
											bacterial aminotransferases
y4eK		+2	098675-099421	248	10-245	Adh	252	U00084	37	53	hyp. short chain type dehydrogenase/reductase
y4eL		+3	099447-100193	248	1-244	Gno	256	X80019	31	47	hyp. short chain type dehydrogenase/reductase
fe4		+	100270-101901			IIvG		M37337			put. fragment; put. frameshifts: 100721 (1 > 2), 101728
											(2 > 1)
fe7		−1	101585-102298	237	1-103	Tnp	398	U08627	91	95	put. truncated transposase-like protein; similar to Y4pO
y4eN		−3	102625-102936	103							hyp. 11.5 kd protein
y4eO		−2	102933-103598	221							hyp. 24.5 kd protein
y4fA		−1	103805-106342	845	327-837	McpA	657	X66502	41	59	prob. methyl-accepting chemotaxis protein
					7-845	Y4sI	756	this work	29	49
y4fB		+3	106620-108614	664							hyp. 73.7 kd protein
y4fC		+3	109884-110618	244	10-163	DszA	453	L37363	38	52	hyp. (fragmentous?) monooxygenase; extended homology
											to DszA in fr.2: 110372 to 110506.
y4fD		−1	110516-111178	220							hyp. 24.6 kd integral membrane protein
y4fE		−2	111195-111677	160							hyp. 17.2 kd protein precursor
y4fF		−1	111803-112348	181							hyp. 19.5 kd protein
y4fG		−2	112338-112727	129							hyp. 14.5 kd protein
y4fH		−1	113474-113782	102							hyp. 11.6 kd protein
ff1		−3	113779-114114	111	61-97	DppF	330	L08399	56	86	hyp. protein fragment, similar to central region of
											oligo/di-peptide ABC transporter ATP-binding proteins
y4fJ		−2	114348-115379	343	3-210	RopA	318	M69214	53	66	put. outer membrane protein (porin) precurser
y4fK		−2	116112-117395	427	275-421	XylS2	157	L02642	31	53	put. transcriptional regulator (AraC family)
y4fL		−3	117385-118212	275	9-243	ORF	268	U39059	32	46	hyp. 29.1 kd integral membrane protein, belongs to the
											inositol monophosphatase family
y4fM		−2	118209-119144	311							hyp. 35.5 kd protein
y4fN		−2	119145-120854	569	11-513	CysU	550	U32807	23	45	prob. ABC transporter permease protein; put. part of
											binding-protein-dependent transport system Y4fNOP
y4fO		−1	120851-121870	339	12-247	PotA	381	U32759	49	68	prob. ABC transporter ATP-binding protein
y4fP		−1	121883-122959	358	32-293	SufA	338	M33815	23	42	prob. ABC transporter periplasmic binding protein precurser
y4fQ		+1	123016-124194	392	9-234	NagC	406	X14135	25	46	hyp. 41.6 kd protein; belongs to “ROK” family
											(transcriptional regulator or transferase)
y4fR		+1	124813-126453	546	88-539	IpaH	532	M32063	38	54	hyp. 60.5 kd protein, hom. to invasion plasmid antigen H
y4gA		−1	126806-127369	187							hyp. 20.9 kd protein; low similarity to Y4rE
y4gB		−2	127485-127904	139							hyp. 16.1 kd protein
y4gC		−1	127901-128479	192	1-178	ORF2	415	L34580	43	58	put. integrase/recombinase (“phage-type)
y4gD		−1	128579-128857	92							hyp. 10.5 kd protein
y4gE		+2	131021-131767	248							hyp. (fragmentous?) 27.7 kd protein;
											put. frameshifts: 131532 (2 > 1), 131892 (1 > 2)
y4gF		+2	132734-133786	350	4-345	RhsB	353	U51197	65	74	prob. dTDP-D-glucose-4,6-dehydratase (Y4gFGH inv. in
											dTDP-L-rhamnose biosynthesis)
y4gG		+2	133790-134680	296	1-290	RhsD	288	U51197	48	66	prob. dTDP-4-dehydrorhamnose reductase
y4gH		+1	134677-135537	286	2-285	RfbA	293	U09876	65	82	prob. glucose-1-phosphate thymidylyltransferase
y4gI		+3	135534-138263	909	276-894	RfbC	1275	U36795	38	55	hyp. 102.8 kd protein (homolog is involved in O-antigen
											biosynthesis)
y4gJ		−1	138737-139315	192							hyp. 21.1 kd protein
y4gK	fixF	+3	142026-143234	402	114-184	KpsS	389	X74567	26	54	necessary for functional nitrogen fixation, hom. to
					203-362				30	53	capsule polysaccharide export protein
y4gL		−3	143473-144060	195	24-192	RhsC	188	U51197	53	65	prob. dTDP-4-dehydrorhamnose-3,5-epimerase (inv. in
											dTDP-L-rhamnose biosynthesis)
y4gM		−2	144147-145907	586	26-581	MsbA	582	Z11796	32	56	prob. ABC transporter ATP-binding protein
y4gN		+2	146075-147226	383	52-297	VirA	304	L08012	29	46	hyp. 45 kd protein
y4hA		−1	147455-148558	367	7-362	ChaA	366	L28709	34	58	put. ionic transporter
y4hB	noeE	−3	148819-150078	419	3-138	F42G9.8	359	U00051	32	49	nodulation protein (put. sulfate transferase)
					197-289				25	50
y4hC	noeG	−3	151051-151782	243	18-229	u0002kb	243	U00024	27	42	nodulation protein (unknown function)
y4hD	nolO	−1	151979-154021	680	1-126	NolN	127	L22756	70	83	inv. in O-carbamoylation of Nod factors (sim. to NodU)
					140-496	NolO	358		78	89
y4hE	nodJ	−3	154120-154908	262	5-261	NodJ	262	J03685	69	84	prob. ABC transporter permease (see nodI)
y4hF	nodI	−3	154912-155943	343	15-343	NodI	339	X55795	69	85	prob. ABC transporter ATP-binding transport protein;
											put. role: together with NodJ export of modified beta-1,4-
											N-glucosamine oligosaccharides
y4hG	nodC	−1	156095-157336	413	1-413	NodC	413	X73362	99	100	N-acetylglucosaminyltransferase
y4hH	nodE	−3	157351-157998	215	1-215	NodB	214	X73362	99	99	chitooligosaccharide deacytelase
y4hI	nodA	−2	157995-158585	196	1-196	NodA	196	X73362	100	100	N-acyltransferase; nodABC involved in synthesis of
											backbone of modified N-acylated glucosamine
											oligosaccharides
y4hJ		−1	158993-159775	260	59-240	ORF2	251	L133618	68	81	hom. to part of coproporphyrinogen III oxidase (lacks C-
											terminus and conserved N-term. domain)
y4hK		+3	160722-161465	247							hyp. 25.4 kd integral membrane protein
y4hL		+1	161569-161826	85							hyp. 9.6 kd protein
y4hM		+1	163042-164253	403	53-169	Gfor	439	M97379	31	54	hyp. 43.9 kd protein (partially hom. to glucose-fructose
											oxidoreductase)
y4hN		+2	164600-165034	144	10-144	ORFA	135	X84099	38	53	hyp. 16 kd protein; partially hom. to Y4jB and Y4rG
y4hO		+1	165037-165384	115	1-115	ORF140	140	X74068	100	100	hyp. 12.8 kd protein
					1-115	ORFC	144	X84099	54	69
					1-115	Y4jC	117	this work	36	62
y4hP		+1	165430-167088	552	1-215	no1265	266	X74068	97	97	hyp. 61.7 kd protein; similar to Y4aQ, Y4jD and Y4qI
					80-328	ORF2	258	M10204	67	79
					362-492	ORF3	163	M10204	47	61
y4hQ		+3	167091-167675	194	5-185	ORF3	237	X51418	35	53	hyp. 21.7 kd protein
					1-52	ORF91	>91	X74068	96	98
y4hR		−3	167710-167934	74
fi1		−	168208-168300								hyp. transposase fragment similar to R. meliloti
											ISRm2011-2
fi2		+1	168430-168792	120	1-130	Y4iO	252	this work	78	87	put. defective transposase (homologous to N-terminal
					1-108	Y4rJ	396	this work	74	87	parts of Y4iO and Y4rJ)
fi3		+2	168798-169190	130	1-109	ORF1A	317	M33159	37	55	put. defective transposase (hom. to C-terminal parts of
					1-130	Y4iO	252	this work	78	87	Y4iO and Y4rJ); additionally weak homology to
					1-130	Y4rJ	396	this work	76	84	Y4pF/Y4sB and Y4qE (<30% identity)
y4iR		−3	169231-169716	161	15-145	PsiB	134	L26581	55	74	hyp. protein (homolog located in a polysaccharide
											biosynthesis inhibition operon
y4iC		−2	169929-110621	230	58-123	ORF	161	Z73419	41	54	hyp. 25.8 kd protein (ORF = MTCY373.06)
y4iD		−3	170563-172551	662	137-342	ORF	495	Z73101	40	59	prob. monooxygenase (ORF = MTCY31.20)
					418-605				28	51
y4iE		+3	173295-173702	135	1-135	Y4rL	155	this work	33	52	hyp. 15.4 kd (fragmentous?) protein; similar to Y4zA
y4iF		−3	174211-175128	305							hyp. 34.1 kd protein
y4iG		−2	175590-175862	90	1-73	Y4aT	266	this work	93	97	hyp. 10.5 kd (fragmentous?) protein
					1-73	Y4bF	457	this work	60	76
y4iH		+2	176045-176764	239	1-236	Y4jT	336	this work	32	53	hyp. 26 kd protein precurser
y4iI		−2	176937-179048	703							hyp. 76.2 kd integral membrane protein
y4iJ		−2	179097-180887	596							hyp. 65.5 kd protein; ;ow similarity to Y4iM
y4iK		−3	180940-181638	232							hyp. 26.8 kd protein; y4iKL: two fragments of one gene?;
											put. frameshift; 181884 (−3 < −2)
y4iL		−2	181692-182990	432							hyp. 47.8 kd protein; y4iKL two fragments of one gene?;
											put. frameshift: 181884 (−3 < −2)
y4iM		−2	183036-184334	432							hyp. 47.1 kd protein; low similarity to Y4iJ; y4iMN two
											fragments of one gene?;
											put. frameshift: 184440 (−2 < −3)
y4iN		−3	184309-184935	208							hyp. 22.1 kd protein precurser; y4iMN two fragments of
											one gene?; put. frameshift: 184440 (−2 < −3)
y4iO		−2	185679-186437	252	17-243	Tnp	334	Z48244	29	46	put. transposase or transposase-fragment; additionally
					1-121	Fi2	120	this work	67	79	weak homology to Y4pF/Y4sB and Y4qE (<30% identity)
					123-252	Fi3	130	this work	78	87
					1-252	Y4rJ	396	this work	71	83
y4iP		−1	186437-186832	131	4-163	Y4rJ	396	this work	58	80	hyp. 14.4 kd protein or fragment hom. to N-term. of Y4rJ
y4iQ		−3	187162-188058	298	13-253	IstB	265	U38187	34	56	identical to Y4nD/Y4sD; put insertion sequence ATP-
					8-283	Y4bM	263	this work	31	56	binding protein; similarity to Y4bM/Y4kI/Y4tA, Y4uH
					5-265	Y4uH	248	this work	31	52	and weakly to Y4pL
y4jA		−2	188055-189569	504	147-494	IstA	507	U38187	25	42	identical to y4nE/y4sE; hyp. 57.2 kd protein with low
					395-504	Fz4	110	this work	72	85	similarity to IS21/IS408/IS1162 transposases
y4jB		+3	190248-190706	152	24-79	ORF1	130	U19148	46	69	hyp. 16.7 kd protein; partially similarity to Y4hN; low
											similarity to Y4rG
y4jC		+2	190703-191056	117	1-115	ORFC	144	X84099	39	58	hyp. 13.1 kd protein; see y4hO
					1-117	Y4hO	115	this work	36	62
y4jD		+2	191105-192640	511	89-298	ORF2	258	M10204	36	53	hyp. 56.7 kd protein; see y4hP
					340-453	ORF3	163	M10204	28	49
					18-183	no1265	266	X74068	32	48
y4jE		+1	192637-193458	273							hypothetical (fragmentous?) 29.4 kd integral membrane
											protein; put. frameshift: 192996 (1 > 2; end of shifted ORF
											at 193183)
y4jF		−1	194771-196330	519							hyp. 55.4 kd integral membrane protein
y4jG		−3	196333-196821	162							hyp. 17.9 kd transmembrane protein
y4jH		−2	196818-197435	205							hyp. 23 kd protein
y4jI		−3	197428-197820	130							hyp. 13.6 kd protein
y4jJ		+1	198043-198300	85	1-85	StbC	103	L48985	67	76	put. plasmid stability protein
y4jK		+3	198297-198719	140	1-138	StbB	139	L48985	57	76	put. plasmid stability protein
y4jL		+3	199002-199664	220							hyp. 25.1 kd protein
y4jM		−2	199746-199958	70	11-58	Y4bF	457	this work	75	79	hyp. 8 kd protein or protein fragment
					15-58	fb1	188	this work	50	64
y4jN		−3	199975-200415	146							hyp. 16.3 kd protein
y4jO		−3	201514-202479	321							hyp. 36.1 kd protein; y4jOP; two fragments of one gene?,
											put. frameshift: 202550 (−3 < −1)
y4jP		−1	202406-203194	262							hyp. 29.5 kd protein; y4jOP: two fragments of one gene?,
											put. frameshift: 202550 (−3 < −1)
y4jQ		+2	203729-206848	1039							hyp. 115.9 kd protein
y4jR		+1	206860-207315	151							hyp. 17.3 kd protein
y4jS		+1	207316-208557	413							hyp. 44.8 kd protein
y4jT		−1	208877-209887	336	17-283	Y4iH	239	this work	32	53	hyp. 36.4 kd protein precurser
y4kA		−3	209917-210885	322							hyp. 36.7 kd protein
y4kB		+1	211663-212088	141							hyp. 15.2 kd integral membrane protein
fk2		−1	212111-212479	122	58-116	ORF14	104	X00493	59	76	hyp. fragment; sim. to Y4hP, Y4jD and Y4qI; additional
											homology to ORF14 in fr. +3/+2: 212331-212509
y4kD		−1	212750-214399	549							hyp. 60.4 kd protein
y4kE		−1	214412-215455	347							hyp. 38 kd protein; y4kEF: two fragments of one gene?,
											put. frameshift: 215616 (−1 < −2)
y4kF		−2	215439-216743	434							hyp. 47.4 kd protein; y4kEF: two fragments of one
											gene?, put. frameshift: 215616 (−1 < −2)
y4kG		−2	216855-217064	69							hyp. 7.7 kd protein
y4kH		−3	217105-217488	127							hyp. 14.1 kd protein
y4kI		−1	217670-218461	263	—	—	—	—	—	—	see y4bM
y4kJ		−3	218458-220008	516	—	—	—	—	—	—	see y4bL
y4kK		−1	220103-221041	312							hyp. 34.9 kd protein
y4kL		−2	221049-222041	330	101-296	ORF300	300	U23723	39	56	hyp. 37.6 kd AAA-family ATPase protein
y4kM		+2	222641-222994	117							hyp. 13.1 kd protein
y4kN		+2	223115-223537	140							hyp. 15.7 kd protein
y4kO		+2	223970-224218	82							hyp. 9.2 kd protein
y4kP		+1	224215-224505	96							hyp. 11 kd protein
y4kQ		−2	224898-225326	142							hyp. (fragmentous?) 15.3 kd protein; homology to hipO
											fragments on the complementary strand
fk1		+3	225094-225473					Z36940			fragments hom. to HipO
y4kR		−3	225535-225666	43	1-36	ORF6	347	M87280	55	66	hyp. 4.8 kd (fragmentous?) protein (smallest ORF
											predicted to be a protein); hom. to N-term. of protein in
											crtE-crtX intergenic region
y4kS		−3	225751-226656	301	1-301	ORF8	300	U12678	93	94	hyp. 33.2 kd protein
y4kT		−2	226653-228203	516	1-516	ORF7	516	U12678	93	94	hyp. 55.1 kd protein
y4kU		−3	228514-229512	332	1-332	ORF6	332	U12678	90	94	prob. geranyltranstransferase
y4kV		−3	229666-231009	447	92-447	CYP117	356	U12678	89	94	cytochrome P-450 BJ-4 homolog
y4lA		−2	231009-231845	278	1-274	ORF4	275	U12678	83	87	short-chain type dehydrogenase/reductase
y4lB		−3	231832-232140	102	1-58	ORF3	94	U12678	93	98	put. P450-system 3Fe-3S ferredoxin
y4lC		−2	232170-233573	467	48-428	CYP114	382	U12678	90	93	cytochrome P-450 BJ-3 homolog
y4lD		−1	233666-234868	400	3-400	CYP112	401	U12678	92	95	cytochrome P-450 BJ-1 homolog
fl3		−2	235704-235904	66	2-54	ORF8	>207	X66124	60	71	hyp. 7.6 kd protein fragment, homology to ORF8
											fragments also upstream of fl3 up to 236048
fl1		−	236796-237416					Z36981			homology to hupK/hupJ fragments (fr. −3/−2)
y4lF		+1	237508-238479	323							hyp. 36.1 kd protein
y4lG		+2	238490-238975	161							hyp. 17.4 kd protein
y4lH		−2	238959-239537	192	3-184	Fic	200	M28363	34	51	hyp. 22.4 kd protein; hom. to cell filamentation/division
											protein
y4lI		−2	239541-239750	69							hyp. 7.3 kd protein
y4lJ		−3	240358-240861	167							hyp. 18.1 kd protein
fl2		−	240920-241040					X65471			fragments fo transposase (ISRm4)
y4lK		+1	241207-241605	132							hyp. 14.3 kd protein
y4lL		−2	241845-244328	827	118-816	SLR0359	1244	D63999	33	50	hyp. 91.8 kd protein (member of E. coli
											YegE/YhdA/YhjK/YjcC family)
fl4		+1	244540-244851	103	19-103	TnpA	990	L14931	39	51	put. truncated transposase; hom. to N-term. of TnpA
					28-81	F15	112	this work	94	98	(transposon Tn163); strong similarity to C-terminus of F15
y4lN		+3	244848-245330	160							hyp. 18.1 kd protein
y4lO		−3	247156-247938	260	11-216	AvrRxv	373	L20423	36	50	hyp. 29.1 kd protein; hom. to avirulence protein; put.
											frameshift according to homolog: 247230-247293 (−2 < −3);
											end of shifted frame: 246960
fl5		+1	248290-248628	112	59-112	F14	103	this work	94	98	hyp. protein fragment; strong similarity to part of F14
fl6		+3	248814-249680	288	8-286	Tnp	988	M97297	27	49	put. fragmentous transposase; homologous to C-term. of
											transposase (Tn1546)
y4lR		+3	249696-251264	522							hyp. 56.8 kd protein
y4lS		+1	251407-251958	183	3-176	PaeR7IN	195	S78872	42	56	put. integrase/recombinase (“resolvase-type”)
