Genetic snapshots of the Rhizobium species NGR234 genome

Abstract

Background:

In nitrate-poor soils, many leguminous plants form nitrogen-fixing symbioses with members of the bacterial family Rhizobiaceae. We selected Rhizobium sp. NGR234 for its exceptionally broad host range, which includes more than I 12 genera of legumes. Unlike the genome of Bradyrhizobium japonicum, which is composed of a single 8.7 Mb chromosome, that of NGR234 is partitioned into three replicons: a chromosome of about 3.5 Mb, a megaplasmid of more than 2 Mb (pNGR234b) and pNGR234a, a 536,165 bp plasmid that carries most of the genes required for symbioses with legumes. Symbiotic loci represent only a small portion of all the genes coded by rhizobial genomes, however. To rapidly characterize the two largest replicons of NGR234, the genome of strain ANU265 (a derivative strain cured of pNGR234a) was analyzed by shotgun sequencing.

Results:

Homology searches of public databases with 2,275 random sequences of strain ANU265 resulted in the identification of 1,130 putative protein-coding sequences, of which 922 (41%) could be classified into functional groups. In contrast to the 18% of insertion-like sequences (ISs) found on the symbiotic plasmid pNGR234a, only 2.2% of the shotgun sequences represent known ISs, suggesting that pNGR234a is enriched in such elements. Hybridization data also indicate that the density of known transposable elements is higher in pNGR234b (the megaplasmid) than on the chromosome. Rhizobium-specific intergenic mosaic elements (RIMEs) were found in 35 shotgun sequences, 6 of which carry RIME2 repeats previously thought to be present only in Rhizobium meliloti. As non-overlapping shotgun sequences together represent approximately 10% of ANU265 genome, the chromosome and megaplasmid may carry a total of over 200 RIMEs.

Conclusions:

'Skimming' the genome of Rhizobium sp. NGR234 sheds new light on the fine structure and evolution of its replicons, as well as on the integration of symbiotic functions in the genome of a soil bacterium. Although most putative coding sequences could be distributed into functional classes similar to those in Bacillus subtilis, functions related to transposable elements were more abundant in NGR234. In contrast to ISs that accumulated in pNGR234a and pNGR234b, the hundreds of RIME elements seem mostly attributes of the chromosome.

Background

Many different Gram-negative bacteria colonize the nutrient-rich rhizospheres of plant roots. Some bacteria are pathogenic, whereas others form beneficial associations. In nitrate-poor soils, strains of Azorhizobium, Bradyrhizobium, Mesorhizobium and Rhizobium (collectively known as rhizobia), form nitrogen-fixing symbioses with leguminous plants. In compatible interactions, invading rhizobia penetrate their hosts through infection threads, which develop centripetally. At the same time, new structures called nodules develop from meristems induced in the cortex of infected roots. When infection threads reach nodule cells, rhizobia are released as symbiosomes into the cytoplasm of infected cells where they eventually enlarge and differentiate into nitrogen-fixing bacteroids. Continuous exchange of chemical signals between the two symbionts coordinates expression of bacterial and plant genes required for a symbiotic development. Flavonoids released by legume roots are amongst the first signals exchanged in this molecular dialog. By interacting with rhizobial regulators of the NodD family, flavonoids trigger the expression of nodulation genes (nod, noe and nol). In turn, most nodulation genes participate in the synthesis and secretion of a family of lipochito-oligosaccharide molecules, the Nod factors that are required for bacterial entry into root hairs. Little is known about how rhizobia migrate inside the infection threads, although it seems likely that genetic determinants of both partners are again involved (see [1,2]). Once within the cortex, the rhizobia differentiate into bacteroids where low free-oxygen tensions help coordinate the expression of genes involved in nitrogen fixation (nif and fix) [3].

Taxonomic proposals based on DNA sequences of highly conserved genes indicate that rhizobia are a group of genetically diverse soil bacteria [4]. Other data suggest that in populations of soil bacteria, natural genetic mechanisms exist which can transform isolates with widely different chromosomal backgrounds into nodulating bacteria (that is, rhizobia) (for review see [1]). Comparisons of genomes of soil bacteria will help define the pools of symbiotic genes. Unfortunately, genomic studies of this kind have been hindered by the relatively large size of rhizobial genomes (6.5 to 8.7 Mb for R. meliloti and B. japonicum, respectively). Instead, as many symbiotic loci are often clustered on large plasmids in Rhizobium strains, or in chromosomal 'symbiotic islands' as in B. japonicum [5] and M. loti [6], physical and genetic analyzes of symbiotic plasmids or 'islands' prevailed. Rhizobium sp. NGR234 was selected for its exceptionally broad host range, which includes more than 112 genera of legumes in addition to the non-legume Parasponia andersonii [7,8]. As in R. meliloti, the genome of NGR234 is partitioned into three replicons, a chromosome of about 3.5 Mb, a megaplasmid of more than 2 Mb (pNGR234b) and pNGR234a, a 536 kb symbiotic plasmid [9,10,11]. Although various experiments have shown that most symbiotic genes are amongst the 416 open reading frames (ORFs) identified in the complete sequence of pNGR234a [9,12,13], others are carried by the chromosome and/or the mega-plasmid [10,14].

Many ways of finding genes exist, but with the rapid advances in genomics, among the most effective are those that involve sequencing parts of or entire genomes. Although contiguous sequences of several symbiotic islands/plasmids will be released in the near future, R. meliloti strain 1021 as well as the phytopathogens Ralstonia solanacearum and Xanthomonas citri are the only plant-interacting microbes currently being sequenced [15,16,17]. The cost of sequencing a complete genome is still well beyond the capability of most laboratories, however. Nevertheless, extensive information on the structure and content of genomes can be gained by randomly sequencing libraries made from total DNA [18,19,20,21]. Here, we have used this approach to analyze the megaplasmid and chromosome of NGR234. A total of 2,275 individual shotgun sequences of ANU265 (a derivative strain of NGR234 cured of its symbiotic plasmid [22]) were searched for protein and/or DNA homologies, and putative coding sequences were grouped into 28 classes according to their putative function. In addition, clones carrying various Rhizobium-specific repeated elements such as RIME1 and RIME2 were also analyzed.