					4-181	Y4cG	305	this work	40	60
y4mA		+3	251955-252380	141							hyp. 15.8 kd protein
fm1		−	254694-254920								fragments hom. to xylitol-dehydrogenase
y4mB		+3	255450-256139	229	59-229	ORF4	212	X13583	33	53	hyp. 24.6 kd outer membrane protein precurser
y4mC		+2	256811-257524	237							hyp. 26.2 kd protein precurser
y4mD		−1	258065-258334	89							hyp. 10 kd protein
y4mE		−3	259030-260292	420	6-334	HipA	440	M61242	32	46	hyp. 45.7 kd protein
				2-417	Y4dM	409	this work	34	56
y4mF		−2	260289-260519	76	11-47	ORF3	90	X06090	37	70	hyp. transcriptional regulator; very low similarity to
											phage repressor proteins
y4mG		+3	261174-261395	73							hyp. 7.8 kd protein
y4mH		−2	261747-262640	297							hyp. 33.9 kd protein
y4mI		−2	262698-263672	324	11-252	RbsB	296	M13169	25	49	prob. ABC transporter periplasmic binding protein
											precurser (transport system Y4mIJK probably transports a sugar)
y4mJ		−3	263716-264717	333	12-323	RbsC	321	M13169	34	55	prob. ABC transporter permease
y4mK		−2	264714-266207	497	8-489	RbsA	501	M13169	34	55	prob. ABC transporter ATP-binding protein
y4mL		−3	266218-267477	419	1-418	HI1029	425	U00079	33	58	put. permease (E. coli YiaN/YgiK family)
y4mM		−2	267474-269099	541	38-360	HI1028	328	U32729	33	54	put. permease (SBR family 7)
y4mN		−1	269096-270133	345	37-340	Tkt	655	U09256	36	54	hyp. transketolase family protein (fragmentous?); hom. to
											C-term. of transketolases
y4mO		−3	270130-270969	279	9-270	Tkt	655	U09256	36	52	hyp. transketolase family protein (fragmentous?); hom. to
											N-term. of transketolases
y4mP		−3	271000-271761	253	4-249	F09E10.3	255	U41749	41	60	put. short-chain type dehydrogenase/reductase
y4mQ		+1	271909-272805	298	1-289	PerR	297	U57080	48	65	hyp. transcriptional regulator (LysR family)
y4nA		−2	273204-275384	726	45-302	ORF	690	D14005	21	36	prob. peptidase; very low similarity to Y4qF and Y4sO
					365-718				38	54	(<25% identity)
y4nB	nodU	−3	276451-278127	558	1-558	NodU	558	X89965	100	100	inv. in 6-O-carbamoylation of Nod factors; similar to Y4hD
y4nC	nodS	−1	278144-278794	216	1-216	NodS	216	J03686	100	100	methyltransferase inv. in Nod-factor synthesis
y4nD		−3	280453-281349	298	—	—	—	—	—	—	see Y4iQ
y4nE		−2	281346-282860	504	—	—	—	—	—	—	see Y4jA
fn1		+	283238-283467				241	M26938			hom. to virG fragments; similar to fq3
y4nF		+3	283809-284501	230							hyp. 25.4 kd protein precurser; low similarity to Y4aO
											(<30% id.)
fn2		−	284752-284923					X79443			fragments hom. to ORF2 (IS-ATP-binding protein) from
											IS1162
y4nG		+2	285407-286597	396	53-365	ORF4	333	U08223	31	47	put. NAD-dep. nucleotide sugar epimerase/dehydrogenase
y4nH		+1	286594-286947	117	5-113	MvrC	110	M62732	30	47	hyp. 12.3 kd integral membrane protein (some similarity
											to ethidium bromide resistance proteins)
y4nI		+2	286964-287326	120							hyp. 13 kd transmembrane protein
y4nJ		+1	287335-288852	505	80-266	BetA	548	U39940	29	44	hyp. GMC-type oxidoreductase
					343-468				32	45
y4nK		−2	288906-290894	662							hyp. integral membrane protein
y4nL		−3	290914-291984	356	14-345	ORF6	328	U47057	26	45	put. NAD-dep. nucleotide sugar epimerase/dehydrogenase
y4nM		−3	292003-293553	516	226-514	NoeC	307	L18897	30	52	put. permease
y4oA		−3	294502-296283	593	328-494	MccB	350	X57583	29	41	hyp. 65.2 kd protein; homolog inv. in production of the
					4-590	Y4qC	583	this work	30	50	translation inhibitor microcin C7
y4oB		+1	296572-296961	129							hyp. 14.7 kd protein
y4oC		+1	296965-297657	230							hyp. 26 kd protein
y4oD		−1	297746-298390	214							hyp. 23.5 kd protein
y4oE		−3	298939-299148	69							hyp. 7.4 kd protein
fo1		−2	299145-299588	147							fo1 and fo2: two fragments of one put. gene; put.
											frameshift: 299664 (−2 < −3)
fo2		−3	299578-299955	125	25-109	ORF11	344	X53264	37	63	homology to 5′part of ORF11;
					1-123	Y4cM	325	this work	25	51	fo1 and fo2: two fragments of one putative gene; put.
											frameshift: 299664 (−2 < −3)
fo3		+3	300015-300815	267	15-252	Tnp	518	L09108	40	59	fo3 and fo7: transposase-like protein interrupted by
											NGRIS-6
fo4		−2	300828-301259	143	1-143	Y4bA	694	this work	77	83	hyp. fragment; fo4/5/6: fragments of one gene similar to
											Y4bA/Y4pH
fo5		−1	301274-301684	136	1-127	Y4bA	694	this work	83	94	hyp. fragment; fo4/5/6: fragments of one gene
fo6		−2	301608-302900	430	1-393	Y4bA	694	this work	89	95	hyp. fragment; fo4/5/6: fragments of one gene
y4oL		−3	302890-303156	88	1-88	Y4bB	98	this work	63	69	hyp. 9.6 kd protein
y4oM		−1	303179-303628	149	1-149	Y4bC	149	this work	79	88	hyp. 16.8 kd protein
y4oN		−2	303810-304022	70	1-70	Y4bD	89	this work	73	84	hyp. 8.1 kd protein
fo7		+2	304118-304453	111	4-103	Tnp	518	L09108	40	59	fo3 and fo7: transposase-like protein interrupted by
											NGRIS-6
y4oP		+1	304861-306156	431	47-429	u1756v	469	U15180	27	42	prob. ABC transporter binding protein (Y4oPQRS: sugar-
											like transport system)
y4oQ		+2	306236-307165	309	31-301	MalF	310	U15180	35	56	prob. ABC transporter permease protein
y4oR		+2	307178-308011	277	12-277	MalG	296	U15180	30	52	prob. ABC transporter permease protein
y4oS		+1	308008-309123	371	7-369	UgpC	369	U00039	50	68	prob. ABC transporter ATP-binding protein
y4oT		−2	309132-309722	196	2-196	Y4pA	609	this work	28	50	hyp. 20.6 kd protein; homologous to N-terminus of
											Y4aA, and weakly to Y4oV
y4oU		+1	309853-311061	402							hyp. 43.1 kd protein precurser
y4oV		+2	311051-311908	285	3-280	Y4pA	609	this work	32	56	hyp. 30.2 kd protein; homologous to N-terminus of
											Y4pA, and weakly to Y4oT
y4oW		+1	311911-312561	216							hyp. 23.7 kd protein
y4oX		+3	312606-313688	360	36-233	MocA	317	X78503	29	44	prob. NAD-dep. oxidoreductase
y4pA		+1	313714-315543	609	310-596	HydG	441	U00006	33	50	put. transcriptional regulator (sigma54-dep.)
					6-290	Y4oV	285	this work	32	56
					35-237	Y4oT	196	this work	28	50
y4pB	otsB	+3	316350-317147	265	30-260	OtsB	266	X69160	41	57	prob. trehalose-6-phosphate phosphatase
y4pC	otsA	+1	317185-318579	464	1-456	OtsA	474	X69160	46	66	prob. trehalose-6-phosphate synthase; similar to fq1/2
fp1		+	318915-319242					U08864			fragments homologous to ORF3; put. frameshift acc. to
											homologue: 319122 (3 > 1)
fp2		+	319236-319670					U08864			fragment homologous to ORF1 from IS1248 (fr. 3);
											similar to fs4
y4pD		−1	319601-320116	171	13-140	Ros	142	M65201	50	71	put. transcriptional regulator (MucR family); missing Zn
											finger motif; similar to Y4aP
y4pE		−1	320606-321013	135	1-135		222	U18764	91	94	identical to y4sA; hyp. 15.5 kd protein hom. to N-term.
											of RFRS9 25kDa protein
y4pF		−2	321297-322460	387	50-374	Tnp	334	Z48244	43	60	identical to y4sB; put. transposase; low similarity to
											Y4qE, Y4iB and Y4iO (<30% aa-id.)
y4pG		−3	322486-323064	192	1-191	ORFA	197	U22323	47	64	identical to y4sC; hyp. 21.1 kd protein
fp3		+2	323189-323956					X79443			“ORF” homologous to ORF1 of IS1162 interrupted by
											stop codon (323444)
y4pH		−1	323969-326053	694	—	—	—	—	—	—	see y4bA
y4pI		−2	326043-326309	88	—	—	—	—	—	—	see y4bB
y4pJ		−3	326329-326778	149	—	—	—	—	—	—	see y4bC
y4pK		−1	326969-327238	89	—	—	—	—	—	—	see y4bD
fp4		+1	327277-328059					L09108	48	65	fragment homologous to put. IS-ATP-binding protein
y4pL		+3	328071-328808	245	1-204	ORF2	231	X79443	51	63	put. insertion sequence ATP-binding protein; similarity to
					1-242	Y4bM	263	this work	55	73	Y4bM/Y4I/Y4tA, Y4uH, and weakly to
					1-245	Y4uH	248	this work	61	77	Y4iQ/Y4nD/Y4sD (<30 aa-id.)
y4pM		+2	329159-329977	272							hyp. 30.9 kd protein
fp5		−	330657-331414								put. frameshift: 331032 (2 < 1)
y4pN	syrM1	−3	332506-333522	338	13-324	SyrM	326	M33495	63	77	probable symbiotic regulator (LysR family)
					1-338	SyrM2	339	this work	62	79
y4pO		+1	335062-336264	400	1-400	Tnp	400	M60971	96	98	prob. transposase (Mutator family); similarity to fe7
fq2		−2	333987-335003	338	1-320	OtsA	474	X69160	44	61	join fq1 + fq2: hom. to trehalose-6-phosphate synthase
											interrupted by ISRm3-like element NGRIS-8; similarity
											to Y4pC (45% aa-id.)
fq1		−1	336311-336694	128	44-174	OtsA	474	X69160	48	67	see fq2
fq3		+	337338-338056					M26938			virG homologous fragments: stop at 37380; put.
											frameshift at 337844 (3 > 2); similar to fn1
y4qB		−1	339053-339547	164							hyp. 18.8 kd protein
y4qC		−3	339535-341286	583	314-489	ORF	401	Z54354	28	46	hyp. 63.6 kd protein
					1-583	Y4oA	593	this work	30	50
y4qD		−3	343216-343950	244	1-244	Y4rO	618	this work	55	74	hyp. 26.8 kd protein, similar to N-terminus of Y4rO
y4qE		+2	344114-345286	390	37-380	Tnp	364	X77623	38	57	prob. transposase; low similarity to Y4pF/Y4sB, Y4iB,
											Y4iO and Y4rJ (<30% aa-id.)
fq4		+3	345798-346130					M38257	34	51	fragments homologous to XerC (integrase)
y4qF		−2	346215-348479	754	41-725	PtrII	707	D10976	31	49	prob. peptidase (S9A family); high similarity to Y4sO;
					32-736	Y4sO	705	this work	70	84	low similarity to Y4nA (<25% id.)
y4qG		−2	348501-349847	448	40-389	YgiG	454	U32722	42	62	prob. aminotransferase (class 3)
y4qH		−1	350294-351274	326	144-326	LasR	239	M59425	37	51	hyp. transcriptional regulator (LuxR family)
y4qI		−2	351837-353456	539	146-419	ORF1	322	M25805	44	63	hyp. 59.7 kd protein; similar to Y4aQ, Y4hP, Y4jD
fq5		−3	353533-353775								fragments fq5 and fr3 represent one put. gene similar to
											Y4hO and Y4jC interrupted by IS elements
y4qJ		−1	354140-355336	398	7-395	TnpA	388	U14952	42	60	put. transposase
y4qK		−2	355344-356270	308	51-293	Int	259	U14952	39	55	put. integrase/recombinase (“phage-type”); similar to
					51-308	Y4eF	251	this work	92	94	Y4rF; low similarity to Y4rABCDE
fq6		−2	356436-356636	66	1-66	Fe5	66	this work	79	94	put. defective integrase/recombinase (“phage-type”); 75%
						Y4rC	332	this work	45	62	nt-identity; 356436-356710 and 94988-95262 [R-20]
y4rA		+1	356803-358032	409	17-397	ORF2	415	L34580	39	55	put. integrase/recombinase (“phage-type”)
y4rB		+3	358029-358973	314	135-267	TnpI	284	X07651	30	51	put. integrase/recombinase (“phage-type”)
y4rC		+2	358970-359968	332	22-294	XerC	295	U32696	31	50	put. integrase/recombinase (“phage-type”)
					267-332	Fe5	66	this work	41	55
					267-332	Fq6	66	this work	45	62
y4rD		−3	360025-360870	281	15-277	XprB	298	M54884	25	46	put. integrase/recombinase (“phage-type”)
y4rE		−2	360867-361799	310	50-288	YqkM	296	D84432	27	48	put. integrase/recombinase (“phage-type”); low similarity
											to Y4gA
y4rF		−1	361796-363073	425	126-414	ORF2	415	L34580	34	49	put. integrase/recombinase (“phage-type”)
y4rG		−1	363287-363694	135	16-109	ORF1	130	U19148	32	48	hyp. 14.8 kd protein (IS866 family); low similarity to
											Y4jB, Y4hN
y4rH		−3	363895-365331	478	62-374	Bcp	598	X63470	26	44	put. ligase; hom. to biotin carboxylases
fr1		−3	366307-366669								85% aa-identity to part of Y4rL
fr2		−	366594-367402								put. frameshift: 367296 (−2 < −1)
fr3		−3	367705-367827								hom. to N-term. of Y4hO; see fq5
y4rI		−3	368503-369675	390							hyp. 44 kd protein
y4rJ		+1	369697-370887	396	152-379	Tnp	339	M80806	28	45	put. transposase; low similarity to Y4qE (<30% aa-id.)
					135-244	Y4iA	120	this work	74	87
					266-396	Y4iB	130	this work	76	84
					135-396	Y4iO	252	this work	71	83
					2-131	Y4iP	131	this work	58	80
y4rK		−1	370976-371350	124							hyp. 14.5 kd protein
y4rL		−2	371454-371921	155	1-99	Y4zA	295	this work	99	99	hyp. 17.7 kd protein; y4rLM: two fragments of one
					17-155	Y4iE	135	this work	33	52	gene?; put. frameshift: 371972 (−2 < −3); 85-99% aa-
											identity to parts of Y4zA and fr1
y4rM		−3	371938-372990	350	258-339	Y4zA	295	this work	98	98	hyp. 39.4 kd protein; see y4rL
y4rN		−2	373578-374795	405	35-368	P43	416	X57470	26	44	hyp. 41.6 kd integral membrane protein
y4rO		+1	375313-377169	618	274-596	H1N0578	366	U32742	25	45	hyp. 69.3 kd protein; N-terminus: hom. to Y4qD; C-
					1-244	Y4qD	244	this work	55	74	terminus: hom. to C-terminus of histidinol-1-phosphate
											transaminase
fr4		+	377185-377534					X66016			sim. to Y4rG; put. frameshift: 377376 (1 > 3); hom. to
											fragment of ORFA3 (377409-377540)
y4sA		−3	377842-378249	135	—	—	—	—	—	—	seey4pE
y4sB		−1	378533-379696	387	—	—	—	—	—	—	see y4pF
y4sC		−2	379722-380300	192	—	—	—	—	—	—	see y4pG
y4sD		−1	380933-381829	298	—	—	—	—	—	—	see y4iQ
y4sE		−3	381826-383340	504	—	—	—	—	—	—	see y4jA
fs5		−3	383593-384054	153	8-150	Tnp	334	Z48244	48	65	put. defective transposase; sim. to fs1
			384210-384493								fragments with 94-84% nt-id. to ISRm6 (R. meliloti;
											acc. no. X95567)
y4sG		+1	384808-385818	336	97-325	Ddl	306	M14029	34	57	hom. to D-alanine:D-alanine ligase; probably different function
y4sH		+3	386505-387890	461	267-337	CapA	411	M24150	42	63	hom. to encapsulation protein A; nearly identical to Y4aA
fs1		−	388138-388586			Tnp		Z48244			fragments of put. transposase; put. frameshift: 388452
											(−3 < −2); sim. to Y4pF, Y4sB, fs5
fs2		+2	388697-388897			ORF1		U19148	43	62	put. transposase fragment; hom. to N-term. of ORF1;
											sim. to Y4jB, Y4rG, Y4hN
fs3		+	388966-390695			AtoC		U17902			put. transcriptional regulator fragment (put. frameshifts:
											389891 (1 > 2); 390170 (2 > 3)); sim. to Y4pA, Y4oV, Y4oT)
y4sI		+2	390971-393241	756	325-741	McpA	657	X66502	41	60	prob. methyl-accepting chemotaxis protein
					1-749	Y4fA	845	this work	29	49
y4sJ	gapD	−3	393202-394677	491	29-489	GabD	482	M88334	58	75	prob. succinate-semialdehyde dehydrogenase
y4sK		−1	394790-395170	126	5-122	C23G10.2	185	U39851	55	71	bel. to the YER057C/YIL051C/YJGF family; probably
											important cellular function
y4sL		−1	395204-395815	203	2-203	DadA	432	L02948	57	74	either functional dehydrogenase or non-functional
											fragment; hom. to small subunit of D-aminoacid
											dehydrogenase
y4sM		+1	395935-396318	127	1-127	ORF1	127	X74314	99	99	put. transcriptional regulator (AsnC/Lrp family; low
											homology to y4tD); missing H-T-H region
y4sN		+1	396523-396900	125	1-123	ORF2	>123	X74314	98	98	similar to ORFs derived from insertion elements (IS6501
											family); low similarity to fu4
fs4		+	396855-397283	(143	8-141	ORF1	186	X53945	48	63	put. IS-derived protein fragment (homology to C-term. of
					1-141	Fp2	145	this work	39	62	ORF1 from IS869)
y4sO		−2	397608-399725	705	10-694	PtrII	706	D10976	32	49	prob. peptidase (S9A family); low similarity to Y4nA
					1-705	Y4qF	754	this work	70	84	(<25% id.)
ft1		+3	400377-400625	(83)	20-83	Y4tE	300	this work	64	78	ft1 and ft2: one put. gene encoding an amino acid ABC
											transporter binding protein interrupted by NGRIS-3c.
y4tA		−3	400732-401523	263	—	—	—	—	—	—	see y4bM
y4tB		−2	401520-403070	516	—	—	—	—	—	—	see y4bL
ft2		+1	403249-403899	(216)	5-195	ArgT	260	V01368	25	48	see ft1
					2-215	Y4tE	300	this work	76	86
y4tD		+1	404182-404691	169	11-161	HIN1362	168	U32817	38	64	put. transcriptional regulator (AsnC/Lrp family; but low
											homology to y4sM)
y4tE		+1	405157-406059	300	31-281	FliY	257	U32734	27	48	prob. aminoacid ABC transporter binding protein
					86-299	Ft2	215	this work	76	86	(periplasmic); prob. part of binding-protein-dep. transport
											system Y4tEFGH
y4tF		+1	406111-406827	238	25-233	YckJ	234	X77636	35	54	prob. aminoacid ABC transporter permease protein
y4tG		+3	406830-407525	231	1-220	GlnP	226	D30762	32	54	prob. aminoacid ABC transporter permease protein
y4tH		+2	407522-408295	257	5-256	GlnQ	242	M61017	52	71	prob. aminoacid ABC transporter ATP-binding protein
y4tI		+1	408745-409953	402	22-391	Slr0072	393	D64004	35	54	put. peptidase (M40 family)
y4tJ		+1	409990-410988	332	7-328	Thd2	329	M21312	35	57	put. threonine dehydratase
y4tK		+3	410988-411983	331	69-326	ArcB	351	U39262	30	44	hyp. cyclodeaminase; (sim. to ornithine cyclodeaminase)
y4tL		+2	412118-413290	390	10-384	ORF	411	D14463	27	45	hyp. hydrolase/peptidase (M24 family)
					1-389	Y4tM	392	this work	34	53
y4tM		+2	413453-414631	392	17-390	PepQ	368	Z34896	24	43	put. hydrolase/peptidase (M24 family)
					1-390	Y4tL	390	this work	34	53
y4tN		+1	414655-415179	174							hyp. 19.6 kd protein
y4tO		+1	415252-416847	531	1-484	OppA	543	M60918	28	46	prob. peptide ABC transporter binding protein presurser;
											prob. part of a binding-protein-dependent transport system
											Y4tOPQRS
y4tP		+2	416852-417793	313	4-313	DppB	339	L08399	36	58	prob. peptide ABC transporter permease protein
y4tQ		+1	417796-418671	291	9-287	AppC	303	U20909	36	56	prob. peptide ABC transporter permease protein;
											418611; C or T possible!
y4tR		+2	418673-419680	335	12-327	OppD	336	X56347	50	68	prob. peptide ABC transporter ATP-binding protein
y4rS		+1	419677-420738	353	3-320	AppF	329	U20909	49	69	prob. peptide ABC transporter ATP-binding protein
y4uA		+3	420774-422159	461	267-337	CapA	411	M24150	42	63	put. cell wall compound biosynthesis protein; almost
											identical to Y4sH
y4uB		+3	422628-424031	467	1-464	BioA	448	U51868	33	57	prob. aminotransferase (class 3)
y4uC		+3	424056-425594	512	58-509	GabD	482	M88334	33	52	prob. aldehyde dehydrogenase
fu1		+2	425699-425779			N15K	238	D45911			put. protein fragment; 67% id. to N15K in 26 aa
fu2		+3	425841-426083			PhbA	393	U17226			fragment 65% identical to C-term. of beta-keto-thiolase
y4uD		+1	426010-426507	165							hyp. 18.7 kd protein
y4uE		−3	426949-428028	359	78-290	Tnp	414	X15942	31	45	put. transposase (IS110 faminly); put. frameshift: between
											427040 and 427180 (−2 < −3; end of shifted ORF: 426699)
y4uF		+3	428292-429623	443	13-440	GLUD1	558	X07674	42	60	prob. glutamate dehydrogenase
fu3		+	429860-430007			Tnp	398	U08627			put. transposase fragment (92% id. in 16 aa); 85% nt-
											identity to 3′term. part of ISRm5
y4uG		+1	430105-430320	71							hyp. 7.8 kd protein
y4uH		−1	430538-431284	248	1-202	ORF2	231	X79443	48	63	put. insertion sequence ATP-binding protein; similarity to
					1-245	Y4pL	245	this work	61	77	Y4pL, Y4bM/Y4kI/Y4tA and Y4iQ/Y4nD/Y4sD
					1-248	Y4bM	263	this work	48	68	(IS21/IS1162 family)
					4-248	Y4iQ	298	this work	31	52
y4uI		−3	431296-432840	514	1-514	Tnp	518	L09108	44	63	put. transposase; similiarity to Y4bL/Y4J/Y4tB
											(IS21/IS1162 family)
fu4		−	433222-433560			Tnp	201	X65471			put. tranposase fragments (74-92% id. in 88 aa); 79% nt-
											identity to 5′term. of ISRm4
y4uJ	fixU	−1	433880-434110	76	1-70	FixU	70	X51963	63	80	hyp. 8.5 kd protein
y4uK	nifZ	−3	434107-434433	108	6-79	ORF2	>78	X07567	52	78	put. nitrogen fixation NifZ protein
y4uL	fdxN	−2	434517-434711	64	1-64	FdxN	64	M21841	79	84	prob. 4Fe4S ferredoxin
y4uM	nifB	−1	434753-436234	493	1-493	NifB	490	M15544	72	81	involved in FeMo cofactor biosynthesis
y4uN	nifA	−1	436460-438244	594	37-594	NifA	584	U31630	62	74	positive regulator of nif, fix, and additional genes
											(sigma54-dep.)
y4uO	fixX	−2	438297-438590	97	2-97	FixX	98	M15546	84	89	prob. 3Fe-3S ferredoxin inv. in nitrogen fixation
y4uP	fixC	−1	438605-439912	435	1-435	FixC	435	M15546	82	89	required for nitrogenase acitivity
y4vA	fixB	−2	439923-441032	369	18-363	FixB	353	M15546	79	87	putatively inv. in a redox process in nitrogen fixation
y4vB	fixA	−2	441042-441899	285	1-280	FixA	292	M15546	75	90	putatively inv. in a redox process in nitrogen fixation
fv1		−1	442181-442252			NifS	384	X68444			put. NifS fragment (70% identity in 24 aa)
y4vC		−1	442316-442636	106	1-106	ORF118	118	X13691	54	72	hyp. 11 kd protein (HesB/YadR/YfhF family);
											homologues located upstream of nifS
y4vD		−2	443313-443879	188	5-173	HIN1693	241	U32848	46	60	put. redox enzyme (hom. to glutaredoxin-like membrane
											protein and peroxysomal membrane proteins)
y4vE	nifQ	+1	444337-445029	230	56-212	NifQ	180	M26323	39	56	putatively involved in Mo cofactor processing
y4vF	dctA1	+2	445088-446602	504	1-443	DctA1	456	S38912	99	99	C4-dicarboxylate transport protein; nt-deletion at 446416
											in comparison to sequence of acc. no. S38912 causing a
											frameshift (DctA1 is 48 aa longer than DctA1 in S38912)
y4vG		+1	446599-447843	414	13-413	CamC	415	M12546	34	50	prob. cytochromeP450
y4vH		+1	447844-448500	218	(32-157	LinA	155	D90355	28	46)	hyp. 24.6 kd protein (with very weak homology to
											gamma-hexachlorocyclohexane-dechlorinase)
y4vI		+3	448557-450203	548	9-250	FabG	244	U39441	38	56	short-chain type dehydrogenase/reductase
					276-513				30	48
y4vJ		+2	450341-451396	351	1-188	LuxA	357	M36597	27	47	put. monooxygenase; similar to Y4wF;
y4vK	nifHl	+1	451993-452883	296	1-296	NifH	296	M26961	99	99	Fe protein of nitrogenase
y4vL	nifDl	+1	452980-454494	504	199-393	NifD	>195	M26962	98	99	alpha-subunit of MoFe protein of nitrogenase
y4vM	nifKl	+3	454590-456131	513	132-195	NifK	>64	M26963	100	100	beta-subunit of MoFe protein of nitrogenase
y4vN	nifE	+1	456187-457677	496	1-469	NifE	547	X56894	62	78	involved in FeMo cofactor biosynthesis
y4vO	nifN	+1	457687-459096	469	1-455	NifN	441	M18272	70	81	involved in FeMo cofactor biosynthesis
y4vP	nifX	+3	459093-459575	160	22-156	NifX	159	X17433	52	68	nitrogen fixation protein
y4vQ		+3	459579-460067	162	22-162	ORF4	156	X17433	49	70	hyp. 17.7 kd protein, similar to proteins of other
					1-162	Y4xD	162	this work	61	75	nitrogen-fixing bacteria and to Y4xD
y4vR		+1	460501-460920	139	1-58	NifH	296	M26961	50	63	similar to N-term. of Fe protein of nitrogenase
y4vS	fdxB	+2	461228-461545	105	1-88	ORF5	102	M26323	52	65	prob. 4Fe-4S ferredoxin
y4wA		+1	463201-464739	512	86-499	PqqE	709	L43135	50	70	hyp. zinc protease (M16 family); sim. to Y4wB
y4wB		+3	464736-466079	447	236-438	PqqF	213	L43135	42	61	put. protease (lacks Zn-binding site; M16 family); sim. to Y4wA
y4wC		+3	466590-467021	143	8-132	ORF3	127	L13845	48	66	put. DNA-binding protein; high similarity to Y4aM
					1-143	Y4aM	143	this work	69	77
y4wD		+1	467758-468891	377	11-370	MosC	407	U23753	29	48	permease-type protein; hom. to membrane protein from
											the rhizopine biosynthesis (mosABC) gene cluster
y4wE		+3	469311-470417	368	20-361	His1	356	D14440	32	53	prob. aminotransferase (class 2)
y4wF		+1	470824-471852	342	40-194	LuxA	354	X06758	27	54	put. monooxygenase; sim. to Y4vJ
y4wG		+2	471890-472435	181							hyp. 19.4 kd protein
y4wH		+3	473343-473780	145	1-145	ORF2	145	M19352	64	76	hyp. 15.6 kd protein
y4wI		−2	473928-475469	513							hyp. 59 kd protein
y4wJ		−2	475503-475880	125							hyp. 13.3 kd protein
y4wK	nifW	−1	476519-476971	150	12-118	NifW	108	M86823	50	63	NifW protein homolog; required for full activity of FeMo protein
y4wL	nifS	−2	477135-478298	387	4-387	NifS	402	M17349	58	73	prob. NifS protein (member of class-5 pyridoxal-
											phosphate-dep. aminotransferase family)
y4wM		−2	479145-481136	663	225-620	YejA	>409	U00008	38	55	put. ABC transporter binding protein (transporter or
											enzymatic function)
fw1		−1	481460-481834	124	1-116	DctA	441	M26531	55	61	hyp. truncated transporter-like protein; hom. to N-term. of
											DctA (see y4vF); two frameshifts acc. to homologue:
											481606 (−3 < −1); 481530 (−2 < −3; homology stops at
											481419)
y4wO		−3	481834-482154	106							hyp. 11 kd protein
y4wP		+2	482540-482947	135							hyp. 14.9 kd protein
y4xA	nifH2	+1	483871-484761	296	1-296	NifH	296	M26961	99	99	Fe protein of nitrogenase
y4xB	nifD2	+1	484858-486372	504	199-393	NifD	>195	M26962	98	99	alpha-subunit of MoFe protein of nitrogenase
y4xC	nifK2	+3	486468-488009	513	132-195	NifK	>64	M26963	100	100	beta-subunit of MoFe protein of nitrogenase
y4xD		+3	488262-488750	162	22-162	ORF4	156	X17433	47	73	hyp. 18 kd protein; similar to proteins of other nitrogen-
					2-162	Y4vQ	162	this work	61	75	fixing bacteria and to Y4vQ
y4xE		+1	488773-488976	67	1-64	ORF1	69	X55450	40	67	hyp. 7.6 kd protein; similar to proteins of other nitrogen-
											fixing bacteria
y4xF		+3	488973-489149	58							hyp. 6.5 kd protein
y4xQ		+2	489281-89583	100	14-83	ExoX	98	M6I751	31	52	put. exopolysaccharide production repressor (intrgal
											membrane protein)
y4xG		+2	490010-491527	505							hyp. 55.5 kd protein
y4xH	nodD2	−2	491655-492593	312	1-312	NodD2	312	L38460	99	99	transcriptional regulator (LysR family); high similarity to
					1-310	NodD1	322	this work	68	83	Y4aL (NoD1)
y4xI		+2	494297-494977	226	1-224	PmrA	222	L13395	39	58	signal transduction-type regulator
y4xJ		+1	495157-496428	423	76-378	GPIV	426	J02451	27	46	hyp. protein hom. to proteins of the general secretion
											pathway (pulD family), sim. to Y4yD (NolW)
y4xK		+1	496438-497004	188							hyp. 20.6 kd protein precurser
y4xL		−1	497444-498460	338							hyp. 37.1 kd protein
y4xM		−1	498719-499933	404	23-403	ORF1	408	X59939	22	49	permease-type protein
						(YceE)
y4xN		−3	499930-501816	628	183-505	IucC	580	X76100	28	43	hyp. 71 kd protein hom. to aerobactin synthetase subunit
y4xO		−2	501816-502955	379							hyp. 40.9 kd protein
y4xP		−1	502952-503962	336	5-304	CysK	308	D26185	40	60	put. cysteine synthase
y4yA		−1	503963-505336	457							hyp. 49.9 kd protein; low similarity to diaminopimelate
y4yB		−3	505336-505800	154							hyp. 17.1 kd protein
y4yC	nolX	−2	505950-507740	596	1-596	NolX	596	L12251	98	99	nodulation protein as in R. fredii USDA257
y4yD	nolW	−3	508021-508725	234	1-234	NolW	234	L12251	99	100	nodulation protein (PulD family); sim. to Y4xJ
y4yE	nolB	+3	508881-509375	164	1-164	NolB	164	L12251	98	99	nodulation protein
y4yF	nolT	+3	509385-510254	289	1-289	NolT	289	L12251	96	97	nodulation protein precurser (YscJ homolog; M74011)
y4yG	nolU	+2	510251-510889	212	1-212	NolU	212	L12251	99	99	nodulation protein
y4yH	nolV	+3	510891-511517	208	1-60	ORF4	65	L12251	100	100	homologous to two (nodulation) proteins of R. fredii
					73-208	NolV	135		96	97	USDA257 (YscL homolog; M74011)
y4yI	hrcN	+2	511514-512869	451	35-450	YscN	439	U00998	55	73	prob. ATPase involved in secretion
					1-80	HrcN	450	L12251	97	97
					105-450				97	98
y4yJ		+1	512845-513381	178	1-178	ORF7	178	L12251	97	98	hyp. 20.4 kd protein
y4yK	hrcQ	+1	513406-514482	358	171-350	YscQ	307	L25667	27	46	prob. translocation protein inv. in secretion processes
					1-358	HrcQ	382	L12251	96	98	(FliN/MopA/SpaO family)
y4yL	hrcR	+2	514475-515143	222	6-216	YscR	217	L25667	46	66	prob. translocation protein inv. in secretion processes
					1-222	HrcR	249	L12251	99	99	(Flip/MopC/SpaP family)
y4yM	hrcS	+1	515143-515418	91	1-66	YscS	88	L25667	34	65	prob. translocation protein inv. in secretion processes
					1-91	HrcS	92	L12251	98	100	(FliQ/MopD/SpaQ family)
y4yN	hrcT	+3	515427-516245	272	28-250	YscT	261	L25667	31	52	prob. translocation protein inv. in secretion processes
					1-272	HrcT	272	L12251	98	99	(FliR/MopE/SpaR family)
y4yO	hrcU	+2	516242-517279	345	5-339	YscU	354	L25667	30	50	prob. translocation protein inv. in secretion processes
					1-340	HrcU	351	L12251	99	99	(FlhB/HrpN/YscU/SpaS family)
y4yP		+1	518077-518892	271	35-262	HipA	295	M19019	88	91	homolog is inducible by root-exudate and diadzein;
											frameshift acc. to homologue: 518855 (1 > 2)
fy1		+	519655-519995			NolJ	148	L26967			nodulation gene homologous fragments (80-100% id. in
											97 aa); frameshifts acc. to homologue: 519789 (1 > 3);
											519900 (3 > 2); 519965 (2 > 3)
y4yQ		+2	520280-521170	296							hyp. 31.3 kd integral membrane protein
y4yR		+2	521360-523453	697	17-677	LcrD	704	M96850	40	65	prob. translocation protein inv. secretion processes
											[Flagella/HR/Invasion proteins export pore (FHIPEP) family]
y4yS		+3	523470-524018	182							hyp. 20.1 kd protein
y4zA		+2	525005-525892	295	34-115	Y4rM	350	this work	98	98	hyp. (fragmentous?) 32.9 kd protein; put. frameshift:
					133-231	Y4rL	155	this work	99	99	525699 (2 > 3); similar to Y4iE
y4zB		+1	526051-527121	356	60-320	Tnp	377	X67862	29	47	put. (fragmentous?) transposase (IS4 family) 526103-
											526200 higher cod. prob. in fr. 2; put. frameshift: 526200
											(2 > 1)
fzl		+	527337-527902			Hdc	378	J02577			fragments homologous to histidine decarboxylases (30-
											45% id. in 134aa); put. frameshift (3 > 2) around 527478
y4zC		+3	529125-529910	261	65-248	AvrPph3	276	M86401	27	41	hyp. 28.3 kd protein; hom. to avirulence protein
y4zD		+3	530145-530294	49							hyp. 5.5 kd protein
fz4		+2	530432-530764	110	1-110	Y4jA	504	this work	72	85	hom. to C-terminus of Y4jA/Y4nE/Y4sE
fz2		+	530761-531250			ORFB	251	X67861			put. IS-ATP-binding protein fragments (32-40% id. in
											137aa); put. frameshift acc. to homolog: 531062 (1 > 2)
y4zF	syrM2	+2	532676-533695	339	1-320	SyrM	326	M33495	69	81	prob. symbiotic regulator (LysR family)
					1-335	SyrM1	338	this work	62	79
fz3		+	534257-534422			ORF	338	M73488			fragments homologous to 1-aminocyclopropane-1-
											carboxylate deaminase (63-83% id. in 56aa); put.
											frameshift: 534291
aopen reading frame (ORF)
bstrand (−/+) or frame (−1; −2; −3; +1; +2; +3)
cnumber (no.)
daminoacids (aa)
eGenBank/EMBL accession numbers
fidentity (I) and similarity (S) have been calculated by the programme BESTFIT (local homology algorithm; Smith and Waterman, 1981) of the WISCONSIN SEQUENCE ANALYSIS PACKAGE (version 8.0, GCG, Madison, USA)
gabbreviations: prob. = probable; cod. prob. = coding probability; acc. = according; inv. = involved; sim. = similar; id. = identical; fr. = frame; acc. no. = accession number; nt = nucleotide; hyp. = hypothetical; put. = putative; hom. = homologous; dep. = dependent; N/C-term. = N/C-terminus