Results and discussion

Random sequencing of the ANU265 genome

Total genomic DNA of ANU265 was used to construct an M13 library with inserts ranging in size from 0.9 to 1.5 kb. Of the 2,856 random clones analyzed, 80% (2,275) produced high-quality DNA sequence with an average read length of 253 bp (Table 1). In this way, more than 575 kb of total nucleotide sequence was collected, which corresponds to approximately 10% of the ANU265 genome [11]. At 61.2 mol%, the mean G+C content of these sequences is similar to that found for the entire genome [23], but is also significantly higher than the value of 58.5 mol% calculated for pNGR234a [9]. This pool of 2,275 sequences was then screened for redundancy. A total of 381 overlapping sequences were identified, and grouped into 195 contigs (sets of overlapping sequences) of two to four elements each: 154 contigs represent pairs of clones, whereas the remaining 73 sequences belong to 23 groups of three elements and one of four clones. Because of the many highly conserved sequences repeated throughout the NGR234 genome [9,11,24], it was not possible to determine if overlapping clones represent contiguous sequences or DNA fragments from distinct repeats. Nevertheless, truly unique sequences represent 92% of the total number of clones. With an average insert size of 1.2 kb, clones tagged with non-overlapping sequences represent more than 40% (2.5 Mbp) of the ANU265 genome.

Table 1.

Major characteristics of the ANU265 shotgun library

M13 insert size (range in bp)		900-1,500
Number of forward-sequencing reactions (M13 primers)		2,856
Number of sequences stored in database		2,275
Average length of the edited reads (bp)		253
Homology searches
Total number of sequences	2,275	(100%)
Sequences matching:
rDNA	3	(0.1%)
tRNA	4	(0.1%)
repeated sequences / intergenic elements only	29	(1.3%)
protein-coding-genes of: known function	922	(40.5%)
unknown function	208	(9.0%)
No database match (pioneer sequences)	1,109	(49.0%)

RIME- and IS-like sequences

Homology searches against nucleotide databases (BLASTN [25]) showed that 35 ANU265 sequences carried Rhizobium-specific intergenic mosaic elements (RIMEs). First identified in R. meliloti, R. leguminosarum bv. viciae and NGR234, RIME1 elements are 108 bp repeats characterized by two large palindromes, whereas RIME2 sequences are 109 bp repeats thought to be present only in R. meliloti [26]. RIMEs have many features of the short interspersed repeated elements that are non-coding, intercistronic sequences of less than 200 bp found in many prokaryotic genomes [27]. Of the 2,275 shotgun sequences of ANU265 collected, 29 contained RIME1 elements and 6 carried RIME2 repeats. Although Southern hybridizations indicated that approximately 20 copies of RIME1 were present in the genomes of. R. meliloti and NGR234 [26], our data indicate that there are many more. Among the 29 clones with RIME1 sequences, most (23) carry repeats that are very similar to the consensus ([26] and Figure 1). In another six (Figure 1, clones 27d06, 29g08, 0lf01, 11b07, 25e07 and 13c06), only one of the two large palindromic structures is conserved, however. This suggests that, in some cases, individual palindromes constitute independent repeats, not necessarily associated to form RIME1 elements. In the eight clones that code for putative proteins (Figure 1), RIME1 sequences are found immediately downstream of predicted ORFs (data not shown), indicating that these elements are probably confined to intergenic regions. Surprisingly, no RIME2 and a single RIME1 repeat were found on pNGR234a [9,11]. If these elements were regularly distributed throughout the NGR234 genome, more than a single RIME1 would have been expected on the 536 kb of pNGR234a. Thus, current data suggest that RIMEs preferentially accumulate on specific replicons, and that NGR234 carries possibly as many as 200 RIME-like elements.

Figure 1.

ANU265 clones that carry RIME1 repeats. ANU265 clones are numbered in bold in the first column and the RIME1 repeat consensus sequence is shown in bold on top row of the alignment. Positions in the ANU265 sequences of the initial and final bases in each alignment are given in the 5' and 3' columns, respectively. Partial alignments represent RIME repeats covering either the end (filled diamonds) or the beginning (filled triangle) of the established sequence. The two palindromic structures of RIME1 are shaded in gray. Internal inverted repeats are shown in matching colors. Gaps (marked with red hyphens) and single-nucleotide deletions (inverted red triangles) were introduced for optimal alignment. Base mismatches are colored in red. Arrows mark clones that encode putative proteins. N, any base; Y indicates C or T, R indicates A or G.

In contrast to pNGR234a, which carries many IS sequences, only 2.2% (51) of the 2,275 ANU265 sequences were predicted to encode transposon-related functions. Although several clones that did not match database homologs may also carry sequences of yet uncharacterized IS elements, these results suggest that in proportion to their size, chromosome and megaplasmid carry fewer transposable elements than pNGR234a. Nevertheless most of the 51 clones (70%) matched ISs that were first identified in pNGR234a [9]. For example, ten sequences highly homologous to NGRIS-4 were found. This 3,316 bp element is duplicated in pNGR234a [9], whereas chromosome and megaplasmid carry two and five copies of NGRIS-4 respectively [11,24].

Identification of putative genes

To assign putative functions to the cloned DNA fragments, sequences were compared to protein and nucleotide databases [25,28]. BLAST analyses showed that about 50% (1,130) of the 2,275 sequences matched protein-coding ORFs, three were homologous to rDNA and four to tRNA loci (see Table 1). Of the 1,130 putative protein-coding sequences, 208 (or 9% of the 2,275 sequences) were similar to hypothetical genes with no known function (pioneer sequences) of rhizobia and other organisms. Thus, together with the 1,109 clones which showed no significant similarity to entries in nucleotide and amino-acid databases (see Table 1), functions could not be assigned to 58% of the shotgun sequences. To provide an overview of the genetic organization of the ANU265 genome, predicted protein-coding sequences were grouped into various classes according to their putative function (Table 2).

Table 2.

Comparison of sequences encoding probable cellular functions of Rhizobium sp. NGR234 strain ANU265 with functional classes of proteins of Bacillus subtilis

Functional categories	ANU265		B. subtilis*
Cell envelope and cellular processes
Cell wall	17	(1.9)	93	(3.9)
Transport/binding proteins and lipoproteins	184	(20.0)	381	(16.0)
Sensors (signal transduction)	21	(2.3)	38	(1.6)
Membrane bioenergetics (electron transport and ATP synthase)	49	(5.3)	78	(3.3)
Surface polysaccharides biosynthesis and export	25	(2.7)	16	(0.7)
Sporulation	1	(0.1)	139	(5.8)
Germination/transformation	0		43	(1.8)
Mobility and chemotaxis	26	(2.8)	55	(2.3)
Cell division	5	(0.5)	21	(0.9)
Protein secretion	13	(1.4)	18	(0.8)
Chaperones/heat-shock proteins	12	(1.3)	15	(0.6)
Cell death	8	(0.9)	5	(0.2)
Intermediary metabolism
Carbohydrates and related molecules	69	(7.5)	261	(11.0)
Amino acids and related molecules	91	(9.9)	202	(8.5)
Nucleotides and nucleic acids	11	(1.2)	83	(3.5)
Lipids	19	(2.1)	77	(3.2)
Cofactors/prosthetic groups	37	(4.0)	99	(4.2)
Phosphate	3	(0.3)	9	(0.4)
Opine-like compounds	8	(0.9)	3	(0.1)
Sulphur	2	(0.2)	8	(0.3)
Information pathways
DNA replication, restriction, modification and repair	26	(2.8)	61	(2.6)
DNA segregation, recombination and transfer	10	(1.1)	27	(1.1)
RNA synthesis and modification	19	(2.1)	50	(2.1)
Protein synthesis and modification	63	(6.8)	123	(5.2)
Regulatory functions	68	(7.4)	213	(8.9)
Other categories
Adaptation to atypical conditions and protection	27	(2.9)	147	(6.2)
Transposon-related functions	51	(5.5)	10	(0.4)
Phage-related functions	5	(0.5)	83	(3.5)
Miscellaneous	52	(5.6)	21	(0.9)
Total	922	(100)	2,379	(100)

^*The functional classification of the B. subtilis protein-coding genes was adapted from Kunst et al. [29]. The number of sequences and of genes in each category is listed for ANU265 and B. subtilis, respectively. The percent of the putatively identified genes devoted to each functional group is indicated in brackets.

A genetic snapshot of the ANU265 genome

In total, 922 of the 2,275 sequences were grouped into 28 functional categories (Table 2). Interestingly, comparison of this data with that derived from the complete sequence of the Bacillus subtilis genome [29] showed a similar distribution of genes in both organisms. Although B. subtilis is a Gram-positive bacterium, it is commonly found in soil, water sources and in associations with plants. Thus, with the exception of one homolog of a sporulation gene (which was not expected in rhizobia), the comparative analysis presented in Table 2 suggests that the number of shotgun sequences is probably sufficiently large to form a representative selection of ANU265 loci. All 1,130 sequences for which significant matches were found in database searches are classified by function in Table 3.

Table 3.

Classification of putative protein-coding genes of Rhizobium sp. NGR234 cured of its symbiotic plasmid (= ANU265)