[0059] role of some ORFs like the luciferase-like ORFs (y4vJ and y4wF; see Table 3) in rhizobia is still not clear. In the 100 kb region, the duplication of a 5 kb sequence (position 451,886 to 456,157 and 483,764 to 488,035) including the genes nifHDK is remarkable. These genes encode the basic subunits of the nitrogenase. Furthermore, the transcriptional regulator nodD2 is very interesting because its role seems not to be identical to a previously identified nodD2 in a closely related strain (Appelbaum et al., 1988; data not shown). Also the pmrA-homologous ORF y4xI putatively plays an important role in regulating symbiotic processes because a nod box (binding region for the basic regulator nodD1; Fisher and Long, 1993) is located upstream of this ORF (position 493,962 to 494,000). Finally, the presence of ORFs (y4yI and y4yK to y4yN; see Table 3) homologous to type III secretion proteins, which have only been known previously in plant or animal/human pathogenic bacteria, shows that there only seems to be a subtle difference between symbiotic and pathogenic abilities of microorganisms.

[0060] In a second stage, the remaining 436 kb of pNGR234a were analyzed. Several ORFs and their deduced proteins were identified that belong to functional groups not previously identified in the analysis of cosmids pXB296, pXB368 and pXB110 (replication of the plasmid, conjugal transfer of the plasmid, functions in oligosaccharide biosynthesis and cleavage, functions in sugar or sugar-derivative metabolism, functions in lipid or lipid-derivative metabolism, functions in chemoperception/chemotaxis, functions in biosynthesis of cofactors, prosthetic groups and carriers, etc.).