Clone		Homolog description	Clone		Homolog description
No.	Name		No.	Name
Cell envelope and cellular processes			53	26d04	sugar transp. ATP-binding prot.
Cell wall			54	26h11	sugar transp. ATP-binding prot.
1	01d07	N-acetylmuramoyl-L-alanine amidase	55	05b03	sugar transp. system permease prot.
2	06g04	N-acetylglucosamine-1-phosphate uridyl Tase	56	30b08	sugar transp. system permease prot.
3	26b06	UDP-N-acetylenolpyruvoylglucosamine RDase MurB	57	26a07	sugar ABC transp., ATP-binding prot.
4	28f02	UDP-N-acetylmuramate-alanine ligase MurC	58	14e06	xylose transp. permease prot.
5	22b10	UDP-N-acetylmuramoylalanine-D-glutamate ligase MurD	59	03f04	xylose transp. permease prot.
6	29g07	UDP-N-acetylmuramyl-tripeptide synthetase MurE	60	19a04	maltose binding prot.
7	08h05	UDP-N-acetylmuramyl-tripeptide synthetase MurE	61	11c09	membrane bound sugar transp. prot.
8	12f10	outer membrane prot.	62	18a12	sugar transp. ATP-binding prot.
9	17a08	outer membrane prot. Omp28	63	28e01	sugar transp. ATP-binding prot.
10	21h07	group 1 outer membrane prot. OMP1 precursor60	64	30f09	sugar transp. prot.
11	18f12	penicillin-binding prot. 1B	65	01c10	galactoside transp. system permease prot.
12	19h11	penicillin-binding prot. 1A	66	19g07	galactoside transp. ATP-binding prot.
13	29f10	D-alanyl-D-alanine carboxypeptidase	67	21e06	branched-chain amino acid transp.
14	19d01	monofunctional biosynthetic peptidoglycan TGase MtgA	68	29a02	amino-acid ABC transp. permease prot.
15	28f09	lysozyme M1 precursor Acm	69	27b03	amino-acid ABC transp. permease prot.
16	03f07	acriflavine resistance prot. E precursor AcrE	70	05c04	ABC transp. permease prot.
16a	09h01	overlaps clone 03f07	71	07b05	amino-acid ABC transp. ATP-binding
Transport/ binding proteins and lipoproteins			72	19h08	amino-acid ABC transp. ATP-binding
17	22a03	sugar-binding transp. ATP-binding prot.	73	06a09	amino-acid ABC transp. ATP-binding
18	08h08	ABC transp. ATP-binding prot.	74	11d03	glutamate/ aspartate transp. system permease prot.
19	29c10	sugar ABC transp., permease prot.	75	21d10	high-affinity branched-chain amino acid transp.
20	03f05	inner membrane prot.of trehalose/ maltose transp.	75a	24a05	overlaps clone 21d10
21	02h10	transp. permease prot.	76	02c01	amino acid ABC transp.
21a	09f05	overlaps clone 02h10	77	08f08	branched-chain amino acid transp. prot.
22	11c04	ABC transp. permease prot.	78	10e04	branched-chain amino acid transp. prot.
23	12d03	inner membrane ABC transp.	79	17b01	branched-chain amino acid transp. prot.
24	18h08	sugar ABC transp. ATP-binding prot.	80	08g08	branched-chain amino acid transp. prot.
25	21b03	ATP-binding transp. prot.	81	04g02	branched-chain amino acid transp. permease prot.
26	01h04	ATP-binding transp. prot.	82	28h02	high-affinity branched-chain amino acid transp.
27	21b11	ATP-binding transp. prot.	82a	08a04	overlaps clone 28h02
28	26g01	maltose/ maltodextrin transp. ATP-binding prot.	82b	11g01	overlaps clone 28h02
29	18g11	sugar ABC transp. ATP-binding prot.	83	16c12	periplasmic dipeptide transp. prot. precursor
30	24e03	ABC transp. ATP-binding prot.	84	03h07	dipeptide ABC transp.
31	02c09	ABC transp. ATP-binding prot.	85	10d11	peptide ABC transp. permease prot.
31a	12f05	overlaps clone 02c09	86	15e08	ABC transp. ATP-binding prot.
32	28g12	ABC transp. ATP-binding prot.	87	04a06	peptide ATP-bind. transp.
32a	01a11	overlaps clone 28g12	88	09a09	peptide ABC transp. permease prot.
32b	09h06	overlaps clone 28g12	89	12c06	peptide ABC transp. permease prot.
33	01a10	ribose transp. ATP-binding prot.	90	03f10	ABC transp.
34	06h03	D-ribose-binding periplasmic prot. precursor	91	22c01	ABC transp. ATP-binding prot.
35	14e04	sugar transp. system permease prot.	92	19b12	ABC transp. ATP-binding prot.
36	18d02	sugar transp. system permease prot.	93	23g09	peptide ABC transp. ATP-binding prot.
37	04e03	sugar transp. system permease prot.	94	18e05	ABC transp. ATP-binding prot.
38	14f09	sugar transp. system permease prot.	95	14d12	oligopeptide ABC transp.
39	08g05	sugar transp. system permease prot.	96	19c05	oligopeptide binding prot.
40	06b09	sugar transp. system permease prot.	97	21g02	peptide ABC transp.
41	19e12	sugar transp. system permease prot.	98	22h09	dipeptide transp. ATP-binding prot.
42	16f12	membrane-spanning permease	99	25c02	oligopeptide transp. ATP-binding prot.
43	26d10	sugar transp. system permease prot.	99a	23c05	overlaps clone 99
44	23b06	sugar transp. system permease prot.	100	27b09	oligopeptide ABC transp. permease prot.
45	27h12	ABC transp. integral membrane prot.	101	27c09	oligopeptide-binding prot. precursor
46	22d10	ribose ABC transp. permease prot.	102	30a09	oligopeptide transp. ATP-binding prot
47	25a11	sugar transp.	103	03b02	ABC transp., y4wM pNGR234a
48	21a06	sugar transp. ATP-binding prot.	104	03g02	ABC transp., y4wM pNGR234a
49	21b02	sugar transp. ATP-binding prot.	105	07c06	ABC transp., y4wM pNGR234a
50	24d10	galactoside transp. ATP-binding prot. MglA	106	30e02	ATP-binding prot.
51	24e10	lactose transp. system permease prot. LacF	107	05c03	ATP-binding prot.
52	11f10	sugar transp. prot.	108	20e03	ATP-binding prot.
109	19f02	ABC transp. ATP-binding prot.	164	01h09	L-asparagine permease AnsP
110	19d07	ABC transp. ATP-binding prot.	165	29d05	C4-dicarboxylate transp. prot. DctA1 pNGR234a
111	16a07	ATP-dependent transp.	166	20a10	C4-dicarboxylate transp. prot. DctA1 pNGR234a
112	17f05	ABC transp. ATP-binding prot.	167	20c09	chelated iron ABCtransp. ATP-binding prot.
113	09d03	ABC transp. ATP-binding prot.	168	29f01	chelated iron ABCtransp. ATP-binding prot.