[0061] Although further functional analyses of selected ORFs in pNGR234a still have to be performed, large-scale sequencing gives a global picture of their genomic organization and possible roles. Determination of putative functions of predicted genes by homology searches and identification of sequence motifs (promoters, nod boxes, nifA activator sequences, and other regulatory elements) will aid in finding new symbiotic genes. High-fidelity sequence data covering long stretches of the genome are a prerequisite for these studies. The use of the dye terminator/thermostable sequenase shotgun approach has allowed the completion of the entire ˜500 kb sequence of pNGR234a and has opened up new avenues for the genetic analysis of symbiotic function.

[0062] Genetic Organization of the Whole Plasmid pNGR234a

[0063] Within the complete nucleotide sequence of pNGR234a, which comprises 536,165 bp, a total of 416 ORFs were predicted to encode proteins. An additional 67 ORF-fragments were detected that seem to be remnants of functional ORFs.

[0064] Thirty four percent (139) of the 416 potential proteins, have no obvious similarities to any known proteins. Of the remaining 277 proteins, 31 (8%) are similar to proteins for which no biochemical or phenotypic role has been assigned, 12 (3%) are similar to proteins for which limited biological data is available, and 234 (56%) are similar to proteins with a more precise biological function: enzymes (95), proteins involved in integration and recombination of insertion elements (44), transporters (32), transcriptional regulators (22), protein secretion/export (21), proteins involved in replication and control of the plasmid (12), electron transporters (6), and proteins involved in chemotaxis (2). A high proportion of enzymes was expected of a symbiotic replicon involved in nodulation (Nod-factor biosynthesis, etc.) and nitrogen fixation. As expected from the observation that NGR234 can be cured of its plasmid (Morrison et al., 1983), no ORFs essential to transcription, translation or to primary metabolism were found.

[0065] A large number of protein families are present in several copies on pNGR234a. This is true even after elimination of the many proteins which are encoded in repeated IS elements, or are involved in transposition, integration or recombination. The most notable examples of highly represented protein families include: five members of the short-chain dehydrogenase/reductase family, one of which (y4vI) contains two homologous domains; five complete and one partial ABC-type transporter operons that each encode for at least one ABC-type permease and an ABC-type ATP-binding protein; four cytochrome P450's; and three members of peptidase family S9A. In total, 85 proteins belong to families that are represented more than once and which do not seem to be linked to insertion or recombination.

[0066] The majority (330, 79%) of the putative proteins are probably located in the cytoplasm of the bacterium, 62 (15%) possibly span membranes, 20 (5%) could be located in the periplasm, 3 are predicted to be lipoproteins that could associate with the outer membrane, and 2 are probably outer membrane proteins. These observations accord well with the dominance of biosynthetic proteins, as well as proteins involved in transcriptional regulation and insertion/recombination, most of which are thought to be cytoplasmic.

[0067] Although other start points cannot be excluded, replication of pNGR234a probably begins at oriV which is located within the intergenic sequence (igs) between the repC and repB-like genes y4cI and y4cJ. This locus (positions 54,417 to 54,570) encodes three proteins with 40-60% amino acid identities to RepABC of pTiB6S3 (a Ti-plasmid of Agrobacterium tumefaciens), pRiA4b (an Ri-plasmid of A. rhizogenes) and pRL8JI (a cryptic plasmid of R. leguminosarum bv. leguminosarum). Amongst replication regions, highest identities (69 to 71% at the nucleotide level) are found in the igs's between repC and repB (FIG. 5). In Agrobacterium, these igs's are the determinants which render parental plasmids incompatible. Two ORF's (position 198,500), which are homologous to pseudomonal genes involved in plasmid stability, may also play a role in replication of pNGR234a. A 12 bp portion of the origin of transfer (oriT) is identical to that of pTiC58 of Agrobacterium tumefaciens (nt 80,162 to 80,173), and highly similar to those of RSF1010 (Escherichia coli) and pTFI (Thiobacillus ferrooxidans). This sequence corresponds to the oriT of plasmids containing the “Q-type nick-region” (FIG. 6).

[0068] Another 24 predicted ORFs show homologies to conjugal transfer genes of Agrobacterium Ti-plasmids. All are located in two large clusters between position 57,000 to 83,000. Since pNGR234a was believed to be non-transmissible (Broughton et al., 1987), the fact that both the nucleotide sequence of the individual ORFs and their order is similar in Agrobacterium and NGR234 came as a surprise. Conjugal transfer of Ti plasmids in A. tumefaciens is controlled by a family of N-acyl-L-homoserine lactone auto-inducers (Zhang et al., 1993). Similar molecules, which are able to interact with the traR gene product of A. tumefaciens, were detected in the supernatants of NGR234 cultures using the assay of Piper et al. (1993).

[0069] Reiterated sequences first became apparent in NGR234 during the construction of an ordered array of cosmid clones (Perret et al., 1991). It is now clear that 97 kbp (18%) of pNGR234a represents insertion-(IS) and mosaic-(MS) sequences (FIG. 7). Homology searches for known IS/MS revealed some of these, while comparison of repeated sequences within pNGR234a, as well as between the plasmid and 2,500 random chromosome sequences (V. Viprey, pers. communication) located the rest. Seventy five putative ORFs (18% of the total) and 40 fragments of ORFs were identified this way, nearly half of which (44) show homologies to integrases and transposases. Many of these IS elements are similar not only to those derived from Rhizobium and Agrobacterium species, but also to those of other, diverse Gram (−) and Gram (+) bacteria (e.g. Bacillus, Escherichia, and Pseudomonas). The shear number and diversity of these IS/MS elements suggests that NGR234 has functioned as a “transposon trap”. This is supported by the fact that their average G,C content (61.5%) is 3% higher than that of pNGR234a (58.5%). Interestingly, many IS/MS are clustered between positions 300,000 to 390,000 (FIG. 7), while some loci are almost unaffected by insertions (oriV, nod-, fix- and nif-ORFs). Small IS/MS clusters divide the replicon into large blocks of often functionally related ORFs (e.g. blocks of nod-ORFs, replication and conjugal transfer ORFs, nif-ORFs and fix-ORFs). A list of all sequences with IS-elment or mosaic sequence character is given in Table 4. Although transposition of these IS/MS elements has not been demonstrated, transfer of plasmids amongst rhizobia in the legume rhizosphere (Broughton et

TABLE 4
Insertion/mosaic sequences in pNGR234α
			put. ORFs/
			ORF-fragments			homologous
start of region	stop of region	name of region	included	similarities within pNGR234α	similarities to chromosome	sequences in other organisms/comments
17000	17600	ISH-10b	y4aQ	33% aa-id. to y4hP (ISH-10a)		geneproducts from IS866 and IS66 from
						Ag. tumefaciens
18900	19661	ISH-11b	fa2	54% aa-id. to part of y4bF (ISH-11a);		Tnp of IS1202 from Str. pneumoniae
				19096-19362: 91% nt-id. to ISH-11c
19666	22981	NGRIS-4a	y4bABCD	identical to NGRIS-4b	many copies on the chromosome
22985	25400	ISH-11a	fb1, y4bF	y4bF: sim. to fb1 and fa2 (ISH-11b)	partially 91% nt-id. to chromosomal sequences	Tnp of IS1202 from Str. pneumoniae
32463	35085	NGRIS-3a	y4bLM	identical to NGRIS-3b/c	copie(s) on the chromosome	62% nt-id. (over 2352 nt) to IS1162 of
						Ps. fluorescens (IS21/IS1162/IS408 family)
49300	50300	ISH-13a	y4cG	similar to y41S (ISH-13b)		DNA invertase
69936	70385	ISH-4c	fd1	70233-70385: 93% nt-id. to part of		ORFA of IS5376 from B. stearothermophilus
				NGRIS-4
93322	96025	ISH-12a	fe2, y4eF, fe5,	93574-94927: 90% nt-id. to ISH-12b1;		Tnp (fe2) and Int (Y4eF, fe3) from Weeksella
			fe3	75% nt-id. to fq6 region (ISH-12b2);		zoohelcum-IS-element; (93322-94586: 57% nt-id. to
				95343-95558: 88% nt-id. to ISH-12b3		IS292 from Ag. radiobacter); “phage” integrase
						family (Y4eF, fe5, fe3)
101939	102394	ISH-8b	fe7			84% nt-id. to ISRm5 of R. meliloti; fe7: mutator
						family of transposases
115881	116004	MSH-14b		partially homologous to ISH-14a	72-73% nt-id. to sequences downstream from	mosaic element
					chvl/upstream from rpoN on the chromosome
124396	124500	MSH-14a		partially homologous to ISH-14b	82% nt-id. to sequence RIME1 downstream from	mosaic element
					chvI on the chromosome; parts of MSH-14a show
					73-89% nt-id. to chromosomal sequences
126806	127369	ISH-12f	y4gA	low, similarity to y4rE
127900	128500	ISH-12e	y4gC			recombinase from pAE1 of Al. eutrophus (“phage”
						integrase family)
131000	131800	ISH-15	y4gE*		partially 87% nt-id. to chromosomal sequences
159781	160564	ISH-16				96% nt-id. to repetitive sequence from R. fredii
						USDA257 (acc. no. M73698)
164600	167700	ISH-10a	y4hNOPQ		99% nt-id. of parts of y4hPQ to chromosomal	different ORFs derived from IS-like sequences;
					sequences	partially known as acc. no. X74068 (“Region2”
						from pNGR234a); 164853-167086; 66% nt-id. to
						IS66 from Ag. tumefaciens
168208	169190	ISH-2c	fi1, fi2, fi3	168343-168659: 72% nt-id. to		168208-168383: 70 nt-id. to ISRm2011-2
				ISH-2f1/ISH-2d1		(R. meliloti); fi2/3: IS1111A, IS1328, IS1533
				168785-169091: 73% nt-id. to ISH-		family of transposases
				2f2/ISH-2d2
173295	173702	ISH-8g	y4iE*	y4iE: sim. to y4rL, y4zA. and fr2
175590	175909	ISH-11c	y4iG*	175643-175909: 91% nt-id. to ISH-11a
185672	186507	ISH-2d	y4iO*/P* (3′-	185672-186075(−): 73% nt-id. to ISH-		Y4iO: Tnp of IS1328 from Y. enterocolitica
			end)	2c2(+)		(IS1111A, IS1328, IS1533 family)
				186208-186507(−): 72% nt-id. to ISH-
				2c1(+)
187112	189752	NGRIS-5a	y4iQjA	identical to NGRIS-5b/c	copie(s) on the chromosome	IstA and B (Tnps) of IS1326 from E coli
						(IS21/IS1162/IS408 family)
190000	193500	ISH-10c	y4jBCD(E*)	38/32 aa-id. of y4jCD to y4hOP		different ORFs derived from IS-like sequences;
				(ISH10a)		partially 60% nt-id. to IS866 (Ag. tumefaciens);
						IS292 (Ag. radiobacter); ISR11 (R. leguminosarum)
193518	193634	MSH-17				76% nt-id. to repetitive sequence RMX6 from
						Myxococcus xanthus (acc. no. M60865)
199746	199958	ISH-11d	y4jM*	similarity to fb1 and y4bF (ISH-11a)
211165	211265	ISH-10g				74% nt-id. to ISR11 (R. leguminosarum),
						IS66/IS866 derivative
212350	212580	ISH-10h	fk2	similar to y4jD (ISH-10c)		74% nt-id. to IS66
217564	220186	NGRIS-3b	y4kIJ	identical to NGRIS-3a/c	copie(s) on the chromosome	62% nt-id. (over 2352 nt) to IS1162 of
						Ps. fluorescens (IS21/IS1162/IS408 family)
224547	224995	ISH-18a	y4kQ	83% nt-id. to ISH18b (427651-428102)		IS110 family
			(3′-end)
240800	241040	ISH-24b	fl2			60% nt-id. to ISR12 from R. leguminosarum
244540	244851	ISH-19a	fl4	244620-244812: 97% nt-id. to ISH-19b		TnpA from Tn163 (R. leguminosarum)
248290	248655	ISH-19b	fl5	248463-248655: 97% nt-id. to ISH-19a
248814	249680	ISH-20	fl6			Tnp of Tn1546 (Enterococcus faecium;
						Tn21/501/1721 family)
251407	252400	ISH-13b	y41SmA	y41S: similar to y4cG (ISH-13a)		y41S: invertase; 58% nt-id. (251409-252211) to
						Tn501 from Ps. aeruginosa (acc. no. Z00027)
258551	258657	MSH-21				mosaic sequence: 82% nt-id. to sequence
						upstream of ropA2 (R. liguminosarum; acc. no.
						X80794)
280403	283043	NGRIS-5b	y4nDE	identical to NGRIS-5b/c	copie(s) on the chromosome	IstA and B (Tnps) of IS1326 from E. coli
						(IS21/IS1162/IS408 family)
284722	284985	ISH-1b	fn2			60% nt-id. to IS1162 (Ps. fluorescens,
						IS21/IS1162/IS408 family)
300017	300819	ISH-1c	fo3			61% nt-id. IS408 (Ps. cepacia;
						IS21/IS1162/IS408 family)
300820	304117	NGRIS-6	fo4/5/6,	77% nt-id. to NGRIS-4
			y4oL/M/N
304118	304434	ISH-1d	fo7			61% nt-id. IS408 (Ps. cepacia;
						IS21/IS1162/IS408 family)
318854	319686	NGRIS-7	fp1-2			66% nt-id. to IS1248 of Pa. denitrificans
320456	328935	NGRRS-1a	fp3/4; y4pL		3 copies on the chromosome	interrupted by NGRIS2a and 4b; fp3/4, Y4pL:
						IS21/IS1162/IS408 family
320590	323147	NGRIS-2a	y4pEFG	identical to NGRIS-2b		partially 88-990% nt-id. to repetitive sequence
						RFRS9 of R. fredii USDA257 (IS1111A/IS1328/
						IS1533 family)
323961	327276	NGRIS-4b	y4pHIJK	identical to NGRIS-4a	many copies on the chromosome (disrupts all 4
					copies of NGRRS-1)
335004	336301	NGRIS-8	y4pO	similar to fe7 (ISH-8b)		88% nt-id. to ISRm3 of R. meliloti: mutator family
						of transposases
342272	342419	ISH-12d		342272-342419: 87% nt-id. to ISH-
				12b4
344100	345300	ISH-2e	y4qE			Tnp (Leptospira borgpetersenii):
						IS1111A/IS1328/IS1533 family
345755	346133	ISH-12c	fq4	345755-346133: 82% nt-id. to ISH-		Int (XerC, E. coli): “phage” integrase family
				12b5
351600	351735	MSH-22				80 nt-id. to sequence from pTiS4 (Ag. vitis; acc. no.
						M91609)
351826	353794	ISH-10d	y4qI, fq5	fq5: 35% aa-id. to y4hQ (ISH-10a)	71-95% nt.-id. of parts of y4qI to chromosomal	67% nt-id. to ISR11 (R. leguminosarum; acc. no.
					sequences	L19650); IS866/66 homolog
354000	363073	ISH-12b	y4qJK, fq6,	354942-356123/356215-356383:	70% nt-id. of parts of ISH14a1 to chromosomal	Tnp and Int from Weeksella zoohelcum -IS-element
			y4rABCDEF	90/91% nt-id. to ISH-12a1;	sequences	(y4qJK), different integrases (y4rAB), integrase
				75% nt-id. to fe5 region (ISH-12a2):		XerC of H. influenzae (y4rC);
				359753-359968. 88% nt-id. to ISH-		y4qK, fq6, y4rABCDEF: “phage” integrase family
				12a3;
				361029-361410: 82% nt-id. to ISH-12c
				362507-362654: 87% nt-id. to ISH-12d
363287	363694	ISH-10i	y4rG	low similarity to y4jB and fr4 (ISH-		unknown protein from IS1312 (A. tumefaciens)/
				10c/i)		IS866
366252	367402	ISH-8f	fr1, fr2	366252-366524: 88% nt-id. to ISH-8e
				366773-366953: 92% nt-id. to ISH-8g
367699	367970	ISH-10c	fr3	56% aa-id. of fr3 to y4hO (ISH-10a)		75% nt-id. to IS66 (Ag. tumefaciens)
368503	369675	ISH-23	y4rI		91-93% nt-id. of parts of y4rI to chromosomal
					sequences
369697	370887	ISH-2f	y4rJ	370012-370328: 72% nt-id. to ISH-2c1		y4rJ: Tnp from IS1111a of Coxiella burnitii
				370479-370785: 73% nt-id. to ISH-2c2		(IS1111A/IS1328/IS1533 family)
371399	372990	ISH-8e	y4rL*M*	371399-371671: 88% nt-id. to ISH-8f
				371474-372228: 97% nt-id. to ISH-8d
377185	377695	ISH-10j	fr4	similar to y4rG (ISH-10i)		377327-377695: 75% nt-id. to ISRm6 (R. meliloti)
377826	380383	NGRIS-2b	y4sABC	identical to NGRIS-2a		partially 88-90% nt-id. to repetitive sequence
						RFRS9 of R. fredii USDA257 (IS1111A/IS1328/
						IS1533 family)
380883	383523	NGRIS-5c	y4sDE	identical to NGRIS-5a/b	copie(s) on the chromosome	IstA and B (Tnps) of IS1326 from E. coli
						(IS21/IS1162/IS408 family)
383593	384054	ISH-2g	fs5			Tnp of IS1328 of Y. enterocolitica
						(IS1111A/IS1328/IS1533 family)
384210	384493	ISH-10k				fragments with 94-84% nt-id. to ISRm6
						(R. meliloti)
388100	388600	ISH-2h	fs1			different Tnps (IS1111A/IS1328/IS1533 family)
388601	388900	ISH-10l	fs2			ORF from IS1312 of Ag. tumefaciens (IS66/866
						family)
396445	397301	NGRIS-9	y4sN and fs4		91-99% nt-id. of NGRIS9-parts to chromosomal	different ORFs derived from IS elements; partially
					sequences	known from acc. no. X74314
400626	403248	NGRIS-3c	y4tAB	identical to NGRIS-3a/b	copie(s) on the chromosome	62% nt-id. (over 2352 nt) to IS1162 of
						Ps. fluorescens (IS21/IS1162/IS408 family)
426525	428102	ISH-18b	y4uE*	427651-428102: 83% nt-id. to ISH-18a	77-96% nt-id. of ISH-18b-parts to chromosomal	Tnp of mini-circle DNA from Str. coelicolor
					sequences	(IS110 family)
429860	430007	ISH-8c	fu3			85% nt-id. to ISRm5 (R. meliloti)
430568	432851	ISH-1e	y4uHI			60% nt-id. to IS408/IS1162
						(Ps. cepacia/Ps. fluorescens)
433222	433560	ISH-24a	fu4	low similarity to y4sN (NGRIS-9)		79% nt-id. to ISRm4 (R. meliloti)/ISR12-like
462554	463053	ISH-10f				fragments with 83-69% nt-id. to IS866
						(Ag. tumefaciens)
524946	525892	ISH-8d	y4zA	525095-525849: 97% ni-id. to ISH-8e		524946-525580: 61% nt-id. to ISRm5 (R. meliloti)
526051	527121	ISH-25	y4zB*			Tnp of IS5376 from B. stearothermophilus (IS4
						family of transposases)
530364	531249	ISH-1f	fz4, fz2	79% nt-id. to part of NGRIS-5		fz4/2: IS21/IS1162/IS408 family
Abbreviations: Tnp = transposase; Int = integrase: nt-id. = nucleotide-identity; aa-id. = aminoacid identity
IS elements with precisely defined borders are designated as NGRRS/NGRIS-1 to 9. Other sequences which show homologies to known mosaic or IS-like sequences (mosaic/insertion sequence homologs) are named MSH and ISH. respectively.