114	17h02	putrescine transp. system permease prot.	169	03c12	chelated iron transp. system membrane prot.
115	22e01	inner membrane prot.	170	19a03	chelated iron transp. system membrane prot.
116	02g07	spermidine/ putrescine transmembrane prot.	171	11d07	chelated iron transp. system membrane prot.
116a	06d04	overlaps clone 02g07	172	26g08	iron transp. prot.
116b	24b12	overlaps clone 06d04	173	20e11	phosphoenolpyruvate-prot. phosphoTase
117	13b12	putrescine transp. prot.	174	20e12	Na+/H+-exchanging prot. system component
118	06d07	putrescine transp. permease prot.	175	22b06	mannopine-binding periplasmic prot. motA
119	09b07	putrescine transp. permease prot.	175a	21g06	overlaps clone 22b06
119a	06b04	overlaps clone 09b07	176	21h10	sulfate transp. system permease prot.
120	24a01	glycine betaine transp. system permease prot	177	29h02	taurin-binding periplasmic prot
121	24f03	glycine betaine transp. system permease prot	178	22d09	cytoplasmic membrane prot. CeoB
122	28c03	glycine betaine / proline transp. prot. ProV	179	28f12	integral membrane prot. (sodium:sulfate symporter)
123	10h02	inner membrane prot.	180	23g03	sulphate transp. system permease prot. CysT
124	03b03	aquaporin Z (bacterial nodulin-like intrinsic prot.)	181	25h07	transp. prot., y4xM pNGR234a
125	03c04	arginine / ornithine antiporter	182	24h07	periplasmic binding prot.
126	03e12	glycerol-3-phosphate-binding periplasmic prot.	183	08c01	lipoprot. LppB/NlpD
127	22g07	glycerol-3-phosphate transp. prot.	183a	10c07	overlaps clone 08c01
128	05a06	acriflavine resistance lipoprot. A precursor	184	24f12	lipoprot.
129	29h11	acriflavine resistance prot. B	185	23h12	lipoprot.
129a	15d06	overlaps clone 29h11	186	06a01	outer membrane lipoprot.
130	14b09	acriflavine resistance prot.	186a	11a05	overlaps clone 06a01
131	14c06	antibiotic resistance prot	186b	21d02	overlaps clone 11a05
131a	05c12	overlaps clone 14c06
132	04d08	Leu/ Ile/ Val/ (Thr/Ala)-binding prot. precursor	Sensors (signal transduction)
133	07e02	cytoplasmic prot. CeoB	187	12f09	sensor histidine kinase ExsG
134	01c07	NolH (AcrB/AcrD/AcrF family prot.)	187a	15d09	overlaps clone 12f09
135	27a10	FixI; E1-E2 type cation ATPase	188	18f03	sensor histidine kinase ExsG
136	12b12	heavy-metal transp.ing P-type ATPase	189	06a07	sensor prot. TctD
137	29f07	cation-transp. ATPase PacS	190	16d05	sensor prot. for potassium transp. KdpD
138	11e02	H+/Ca2+ exchanger	191	06d10	sensor prot. for potassium transp. KdpD
139	01g05	tonB-dependent outer membrane heme receptor HemR	191a	25f03	overlaps clone 06d10
140	02b10	inner membrane prot., energy transducer TonB	191b	26d08	overlaps clone 06d10
141	27h11	TonB-dependent transp. ExbD	192	09c11	two-component sensor histidine kinase
142	02b11	nitrite extrusion prot.	192a	26a04	overlaps clone 09c11
143	08f10	nitrate transp. permease prot. nrtB	193	10f06	C4-dicarboxylate sensor prot. DctB
144	16d07	nitrate transp. prot. NrtD	194	13b09	C4-dicarboxylate sensor prot. DctB
145	09g09	phosphate transp. prot. PhoE	195	14c01	sensor of two-component system FlhS
146	27h09	phosphate transp. prot. PhoT	196	01g04	sensor of two-component system FlhS
146a	11g03	overlaps clone 27h09	197	15f11	prokaryotic sensory transduction prot.
147	17e11	phosphate transp. prot. PhoT	198	15g02	sensory transduction histidine kinase
148	17c11	phosphate transp. prot. Pit	199	19a06	sensory transduction histidine kinase
149	21a10	phosphate transp. prot. Pit linked to RIME 2	199a	22d04	overlaps clone 19a06
150	04d06	Pit accessory protein orfA	200	22g10	histidine kinase sensory prot. ExoS
151	12d06	macrolide-efflux determinant	201	23e05	histidine prot. kinase ActS
152	13d04	cation efflux system prot.	202	29f03	sensor kinase NwsA
153	17e08	cation efflux system prot.
154	21e03	ferric siderophore receptor	Membrane bioenergetics (electron transport, etc)
154a	14c02	overlaps clone 21e03	203	09f04	pyridine nucleotide transhydrogenase sub. α PntA
155	29b02	ferric siderophore receptor	204	02h05	pyridine nucleotide transhydrogenase sub. α PntA
155a	18g03	overlaps clone 29b02	205	09d11	pyridine nucleotide transhydrogenase sub. α PntA
156	14e12	potassium uptake prot. Kup	206	20h04	pyridine nucleotide transhydrogenase sub. β PntB
157	14f07	phosphoenolpyruvate-prot. phosphoTase	207	25g07	pyridine nucleotide transhydrogenase sub. β PntB
158	15f09	ABC transp. ATP-binding prot.	208	13b05	FixN cytochrome CBB3 sub. 1
159	16d04	molybdenum transp. prot.	209	01b04	FixN cytochrome CBB3 sub. 1
160	16g11	periplasmic sulphate binding prot. Sbp	210	08a07	FixS cbb3-type cytochrome oxidase formation prot.
161	04f12	periplasmic sulphate binding prot. Sbp	211	24d07	cytochrome-c oxidase chain IIIB CoxP
162	18b09	drug efflux pump (AcrB/AcrD/AcrF family)	212	05f03	cytochrome BB3 sub. 1 CoxN
163	18d12	tartrate transp. TtuB	212a	03h10	overlaps clone 05f03
212b	12g07	overlaps clone 05f03	268	13h11	capsular polysaccharide biosynthesis prot.
213	05f06	cytochrome C oxidase assembly prot. CoxZ	269	08c05	spore coat polysaccharide biosynthesis prot.E
214	05e03	cytochrome C-type biogenesis prot. CycJ/K	270	14d02	β-(1,2)-glucans production inner-membrane prot. NdvB
215	06d08	cytochrome C-type biogenesis prot. CycH
216	11f02	cytochrome c-type biogenesis prot. CcdA	Mobility / chemotaxis
217	12e03	cytochrome oxidase δ, sub. II	271	17a10	(MCP)-glutamate methylesterase CheB
218	11g05	ubiquinol-cytochrome C RDase iron-sulfur sub.	272	05c06	flagellar basal-body (proximal) rod prot. FlgB
219	15e06	cytochrome o ubiquinol oxidase sub. III	273	29g09	flagellar biosynthetic prot. FlhB
220	29e05	cytochrome c small sub. of nitric oxide RDase	273a	08g10	overlaps clone 29g09