[0070] al., 1987) and to other non-symbiotic bacteria in fields (Sullivan et al., 1995) suggests that lateral transfer of genetic information has helped shape symbiotic potential.

[0071] Carbohydrates are constituents of the rhizobial cell wall as well as morphogens called Nod-factors (short tri- to penta-mers of N-acetyl-D-glucosamine, substituted at the non-reducing terminus with C16 to C18 saturated or partially unsaturated fatty acids). Elements of the biosynthetic pathways leading to cell walls or to lipo-chito-oligosaccharides (Nod-factors) are common. Most differences are found in the later stages of the pathways that lead to specific cell-wall components or to Nod-factors.

[0072] As befits a symbiotic replicon, only 13 ORF's with homology to polysaccharide synthesis genes (house-keeping genes senso stricto) are located on the plasmid (Table 3). Sequences homologous to exoB, exoF, exoK, exoL, exoP, exoU, and exoX (X. Perret and V. Viprey, unpublished), and exoY (Gray et al., 1990) are clearly located on the chromosome. Although loci with weak homologies to nod-box:-psiB of R. leguminosarum, and exoX of R. meliloti exist on the plasmid (y4iR, and y4xQ respectively), these are regulatory rather than structural genes, suggesting that almost all cell wall polysaccharide synthesis ORFs are chromosomally located.

[0073] Except for nodPQ and nodE, at least one copy of all the regulatory and structural ORFs involved in Nod-factor biosynthesis seem to be located on the plasmid. The activity of most nodulation genes is modulated by four transcriptional regulators of the lysR family. These are nodD1 (y4aL), syrM1 (y4pN), nodD2 (y4xH), and syrM2 (y4zF). NodC, which is an N-acetylglucosaminyltransferase, the first committed enzyme in the Nod-factor biosynthetic pathway, is part of an operon which includes nodABCIJnolOnoeIE (y4hI to y4hB, Table 3). Together, these genes, which form the hsnIII locus, are responsible for the synthesis of the core Nod-factor molecule, and the adjunction of 3- (or 4)-O-carbamoyl, 2-O-methyl, and 4-O-sulfate groups (Hanin et al., unpublished). nodZ (y4aH), which encodes a fucosyltransferase, is part of the hsnI locus, which includes noeJ (y4aJ), noeK (y4aI), noeL (y4aG), nolK (y4aF), all of which are involved in the fucosylation of NodNGR factors (Fellay et al., 1995a). Wild-type NodNGR factors are also N-methylated and 6-O-carbamoylated, adjuncts which are added by the transferases encoded by nodS and nodU respectively [y4nC and y4nB; hsnII (Lewin et al., 1990)]. Possibly the only other enzyme which may be directly involved in Nod-factor biosynthesis is that encoded by nolL (y4eH, Table 3). As the 2-O-methylfucose residue of NGR234 Nod-factors is either 3-O-acetylated, or 4-O-sulphated, an acetyltransferase is obviously required. Since NolL shows only limited homology to acetyltransferases, experimental proof of the transferase activity will be required however.

[0074] In contrast to R. leguminosarum and R. meliloti harbouring pNGR234a, A. tumefaciens (pNGR234a) transconjugants are incapable of nitrogen fixation (Broughton et al., 1984), suggesting that some essential fix-ORFs are also carried by the chromosome. Nevertheless, more than 40 nif- and fix-ORFs are plasmid borne. Included amongst these are nifA (y4uN) which encodes for a sigma-54 dependent regulator. Mutation of rpoN (which encodes sigma 54) causes a Fix− phenotype on NGR234 hosts (van Slooten et al., 1990). Similarly, mutation of fixF (y4gN) disrupts synthesis of a rhamnose-rich extra-cellular polysaccharide, and results in a Fix− phenotype on Vigna unguiculata, the reference host for NGR234 (unpublished). In fact, loci adjacent to fixF are probably responsible for the synthesis of dTDP-rhamnose from glucose-1-phosphate. Enzymes involved in this biosynthetic pathway include glucose-1-phosphate thymidylyltransferase (y4gH), dTDP-glucose-4,6-dehydratase (y4gF), dTDP-4-dehydrorhamnose-3,5-epimerase (y4gL), and dTDP-4-dehydrorhamnose reductase (y4gG). Rhamnose-rich lipopolysaccharides (LPS) seem to be necessary for complete bacteroid development and nitrogen fixation (Krishnan et al., 1995). Perhaps the enzyme encoded by y4gI is needed for the synthesis of the rhamnose rich LPS's from dTDP-rhamnose.

[0075] Although not directly involved in the fixation process, mutation of the plasmid borne copy of dctA (=dctA1, y4vF) also impairs nitrogen fixation (van Slooten et al., 1992). Other nif- and fix-ORFs are involved in elaboration of the electron-transfer complex (fixAB), in various cofactors required for nitrogen fixation (e.g. fixC, nifB, nifE, niJN, etc.), and in the synthesis of ferrodoxins (fdxB, fdxN, fixX). Finally, those ORFs involved in the synthesis of the nitrogenase complex are also present. Amongst these are two functional copies of the nifKDH ORFs (y4vM to y4vK and y4xC to y4xA) (Badenoch-Jones et al., 1989). Additionally, 17 new ORFs located within the nitrogen fixation cluster (see FIG. 7; ORFs y4vC to y4vJ with the exception of dctAl, y4wA to y4wG, y4wI, y4wJ and y4xQ) are co-transcribed together with the ORFs homologous to known nif and fix genes. It thus seems likely that most ORFs necessary for bacteroid development and synthesis of the nitrogen-fixing complex, are carried by pNGR234a.

[0076] Two types of regulatory elements which frequently occur in pNGR234a are the NodD- and NifA/sigma-54-dependent promoters. NodD-dependent promoter-like sequences known as nod boxes have been identified by homology search within intergenic regions, using the following consensus sequence: 5′-YATCCAYNNYRYRGATGNNNNYNATCNAAACAATCRATTTT ACCAATCY-3′ [12 mismatches allowed (van Rhijn and Vanderleyden, 1993); Y=C or T, R=A or G, N=A, C, G or T]. Putative NifA-dependent promoters (Fischer, 1994) have been predicted by screening for the NifA activator sequence (5′-TGT-N10-ACA-3′) together with the sigma-54 promoter consensus sequence (5′-TGGCAC-N5-TTGCA/T-3′ with GG and GC as the most conserved doublets; 3 mismatches allowed) separated by 60 to 150 nucleotides. The identified conserved promoter-like sequences in pNGR234a are listed in Tables Sand 6.

TABLE 5
nod box-like sequences in pNGR234a
			number of
			mismatches to the
nod	position in	orien-	consensus	distance to the	name of the
box	pNGR234a	tation	sequence	following ORF	following ORF
1	4514-4562	−	11	504	(fa1)
2	8481-8529	−	8	87	nodZ
3	12322-12370	−	7	—	?#
4	97470-97518	−	6	277	nolL
5	129615-129663	+	10	1358	y4gE
6	141088-141136	+	8	890	fixF
7	150280-150327	−	11	202	noeE
8	158820-158868	−	4	235	nodA
9	161891-161939	+	11	1103	y4hM
10	169833-169881	−	7	117	y4iR
11	278947-278995	−	7	153	nodS
12	279821-279869	+	7	—	?#
13	443101-443149	−	10	465	y4vC
14	473059-473107	+	9	236	y4wH
15°	481253-481301	−	16	117	y4wM
16	493961-494009	+	6	288	y4xI
17	532039-532087	+	5	589	syrM2
18	256434-256482	+	12	329	y4mC
19	469151-469199	+	12	112	y4wE
°The majority of the mismatches is located in the 3′-terminal part of the sequence.
#No predicted ORF can be found downstream of the putative nod box.