221	06h06	glycolate oxidase iron-sulfur sub. (Fe-S prot.)	273b	24g01	overlaps clone 08g10
221a	07e07	overlaps clone 06h06	274	13b08	flagellar biosynthetic prot. FliQ
222	08h10	ATP synthase α-chain	275	12f06	flagellar basal-body MS-ring prot. FliF
222a	14d10	overlaps clone 08h10	276	14e10	flagellar hook prot. FlgE
222b	22c06	overlaps clone 14d10	277	14f11	flagellar basal-body (distal) rod prot. FlgG
223	17f07	ATP synthase γ-chain	278	17c05	flagellar C-ring prot. FliG
224	09a12	NADH-ubiquinone oxidoRDase (CI-40 kDa)	279	26d03	flagellar biosynthetic prot. FliP
225	09h02	NADH-ubiquinone oxidoRDase (CI-51kDa)	280	18g10	flagellar basal-body rod prot. FlgF
226	13d12	cyanide insensitive terminal oxidase CioAB	281	27a08	flagellin prot. FlaD
227	01g02	NADH DHase (ubiquinone), sub. 1	282	20h07	new class of flagellar prot. FlmD
228	19b04	NADH DHase	282a	19b08	overlaps clone 20h07
229	12a01	NADH ubiquinone oxidoRDase	282b	30a08	overlaps clone 20h07
230	20c11	NADH ubiquinone oxidoRDase	283	07h07	chemotactic transducer for amino acids
231	06e08	NADH ubiquinone oxidorRDase	284	13g03	methyl-accepting chemotaxis prot
232	26e10	NADH ubiquinone oxidoRDase	285	11b02	methyl-accepting chemotaxis prot
233	08g12	NADH ubiquinone oxidoRDase	286	11c06	methyl-accepting chemotaxis prot
234	21h02	NADH ubiquinone oxidoRDase	287	15b10	methyl-accepting chemotaxis prot
235	22a06	NADH ubiquinone oxidoRDase	288	16b03	methyl-accepting chemotaxis prot
236	27c06	NADH ubiquinone oxidoRDase	289	16f10	methyl-accepting chemotaxis prot
237	24b11	electron transfer flavoprotein-ubiquinone oxidoRDase	290	28h04	methyl-accepting chemotaxis prot
238	25a08	glutathione RDase	291	28a12	methyl-accepting chemotaxis prot
239	01h05	thioredoxin	292	27f01	chemotaxis prot. methylTase CheR
240	29a01	thioredoxin RDase	292a	15e07	overlaps clone 27f01
241	16d03	ferredoxin [2Fe-2S] II	Cell division
242	19f07	ferredoxin-type prot. [4Fe-4S]	293	05f02	cell division prot. FtsH
243	09c06	ferredoxin [3Fe-4S] [4Fe-4S]	294	08d04	cell division prot. FstK
244	26a10	ferredoxin, [2Fe-2S]	295	16b04	cell division prot. FtsK
244a	05c08	overlaps clone 26a10	296	16e07	septum formation prot. Maf
244b	18a04	overlaps clone 26a10	297	25f06	cell division inhibitor MinC
Surface polysaccharide - biosynthesis and export			Protein secretion
245	01c04	ExoB UDP-galactose 4-epimerase	298	14c09	ABC transp. type I PrsD
246	03a05	ExoB UDP-galactose 4-epimerase	298a	06a04	overlaps clone 14c09
247	06a08	ExoN UDP-glucose pyrophosphorylase	299	24e06	ABC transp. type I PrsD
248	07g05	ExoN UDP-glucose pyrophosphorylase	300	11h03	membrane fusion prot. type I PrsE
249	10e08	ExoF exopolysaccharide production prot. precursor	301	29e03	membrane fusion prot. type I PrsE
250	10c12	ExoK endo-β-1,3-1,4-glucanase	302	06h10	general secretion pathway prot. D precursor XpsD
251	18b08	ExoP succinoglycan transp. prot.	302a	03e03	overlaps clone 06h10
252	22e05	ExoU succinoglycan biosynthesis glycosylTase	303	12h06	general secretion prot. F XcpS
253	27h01	ExoU succinoglycan biosynthesis glycosylTase	303a	10c03	overlaps clone 12h06
254	18h12	ExoY exopolysaccharide production prot.	304	29d12	general secretion prot. D GspD
255	25h10	ExoL succinoglycan biosynthesis glycosylTase	304a	12e11	overlaps clone 29d12
256	17b10	exopolysaccharide production prot. Pss	304b	10e05	overlaps clone 12e11
257	11a12	KPS production, fatty acid synthase RkpC	305	22g03	general secretion prot. E GspE
258	27f08	KPS modification / export prot. RkpI	306	28g09	pNGR234a, probable translocation prot. RhcT
259	11d09	KPS modification / export prot. RkpJ	307	29b12	preprotein translocase SecA sub.
260	17h04	polysialic acid transp. prot. KpsM
261	21b04	specialized small acyl carrier prot. (lipid A)	Chaperones
262	10c11	N-acetylglucosamine deacetylase (lipid A)	308	10d03	heat shock prot. cnp60 GroEL
263	10b05	3-deoxy-D-manno-octulosonic-acid (Kdo) Tase KdtA	309	21a09	heat shock prot. cnp60 GroEL
264	18f02	3-deoxy-D-manno-octulosonic-acid (Kdo) Tase KdtA	310	26d02	heat shock prot. cnp10 A GroES
265	26h01	3-deoxy-manno-octulosonate cytidylylTase KpsU	311	21a03	heat shock prot. cnp60 GroEL
266	12c02	membrane bound galactosylTase RfpB	312	07c04	small heat shock prot. HspF
267	08h09	O-antigen acetylase	313	14e03	small heat shock prot. (hsp20-2)
313a	18g01	overlaps clone 14e03	365	21f02	mannonate DTase
314	28f11	18 kd antigen2 (small heat shock prot. Hsp20 family)	366	16g05	alcohol DHase
315	15g09	small heat shock prot. HspE	367	17c02	phosphomannomutase AlgC
316	17b02	DnaJ-like heat shock chaperone prot.	368	17f03	glycogen phosphorylase
317	01e06	heat shock prot. 90 HtpG	369	18d11	phosphoglucomutase
318	23g01	heat shock prot. X HtpX	370	18f01	L-ribulose-P-4-epimerase (AraD/FucA family)
Cell death			371	18h09	triosephosphate isomerase
319	08h11	hemolysin-like prot. TlyC	372	19d03	starch (bacterial glycogen) synthase
319a	05b05	overlaps clone 08h11	373	20d06	zinc-type alcohol DHase-like prot
320	04h12	cyclolysin (haemolysin-adenylate cyclase toxin)	374	20d08	glutathione-dependent formaldehyde DHase
321	22b07	cyclolysin (haemolysin-adenylate cyclase toxin)	375	20e05	succinate DHase (iron-sulfur prot.)
322	10b08	cyclolysin (haemolysin-adenylate cyclase toxin)	376	20h10	tartrate DHase
323	21g08	cyclolysin (haemolysin-adenylate cyclase toxin)	377	21b06	lactaldehyde DHase
324	12b07	iron-regulated prot. (cytotoxins Ca2+ binding domain)	378	02d12	D-lactate DHase
325	20c02	hemolysin	379	22b08	D-lactate DHase
			380	22c08	dihydrolipoamide acetylTase
Intermediary metabolism			381	28b09	dihydrolipoamide DHase
Metabolism of carbohydrates and related molecules			382	30a11	dihydrolipoamide DHase
326	01b09	glucose-6-phosphate isomerase	383	23f06	transketolase
327	24c09	glucose-6-phosphate isomerase	384	23h07	α-glucosidase
328	18g07	glucose-6-phosphate isomerase	385	28a07	D-mannonate oxidoreductase
328a	20e09	overlaps clone 18g07	386	28c08	glutathione-independent formaldehyde DHase
329	09a03	glyoxylate carboligase	387	29a04	y4uC, pNGR234a, aldehyde-DHase-like prot
330	01c12	α-ketoglutarate DHase	388	29h05	fumarate hydratase
330a	24d12	Overlaps clone 01c12	389	30b10	mannitol 2-DHase
331	16g02	acetoin:DCPIP oxidoRDase α	390	05d12	isocitrate lyase
332	02e09	acetoin:DCPIP oxidoRDase β	Metabolism of amino acids and related molecules
333	02e11	succinyl-coA synthetase β chain	391	27a11	α-isopropylmalate synthase LeuA
334	03e11	ribulose-bisphosphate carboxylase, large sub.	392	27d07	α-isopropylmalate synthase LeuA
335	03h09	citrate synthase	393	14d11	α-isopropylmalate synthase LeuA
336	05b01	L-xylulose kinase	394	14b07	3-isopropylmalate dehydratase small sub.LeuD
337	06c08	dihydoxyacetone kinase	395	25g10	aspartate ammonia-lyase AspA
338	18g08	dihydoxyacetone kinase	396	02c06	aspartate ATase (AspC family)
339	06g05	lipoamide DHase E3 subunit of α-ketoacid DHase complex.	397	02h01	5-methyltetrahydrofolate-homocysteine Tase MetH
340	04g03	alcohol DHase(acceptor) precursor	398	06d09	3-dehydroquinate synthase AroD
341	04h06	malate DHase	399	03c03	3-dehydroquinate synthase AroD
342	09d09	malate DHase	400	18c04	shikimate 5-dehydrogenase AroE
343	07e09	glycogen operon protein (glycosyl hydrolases family)	401	28a08	shikimate 5-dehydrogenase AroE
344	08b07	alcohol DHase	402	02h02	3-dehydroquinate DTase AroQ
345	08e03	2-hydroxyhepta-2,4-diene-1,7-dioate isomerase	402a	20f02	overlaps clone 02h02
346	13a05	glycolate oxidase sub	403	03b05	aspartate aminoTase B
347	09d08	glycolate oxidase sub	404	29b06	aspartate aminoTase B
348	10c06	acetyl-CoA synthetase	405	26d07	aspartate aminoTase
349	11b03	aconitate hydratase (citrate hydro-lyase)	406	11e06	aspartate aminoTase
350	25a09	aconitate hydratase (citrate hydro-lyase)	407	03e05	y4sL pNGR234a, D-amino-acid DHase
351	01b12	2-keto-3-deoxygluconokinase	408	03g10	adenylosuccinate Sase (IMP-aspartate ligase) PurA
352	15a09	ribitol kinase	409	04a04	glutamate 5 -kinase
353	11c02	glucose DHase (pyrroloquinoline-quinone)	410	05d06	N-acyl-L-aminoacid amidohydrolase (aminoacylase)
354	25b01	formaldehyde DHase (glutathione)	411	05e10	assimilatory nitrate RDase sub. NirB
354a	11c11	overlaps clone 25b01	411a	05h01	overlaps clone 05e10
355	09a10	β-glucosidase (cellulose degradation)	412	05g02	3-isopropylmalate DTase large sub. LeuC
356	07a05	β-glucosidase (cellulose degradation)	413	05g05	class III pyridoxal-phosphate-dependent ATase
357	18b12	β-glucosidase (cellulose degradation)	414	06a10	threonine deaminase IlvA
357a	12b03	overlaps clone 18b12	415	26f02	threonine deaminase IlvA
357b	07f11	overlaps 12b03	416	13g01	acetolactate Sase (acetohydroxy-acid Sase) IlvB
358	12c07	NADP-dependent malic enzyme	417	01b11	acetolactate Sase (acetohydroxy-acid Sase) IlvB
359	12g01	phosphogluconate DHase	418	08b10	dihydroxy-acid DTase IlvD
360	28c02	glutathione-dependent formaldehyde DHase	419	18c03	dihydroxy-acid DTase IlvD
361	04h08	glycerol-3-phosphate DHase	420	06b12	histidinol DHase HisD
362	14g01	glycerol-3-phosphate DHase	421	07a04	N-acetylornithine ATase
363	30f10	glycerol-3-phosphate DHase	422	07b06	low specificity D-threonine aldolase
364	15e12	dTDP-glucose 4-6-DTase	423	08f03	branched-chain α-keto acid DHase component E1
424	30a04	serine acetylTase (CysE/LacA/LpxA/NodL family)	480	12f08	adenylate kinase (ATP-AMP transphosphorylase)
424a	10c10	overlaps clone 30a04	480a	09b05	overlaps clone 12f08
425	10e02	anthranilate synthase (tryptophan biosynthesis) TrpE/G	481	11c12	deoxyuridine 5′ triphosphate nucleotidohydrolase
426	30e09	anthranilate synthase (tryptophan biosynthesis) TrpE/G	482	14g07	cytosine deaminase CodA
427	11e05	serine hydroxymethylTase GlyA	483	05e12	phosphoribosylformylglycinamidine synthase PurQ
428	29h12	tryptophan synthase TrpA	484	17b06	phosphoribosylformylglycinamidine synthase PurQ
429	11f03	homoserine DHase	485	15f05	formyltetrahydrofolate deformylase-like prot. PurU
430	11h02	5,10-methylenetetrahydrofolate RDase MetF	486	23e07	phosphoribosylformylglycinamidine PurL
430a	15a08	overlaps clone 11h02	487	29g10	thymidine kinase
431	11h04	proline DHase PutA
432	18d05	proline DHase PutA	Metabolism of lipids
433	25c05	proline DHase PutA	488	09a01	Nod Factor fatty acyl chain modification NodG
434	12b02	glutaryl-CoA DHase (acyl-coA DHase. family)	489	17c10	3-hydroxydecanoyl-acyl-carrier-prot. DTase FabA
435	12f03	glycine acetylTase	490	03c07	fatty oxidation complex α sub. FadB
436	13h10	homoserine DHase	491	05h03	3-oxoacyl-acyl-carrier-prot. synthase I FabB
437	14f01	ethanolamine ammonia-lyase heavy chain EutB	492	19g02	malonyl CoA-acyl carrier prot. transacylase FabD
438	15b01	2-oxoisovalerate DHase α sub.	493	10b03	3-oxoacyl-acyl carrier prot.synthase II FabF
439	15d01	methionine gamma-lyase Megl	494	22d03	3-oxoacyl-acyl carrier prot. synthase II FabF / NodE
440	19g12	methionine gamma-lyase MegL	495	15c04	3-oxoacyl-acyl-carrier-prot. synthase III FabH
441	16h06	4-hydroxyphenylpyruvate dioxygenase	496	30f01	enoyl-acyl-carrier-prot. Rdase (NADH) FabI
442	19g03	arginine deiminase ArcA	497	01c09	enoyl-CoA hydratase
443	29h06	arginine deiminase ArcA	498	05d10	3-hydroxyisobutyrate DHase
444	29c04	ornithine cyclodeaminase ArcB	499	10h05	long-chain-fatty-acid--CoA ligase RpfB
444a	17g07	overlaps clone 29c04	500	05g09	acyl-coA DHase