[0077]

TABLE 6
Putative NifA-dependent promoters in pNGR234a
		sigma-54 promoter		distance to the
	NifA-dep. UAS*:	(−12/−24 region#):	orien-	following ORF	name of the
Nr.	position	position	tation	(nt)	following ORF
1	90812-90827	90910-90924	+	127	y4eD
2	162727-162742	162788-162802	+	240	y4hM
3	235036-235051	234934-234948	−	66	y4lD
4	255021-255036	255130-255144	+	306	y4mB
5	285265-285280	285343-285357	+	50	y4nG
6	436363-436378	436275-436289	−	41	nifB
7	442046-442061	441955-441969	−	56	fixA
8	442735-442750	442676-442690	−	40	y4vC
9	444109-444124	443983-443997	−	104	y4vD
10	444137-444152	444241-444299°	+	38°	nifQ
11	451782-451799	451891-451905	+	88	nifH1
12	460319-460334	460424-460438	+	63	y4vR
13	463063-463078	463139-463153	+	48	y4wA
14	478839-478854	478761-478775	−	463	nifS
15°	483663-483678	483769-483783	+	88	nifH2
*“Upstream Activator Sequence”: NifA-binding site located 80 to 150 nt upstream of the transcription start point (5′-TGT-N10-ACA-3′).
#sequence corresponding to the consensus sequence of conserved sigma-54-promoters 12 nt upstream of the transcription start point: 5′-TGGCAC-N5-TTGC-3′ (2 mismatches allowed).
°3 possibilities for a promoter (in two cases only corresponding to the minimal consens: 5′-GG-N10-GC-3′)

EXAMPLES

Example 1

GENERAL METHODS

[0078] Bacteria and Plasmids

[0079] Escherichia coli was grown on SOC, in TB or in two-fold YT medium (Sambrook et al., 1989). The cosmid clones pXB296 and pXB110 (Perret et al., 1991) were raised in E. coli strain 1046 (Cami and Kourilsky, 1978). Subclones in M13mp18 vectors (Yanisch-Perron et al., 1985) were grown in E. coli strain DH5αF′IQ (Hanahan, 1983).

[0080] Construction of Cosmid Libraries

[0081] Cosmid DNA was prepared by standard alkaline lysis procedures followed by purification in CsCl gradients (Radloff et al., 1967). DNA fragments sheared by sonication of 10 μg of cosmid DNA were treated for 10 min at 30° C. with 30 units of mung bean nuclease (New England Biolabs, Beverly, Mass., USA), extracted with phenol/chloroform (1:1), and precipitated with ethanol. DNA fragments, ranging in size from 1 to 1.4 kbp, were purified from agarose gels using Geneclean II (Bio101, Vista, Calif., USA) and ligated into SmaI-digested M13pm18. Electroporation of aliquots of the ligation reaction into competent E. coli DH5αF′IQ was performed according to standard protocols (Dower et al., 1988; Sambrook et al., 1989).

[0082] M13 Template Preparation

[0083] Fresh 1 ml E. coli cultures in twofold YT held in 96-deep-well microtiter plates (Beckman Instruments, Fullerton, Calif., USA) were infected with recombinant phages from white plaques grown on plates containing X-gal (5-bromo-4-chloro-indoyl-β-D-galactoside) and IPTG (isopropyl-β-thiogalactopyranoside). Rapid preparation of -0.5 Mg of single-stranded M13 template DNA was carried out as follows: 190 μl portions of the phage cultures grown for 6 hr at 37° C. were transferred into 96-well microtiter plates. Lysis of the phages was obtained by adding 10 μl of 15% (w/v) SDS followed by 5 min incubation at 80° C. Template DNA was trapped using 10 μl (1 mg) of paramagnetic beads (Streptavidin MagneSphere Paramagnetic Particles Plus M13 Oligo, Promega, Madison, Wis., USA) and 50 μl of hybridization solution [2.5 M NaCl, 20% (w/v) polyethylene glycol (PEG-8000)] during an annealing step of 20 min at 45° C. Beads were pelleted by placing microtiter plates on appropriate magnets and washing three times with 100 μl of 0.1-fold SSC. The DNA was recovered in 20 μl of water by a denaturation step of 3 min at 80° C. When required, larger amounts of single-stranded recombinant DNA (>10 μg) were purified using QIAprep 8 M13 Purification Kits (Qiagen, Hilden, Germany) from 3 ml of supernatant of phage cultures grown for 6 hr at 37° C.

[0084] Sequencing

[0085] Two sequencing methods were used: dye terminator and dye primer cycle sequencing, each in combination with AmpliTaq DNA polymerase (Perkin-Elmer) and Thermo Sequenase (Amersham). All reactions, including ethanol precipitation, were performed in microtiter plates. Reagents were pipetted using 12-channel pipettes. Where necessary, sequencing reaction mixtures, including enzymes, were pipetted into the plates in advance and held at -20° C. until needed.

[0086] Dye Terminator Cycle Sequencing

[0087] For dye terminator/AmpliTaq DNA polymerase sequencing, 0.5 μg of template DNA, and the PRISM Ready Reaction DyeDeoxy Terminator cycle sequencing Kit (Perkin-Elmer) were used. Cycle sequencing was performed in microtiter plates using 25 PCR cycles (30 sec at 95° C., 30 sec at 50° C., and 4 min at 60° C). Prior to loading the amplified products on electrophoresis gels, unreacted dye terminators were removed using Sephadex columns scaled down to microtiter plates (Rosenthal and Charnock-Jones, 1993).

[0088] Dye terminator/Thermo Sequenase sequencing was performed using the same experimental conditions except that the reaction mix contained 16.25 mM Tris-HCl (pH 9.5), 4.0 mM MgCl2, 0.02% (v/v) NP-40, 0.02% (v/v) Tween 20, 42 μM 2-mercaptoethanol, 100 μM dATP/dCTP/dTTP, 300 μM dITP, 0.017 μM A/0.137 μM C/0.009 μM G/0.183 μM T from Taq Dye Terminators (Perkin-Elmer; no. A5F034), 0.67 μM primer, 0.2-0.5 μg of template DNA, and 10 units of Thermo Sequenase (Amersham) in a 30 μl reaction volume. Unincorporated dye terminators were removed from reaction mixtures by precipitation with ethanol.

[0089] Dye Primer Cycle Sequencing

[0090] Dye primer/AmpliTaq DNA polymerase sequencing reactions were performed according to the instructions accompanying the Taq Dye Primer, 21M13 Kit (Perkin-Elmer). Cycle sequencing was carried out on 0.5 μg of template DNA with 19 PCR cycles (30 sec at 95° C., 30 sec at 50° C., and 90 sec at 72° C.) followed by six cycles, each consisting of 95° C. for 30 sec and 72° C. for 2.5 min. Prior to electrophoresis, the four base-specific reactions were pooled and precipitated with ethanol.

[0091] Identical PCR conditions and the Thermo Sequenase Fluorescent Labelled Primer Cycle Sequencing Kit (Amersham) were used for dye primer/Thermo Sequenase sequencing reactions.

[0092] Sequence Acquisition and Analysis

[0093] Gel electrophoresis and automatic data collection were performed with ABI 373A DNA sequencers (Perkin-Elmer). After removing cosmid vector and M13mp18 sequences from the shotgun sequence data, the data were assembled using the program XGAP (Dear and Staden, 1991) and edited against the fluorescent traces. To close remaining gaps, to make single-stranded regions double-stranded, and to clarify ambiguities, additional cycle sequencing reactions with selected shotgun templates were carried out using either custom-made primers (primer-walks) or universal primer.

[0094] The complete double-stranded DNA sequence of cosmid pXB296 was analyzed using programs from the Wisconsin Sequence Analysis Package (version 8, Genetics Computer Group, Madison, Wis., USA). Homology searches were performed with BLAST (version 1.4; Altschul-et al., 1990) and FASTA (version 2.0; Pearson and Lipman, 1988). Several nucleotide and protein databases were screened (GenBank/Genpept, SwissProt, EMBL, and PIR). Identities and similarities between homologous amino acid sequences were calculated with the alignment program BESTFIT (Smith and Waterman, 1981).

Example 2

[0095] Comparison of Fluorescent Traces Created by Different Cycle Sequencing Methods

[0096] When using a thermostable sequenase [Thermo Sequenase (Amersham)], the concentrations of dye terminators (Perkin-Elmer) can be reduced by 20- to 250-fold in comparison to the concentrations needed for Tag DNA polymerase without compromising the quality of the sequencing results (Table 7).

[0097] To compare the dye terminator and dye primer cycle sequencing procedures, representative templates derived from the pXB296 library were sequenced by both methods, each performed with Thermo Sequenase and Taq DNA polymerase

TABLE 7
Concentrations (in μM) of dye terminators in each cycle sequencing
reaction with two different thermostable DNA polymerases
Dye	AmpliTaq DNA	Thermo Sequenase	Dilution factor for
terminator	polymerase	DNA polymerase	dye terminatorsa
A Taq	0.751	0.017	40
C Taq	22.500	0.137	160
G Taq	0.200	0.009	20
T Taq	45.000	0.183	250
aThermo Sequenase vs. AmpliTaq.

[0098] (FIG. 1). In general, dye terminator traces do not contain the many compressions (on average, one compression every 50 bases in single reads) that are common with dye primers if mixes do not contain nucleotide analogues like deoxyinosine or 7-deaza-deoxyguanosine triphosphates or if sequencers are used without active heating systems. In addition, dye terminator traces obtained with Thermo Sequenase show more uniform signal intensities over those obtained with Taq DNA polymerase, thus resulting in a reduced number of weak and missing peaks (e.g. a weak G-signal following an A-signal in Thermo Sequenase traces or a weak C-signal following a G-signal in Taq DNA polymerase traces). Using ABI 373A sequencers, errors in automatic base-calling of Thermo Sequenase/dye terminator scans only arise after 300-350 bases. The average number of resolved bases in dye primer gels (378 bases) is 46 bases longer than in those produced with dye terminators (332 bases). Furthermore, in Thermo Sequenase/dye primer sequences the peaks are very regular and the number of stops and missing bases decreases in comparison to Tag DNA polymerase/dye primer electropherograms. The number of compressions, however, is not significantly reduced.

Example 3

[0099] Shotgun Sequencing of Entire Cosmids Using Dye Terminators or Dye Primers

[0100] To compare the efficiency of both methods, cosmid pXB296 of pNGR234a was shotgun sequenced using a combination of dye terminators and thermostable sequenase (Thermo Sequenase), whereas another cosmid, pXB110, was sequenced using a combination of dye primers and Taq DNA polymerase (Table 1). Over 93% (736 clones) of 786 dye terminator reads of pXB296 were accepted by XGAP with a maximal alignment mismatch of 4%. By increasing this level to 25%, so that most of the remaining data could be included in the assembly, 775 reads led to three 6 to 10 kbp stretches of contiguous sequence (contigs), two of which were joined after editing. To close the last gap and to complete single-stranded regions with data derived from the opposite strand, only 32 additional dye terminator reads using custom-made primers were required. It took <1 week to assemble and finalize the 34,010 bp DNA sequence of pXB296 (EMBL accession no. Z68203; eight-fold redundancy; GC content, 58.5 mol %).

[0101] In contrast, only 308 (34%) of 899 shotgun reads obtained by Taq DNA polymerase/dye primer cycle sequencing of pXB110 were included in the first assembly (4% alignment mismatch). At the 25% alignment mismatch level, 879 reads were assembled, leading to 25 short contigs (1-2 kbp). These contigs had to be edited extensively in order to join most of them. “Primer walks”, covering gaps and complementing single-stranded regions, were not sufficient to clarify all the remaining ambiguities in the assembled sequence. Every 100-150 bp, a compression in one strand could not be resolved by sequence data from the complementary strand. Therefore, it was necessary to resequence clones using dye terminators and universal primer. In total, 191 additional dye terminator reads had to be created. As a result, assembling and finalizing the 34,573 bp sequence of pXB110 (10.5-fold redundancy; GC content, 58.3 mol %) took much more time than pXB296 did.

Example 4

[0102] Analysis of Cosmid pXB296

[0103] Putative ORFs were located on the 34,010 bp sequence of pXB296 using the programs TESTCODE (Fickett, 1982) and CODONPREFERENCE (Gribskov et al., 1984), the latter in combination with a codon frequency table based on previously sequenced genes of Rhizobium sp. NGR234 (as well as the closely related R. fredii). All 28 ORFS and their deduced amino acid sequences exhibited significant homologies to known genes and/or proteins. The positions of the ORFs along pXB296, as well as the best homologues, are displayed in Table 2 and FIG. 2. Ribosomal binding site-like sequences (Shine and Dalgarno, 1974) precede each putative ORF except for ORF9 (position 11,214-12,455). If one disregards the homology to known glutamate dehydrogenases in the first 32 amino acids deduced from this ORF, a downstream alternative start codon (position 11,220) preceded by a Shine-Dalgarno sequence can be identified. Most of the ORFs are organised in five clusters (ORFs with only short intergenic spaces or overlaps between them). Cluster I, containing ORF1 to ORF5, encodes proteins homologous to trans-membrane and membrane-associated oligopeptide permease proteins and to a Bacillus anthracis encapsulation protein. Cluster II, includes ORF6 and ORF7, which are homologous to aminotransferase and (semi)aldehyde dehydrogenase genes. Homologies to transposase genes [ORF8; cluster III (ORF10 and ORF11)] and to various nif and fix genes [cluster IV (ORF12 to ORF20); ORF23, part of cluster V] are also reported.

[0104] Presumed promoter and stem-loop sequences that might represent ρ-independent terminator-like structures (Platt, 1986) are shown in FIG. 2. Significant σ54-dependent promoter consensus sequences (5′-TGGCACG-N4-TTGC-3′; Morett and Buck, 1989), as well as nifA upstream activator sequences (5′-TGT-N10-ACA-3′; Morett and Buck, 1988), are found upstream of the nifB homologue ORF15, the fixA homologue ORF20, ORF21, ORF22, and ORF23. ORF23 is part of cluster V in pXB296, which includes the dctA gene of Rhizobium sp. NGR234 (van Slooten et al., 1992). Surprisingly, the published dctA sequence shows important discrepancies. Therefore, a fragment encompassing this locus was amplified by PCR using NGR234 genomic DNA as template. By sequencing this fragment, the cosmid sequence of the present invention was confirmed.

Example 5

[0105] Analysis of the Complete Plasmid pNGR234a

[0106] Using the thermostable sequenase/dye terminator cycle sequencing method herein described, 20 overlapping cosmids (including pXB296) of the symbiotic plasmid pNGR234a of Rhizobium sp. NGR234 were sequenced, together with two PCR products and a subcloned DNA fragment derived from cosmid pXB564 that cover two remaining gaps (position 276,448-277,944 and position 480,607-483,991). The map of the sequenced cosmids is shown in FIG. 4. The entire assembled 536 kb sequence of pNGR234a is given in FIG. 3 (deposited in EMBL/GenBank under accession no. U00090).

[0107] The analysis of the complete nucleotide sequence revealed few regions of 98-100% identity to already published sequences in public databases. These sequences are listed in Table 8. These sequences had been derived either from Rhizobium sp. NGR234, derivatives of it or closely related strains of it. Therefore, the ORFs and their deduced proteins, 98-100% homologous to nifH, nodA, nodB, nodC, nodD1, nodS, nodU, nolX, nolW, nolB, nolU and “ORF1”, represent already known genes/proteins (Table 8 and References). Some other ORFs and their deduced proteins, nearly identical to public database entries, were either only partially known before the disclosure of the present invention or exhibited significant differences, for instance, dctA, host-inducible gene A, nifD, nifK, nodD2, nolT, nolX, nolV, “ORF140”, “ORF91”, “RSRS9 25 kDa-protein gene” (Table 8 and References).

[0108] As a first step, approximately 100 kb of pNGR234a was analyzed between position 417,796 to 517,279 using the programs TESTCODE (Fickett, 1982) and CODONPREFERENCE (Gribskov et al., 1984). In this initial ˜100 kb of sequence, 76 ORFs were found and ascribed putative functions

TABLE 8
All ORFs that show 98-100% identity in the nucleotide sequence to
ORFs located in pNGR234a and that have already been published in databases:
		EMBL/GeneBank	+	claimed in the patent application/
ORF	organism	accession no.	−	not claimed in the patent application
dctA	Rhizobium sp. NGR234	S38912	+	sequencing mistakes in the database entry: the
				real dctA in pNGR23a is 144 bases longer (see
				table 4)
host inducible geneA	Rhizobium fredii USDA 201#	M19019 RFIND	+	significant difference in pNGR234a (frameshift;
				see table 4)
nifH	Rhizobium sp. ANU 240*	M26961 RHMNIFKDH3	−
nifD (partially)	Rhizobium sp. ANU 240*	M26961 RHMNIFKDH2	+	only part of nifD is in the public database
nifK (partially)	Rhizobium sp. ANU 240*	M26961 RHMNIFKDH1	+	only part of nifK is in the public database
nodABC	Rhizobium fredii USDA 257#	M73362 RSNOD2	−
nodD1	Rhizobium sp. mpik 3030*	Y00059 RSNODD1	−
nodD2	Rhizobium japonicum USDA 191#	M18972 RHMNODD2M	+	significantly different function of NodD2 in
				NGR234 than in USDA 191 (despite of 98%
				identity °)
nodS	Rhizobium sp. NGR234	J03686 NGRNOIDSU	−
nodU (partially)	Rhizobium sp. NGR234	J03686 NGRNODSU	−
nodU (full)	Rhizobium sp.*	X89965 RSNODUGEN
nolXWBTUV	Rhizobium fredii USDA 257#	L12251 RHMNOLBTU	−	nolXWB, nolU
			+	NolT: 97% identical (amino acid sequence level)
			+	NolX, NolV + ORF4 of pNGR234a show
				significant differences to USDA 257 (see table 4)
ORF1; ORF2(partially)	Rhizobium sp. NGR234	X74314 RSORF	−
ORF140 nodulation gene;	Rhizobium sp. NGR234	X74068 RSPLAS	+	database entry includes sequencing mistakes
ORF91(partially)				causing frameshifts
RFRS9 25kDa protein gene*	Rhizobium fredii USDA 257#	U18764 RFU18764	+	repetitive element in pNGR234a showing
				insertions, deletions of nucleotides in
				comparison to the database entry
*strains representing derivatives of NGR234: Rhizobium sp. ANU 240, Rhizobium sp. mpik 3030, Rhizobium sp.
#strains closely related to NGR234: Rhizobium fredii USDA 257, Rhizobium japonicum USDA 191, Rhizobium fredii USDA 201.
° identity in nucleotide sequence as well as amino acid sequence

[0109] (=ORFs y4tQ to y4yO (excluding ORFs y4uD, y4uG, y4wG, y4wO, y4wP, y4xF, y4xQ, y4xG and y4yB and excluding ORF-fragments fu1, fu2, fu3, fu4, fv1 and fw1); see Table 3). It should be noted that since the sequence of cosmid pXB296 forms part of this 100 kb region, all of the ORFs identified in Table 2 (except “ORF1”) are reproduced (albeit with minor, but definitive, revisions) in Table 3. Most of the 76 ORFs and their deduced proteins showed homologies to public database entries that could help identify their putative functions. Only ORFs y4vK and y4xA (duplicated nifH) as well as y4yD, y4yE and y4yG (nolW, nolB and nolU) were identical to database entries (98-100% homology). In the case of 7 ORFs and their deduced proteins, no homologous sequences in public databases have been found.

[0110] As a second step, the remaining 436 kb of pNGR234a were analyzed using the methods noted above. The results of this analysis are discussed in Example 6.

Example 6

[0111] Genetic Organization of the Complete Plasmid pNGR234a

[0112] In order to confirm and to improve the identification of probable coding regions in pNGR234a, the program GeneMark was used which is based on matrices developed for related organisms of Rhizobium sp. NGR234 (R. leguminosarum and R. meliloti (Borodovsky et al., 1994)). The use of this program currently represents the most frequently applied method to distinguish coding and non-coding regions in newly sequences DNA of prokaryotes. Further analysis of the putative ORF products was carried out using methods to detect signal sequences, transmembrane segments and various other domains (PROSITE database search (Bairoch et al., 1995); PSORT program (Nakai et al., 1991)).

[0113] In total, 416 ORFs were predicted to encode putative proteins (Freiberg et al., 1997). Additionally, 67 fragments were detected that seemed to be remnants of functional ORFs. Some of these were disrupted by insertion of mobile elements. All identified functional ORFs and fragments of former functional ORFs are listed in Table 3.

[0114] Within the initial ˜100 kb region (position 417,796 to 517,279) first analyzed in this study, 9 ORFs (y4uD, y4uG, y4wG, y4wO, y4wP, y4xF, y4xQ, y4xG and y4yB) and 6 ORF-fragments (fu1, fu2, fu3, fu4, fv1 and fw1) were predicted in addition to the 76 ORFs (y4tQ to y4yO) listed within Table 3.

[0115] According to Table 8, 12 ORFs of the 416 predicted coding regions were identical to public database entries (98% to 100% homology at the amino acid level), namely: y4hI (nodA), y4hH (nodE), y4hG (nodC), y4aL (nodD1), y4nC (nods), y4nB (nodU), y4sM (ORF1), y4vK (niftH), y4xA (nifH2), y4yD (nolW), y4yE (nolB), y4yG (nolU). In addition, the database entry of the homologue to y4yC (nolX) has been corrected to 98% identical to y4yC. Furthermore, the sequence of the ORF y4hB (noeE) has been available to the public since October 1996. Except the 14 ORFs mentioned above, the remaining 402 ORFs are new. 139 of them show no homology to any known ORF/protein. The others exhibit less than 98% amino acid identity to public database entries over their whole length.

[0116] INDUSTRIAL APPLICABILITY

[0117] The present invention provides a detailed analysis of the symbiotic plasmid pNGR234a of Rhizobium sp. NGR234. The plasmid pNGR234a (including any ORFs encoded therein, or any part of the nucleotide sequence of the plasmid, or any proteins expressible from any of said ORFs or any part of said nucleotide sequence) has industrial applicability which can include its use in, inter alia, the following areas:

[0118] (a) the analysis of the structure, organisation or dynamics of other genomes;

[0119] (b) the screening, subcloning, or amplification by PCR of nucleotide sequences;

[0120] (c) gene trapping;

[0121] (d) the identification and classification of organisms and their genetic information;

[0122] (e) the identification and characterisation of nucleotide sequences, amino acid sequences or proteins;

[0123] (f) the transportation of compounds to and from an organism which is host to at least to one of said nucleotide sequences, ORFs or proteins;

[0124] (g) the degradation and/or metabolism of organic, inorganic, natural or xenobiotic substances in a host organism;

[0125] (h) the modification of the host-range, nitrogen fixation abilities, fitness or competitiveness of organisms;

[0126] (i) obtaining a synthetic minimal set of ORFs required for functional Rhizobium-legume symbiosis;

[0127] (j) the modification of the host-range of rhizobia;

[0128] (k) the augmentation of the fitness or competitiveness of Rhizobium sp. NGR234 in the soil and its nodulation efficiency on host plants;

[0129] (l) the introduction of desired phenotype(s) into host plants using said plasmid as a stable shuttle system for foreign DNA encoding said desired phenotype(s); or

[0130] (m) the direct transfer of said plasmid into rhizobia or other microorganisms without using other vectors for mobilization.

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Claims

1. The nucleotide sequence as shown in FIG. 3 or degenerate variants thereof.

2. The nucleotide sequence of claim 1 which has been altered by mutation, deletion or insertion.

3. ORFs derivable from the nucleotide sequence of claim 1 or claim 2; excluding the ORFS identified as y4aL, y4hB, y4hG, y4hH, y4hI, y4nB, y4nC, y4sM, y4vK, y4xA, y4yC, y4yD, y4yE, y4yG in Table 3.

4. ORFs y4aA to y4aS, y4bA to y4bO, y4cA to y4cQ, y4dA to y4dX, y4eA to y4eO, y4fA to y4fR, y4gA to y4gN, y4hA to y4hR, y4iC to y4iR, y4jA to y4jT, y4kA to y4kV, y4lA to y4lS, y4mA to y4mQ, y4nA to y4nM, y4oA to y4oX, y4pA to y4pO, y4qB to y4qK, y4rA to y4rO, y4sA to y4sL, y4sN to y4sO, y4tA to y4tS, y4uA to y4uP, y4vA to y4vS, y4wA to y4wM, y4xA to y4xQ, y4yA to y4yS as identified in Table 3; excluding the ORFs identified as y4aL, y4hB, y4hG, y4hH, y4hI, y4nB, y4nC, y4vK, y4xA, y4yC, y4yD, y4yE, y4yG in Table 3.

5. The ORFs of claim 3 or claim 4 which encode the functions of: (a) nitrogen fixation, (b) nodulation, (c) transportation or permeation, (d) synthesis and modification of surface poly- or oligosaccharides, lipo-oligosaccharides or secreted oligosaccharide derivatives, (e) secretion (of proteins or other biomolecules), (f) transcriptional regulation or DNA-binding, (g) peptidolysis or proteolysis, (h) transposition or integration, (i) plasmid stability, plasmid replication or conjugal plasmid transfer, (j) stress response (such as heat shock, cold shock or osmotic shock), (k) chemotaxis, (l) electron transfer, (m) synthesis of isoprenoid compounds, (n) synthesis of cell wall components, (o) rhizopine metabolism, (p) synthesis and utilization of amino acids, rhizopines, amino acid derivatives or other biomolecules, or (q) degradation of xenobiotic compounds, or which encode: (r) proteins exhibiting similarities to proteins of amino acid metabolism or related ORFs, or (r) enzymes (such as oxidoreductase, transferase, hydrolase, lyase, isomerase or ligase).

6. The ORFs of any one of claims 3 to 5 which are under the control of their natural regulatory elements or under the control of analogues to such natural regulatory elements.

7. Intergenic sequences derivable from the nucleotide sequence of claim 1 or claim 2.

8. The intergenic sequences of claim 7 which are regulatory DNA sequences or repeated elements.

9. The intergenic sequences of claim 7 which are ORF-fragments.

10. Mobile elements (insertion elements or mosaic elements) derivable from the nucleotide sequence of claim 1 or claim 2.

11. Proteins expressible from the nucleotide sequences or ORFs of any one of claims 1 to 6.

12. Use of the nucleotide sequences or ORFs of any one of claims 1 to 10 or the proteins of claim 11 in the analysis of the structure, organisation or dynamics of other genomes.

13. Use of the nucleotide sequences or ORFs of any one of claims 1 to 10 or the proteins of claim 11 in: (a) screening nucleotide sequences, (b) subcloning nucleotide sequences, (c) amplifying nucleotide sequences by PCR, or (d) gene trapping.

14. Use according to claim 13, wherein said nucleotide sequences are coding sequences or non-coding sequences.

15. Use according to claim 14, wherein said coding sequences are regulatory sequences, repeated elements, mosaic sequences or insertion elements.

16. Use according to any one of claims 12 to 15, wherein said nucleotide sequences or ORFs are oligonucleotide primers or hybridization probes.

17. Use of the nucleotide sequences, individual ORFs or groups of ORFs of any one of claims 1 to 10 or the proteins of claim 11 in: (a) the identification and classification of organisms and their genetic information, (b) the identification and characterisation of nucleotide sequences, amino acid sequences or proteins, (c) the transportation of compounds to and from an organism which is host to at least one of said nucleotide sequences, ORFs or proteins, (d) the degradation and/or metabolism of organic, inorganic, natural or xenobiotic substances in a host organism, or (e) the modification of the host-range, nitrogen fixation abilities, fitness or competitiveness of organisms.

18. The plasmid comprising the nucleotide sequence of claim 1 or claim 2.

19. A plasmid which harbours at least one ORF of any one of claims 1 to 10 or any degenerate variant thereof or which harbours at least one ORF or any degenerate variant thereof which encodes one or more of the proteins of claim 11.

20. The plasmid of claim 18 or claim 19 produced recombinantly.

21. The plasmid of any one of claims 18 to 20 or any variant thereof produced by mutation, deletion, insertion or inactivation of an ORF, ORFs or groups of ORFs.

22. Use of the plasmid of any one of claims 18 to 21 in: (a) obtaining a synthetic minimal set of ORFs required for functional Rhizobium-legume symbiosis, (b) the modification of the host-range of rhizobia, (c) the augmentation of the fitness or competitiveness of Rhizobium sp. NGR234 in the soil and its nodulation efficiency on host plants, (d) the introduction of desired phenotype(s) into host plants using said plasmid as a stable shuttle system for foreign DNA encoding said desired phenotype(s), or (e) the direct transfer of said plasmid into rhizobia or other microorganisms without using other vectors for mobilization.