445	23c08	ornithine cyclodeaminase ArcB	501	15e01	acyl-coA DHase
446	18e07	hydroxypyruvate RDase	502	17h07	3-hydroxybutyryl-CoA DHase
447	19d12	asparagine synthetase	503	19c09	3-hydroxybutyryl-CoA DHase
448	19e03	agmatine ureohydrolase SpeB	504	21h04	sulfolipid biosynthesis prot. SqdA
449	19h06	alanine racemase	505	22d05	sub. A of acetyl-coA carboxylase
450	20d07	ornithine/acetylornithine aminoTase	506	01h11	acetyl-CoA carboxylTase β-sub.
451	21e02	urocanate hydratase HutU
452	21f08	adenosylhomocysteinase	Metabolism of cofactors / prosthetic groups
453	22d08	adenosylhomocysteinase	507	02c03	coenzyme PQQ synthesis prot. C
454	09b12	phosphoglycerate DHase SerA	508	05e02	coenzyme PQQ synthesis prot. E
455	25b12	D-3-phosphoglycerate DHase	508a	03a08	overlaps clone 05e02
456	22e04	aminotripeptidase PepT	509	03c06	DNA / panthotenate metabolism flavoprot.
457	22e08	4-hydroxybenzoate hydroxylase PobA	510	03e04	dihydroxybenzoate DHase EntA
458	22h10	chorismate mutase / prephenate dehydratase PheA	510a	10a12	overlaps clone 03e04
459	25c08	diaminopimelate decarboxylase LysA	511	03g11	glutathione Tase
460	06f04	sarcosine oxidase α SoxA	512	05b08	thiamine biosynthesis prot. ThiC
461	25e04	sarcosine oxidase α SoxA	513	03g09	thiamine biosynthesis prot. ThiG
462	01e05	sarcosine oxidase β SoxB	514	05b12	S-adenosylmethionine: 2-demethylmenaquinonemethylTase
463	03c10	sarcosine oxidase δ SoxD	515	12e09	cobyrinic acid a,c-diamide synthase CobB
464	21h08	sarcosine DHase	516	11b11	precorrin isomerase CobH
465	26a01	sarcosine DHase	517	02b03	cobalamin synthesis prot. CobN
465a	07f10	overlaps clone 26a01	518	05d05	cobalamin/porphyrin biosynthesis prot. CobS
466	21b09	ferredoxin-dependent glutamate Sase GltB	519	28e08	cobalamin synthesis prot. CobT
467	24d04	NADH-glutamate synthase small sub. GltD	520	10d09	glutathione S-Tase Gst
468	13e08	NADPH dependent glutamate synthase small sub. GltX	521	21d03	glutathione synthetase GshB
469	01h06	glutamine synthetase II GlnII	521a	06b03	overlaps clone 21d03
470	26f03	dihydrodipicolinate synthase DapA	522	06e11	ferrochelatase (protoheme ferro-lyase) HemH
471	27g06	malyl-coA lyase	523	10f10	γ-glutamyltranspeptidase precursor
472	28b05	argininosuccinate synthase ArgG	524	10g02	NH (3)-dependent NAD+ Sase NadE
473	30a12	urease accessory prot. (UreD homolog)	525	11e08	riboflavin synthase, β sub. RibH
474	12h11	4-aminobutyrate aminoTase	526	13e09	pu. amino acid oxidase flavoprot. ThiO
475	30e05	w-aminoTase-like prot	527	13e11	1-deoxyxylulose-5-phosphate Sase
476	15h11	uridylyltransferase/uridylyl-removing enzyme GlnD	528	14d08	4-hydroxybenzoate octaprenylTase (polyprenylTase)
476a	08e06	overlaps clone 15h11	529	18a08	7,8-diamino-pelargonic acid ATase (DAPA ATase) BioA
477	01e08	hydantoin racemase HyuE	530	19b10	dihydroneopterin aldolase (DHNA) FolB
			531	14g12	porphobilinogen deaminase precursor HemC
Metabolism of nucleotides and nucleic acids			532	20a12	uroporphyrinogen decarboxylase HemE
478	02b08	uracil phosphoribosylTase Upp	533	30c03	NH(3)-dependent NAD(+) Sase NadE
479	02e04	formyltetrahydrofolate synthase FthfS	533a	26c07	overlaps clone 30c03
534	28d07	NH(3)-dependent NAD(+) Sase NadE	582b	24b02	overlaps clone 21d09
535	22a09	pyridoxal phosphate biosynthetic prot. PdxA	583	15d08	VirB4-like prot., sim. to TrbeB pNGR234a
536	06a02	pyridoxamine kinase	584	06h12	DNA- binding prot. HRm / HU (histone-like prot.)
537	24d01	glutamate 1-semialdehyde 2,1-aminomutase
538	24g03	coenzyme F390 synthetase II	RNA synthesis and modification
539	26a02	molybdopterin biosynthesis prot.	585	07b10	transcription elongation factor GreA
540	29e04	pantothenate synthetase PanC	586	27d08	transcription elongation factor GreA
			587	27e10	RNA polymerase α sub. RpoA
Metabolism of phosphate			588	17b03	ribonuclease HII RnhB
541	04f07	inorganic pyrophosphatase Ppa	589	02a01	RNA polymerase β sub RpoB
541a	29c05	overlaps clone 04f07	590	03e09	RNA polymerase β sub RpoB
542	25h01	phosphonate utilization PhnJ	591	06d05	RNA polymerase β sub RpoB
Metabolism of rhizopine			592	22h12	RNA polymerase β sub RpoB
543	05a11	MocA oxidoreductase	593	28d10	RNA polymerase β sub RpoB
544	07d03	MocB rhizopine-binding prot. precursor	594	16b02	RNA polymerase β ′ sub RpoC
545	15b08	MocB rhizopine-binding prot. precursor	595	04h05	RNA polymerase primary sigma-70 factor RpoD
546	19a12	MocB rhizopine-binding prot. precursor	596	03a11	RNA polymerase sigma-E factor SigH
547	15b06	MocC rhizopine catabolism	597	21b10	RNA polymerase sigma-E factor SigC
548	18c11	MosA rhizopine biosynthesis (dihydrodipicolinate Sase)	598	27f09	RNA polymerase sigma-32 factor RpoH
548a	04a05	overlaps clone 18c11	599	12c03	probable sigma factor SigI
549	11g06	MocB opine catabolism (phosphogluconate DTase)	600	25f08	probable sigma factor
			600a	25h04	overlaps clone 25f08
Metabolism of sulphur			601	27c10	transcription accessory prot. Tex
550	29b11	phospho-adenylylsulfate sulfoTase CysH	602	25d11	VacB ribonuclease II family
551	17a09	sulfite reductase (hemoprot. sub.) CysI	603	24a12	reverse transcriptase/maturase
Information pathways			Protein synthesis and modification
DNA replication, restriction, modification and repair			604	29h08	GTP-binding prot. (protease) HflX
552	02f12	ribonuclease H RnhA	605	01b01	GTP-binding prot. LepA
553	05e01	DNA polymerase III α sub. DnaE	605a	20h09	overlaps clone 01b01
554	28f04	DNA polymerase ε chain DnaQ	605b	25g09	overlaps clone 01b01
555	06d12	DNA polymerase III sub. gamma and tau DnaZX	606	01b02	alanyl-tRNA synthetase AlarS
556	08e04	DNA topoisomerase IV sub. A ParC	607	06a11	cystein- tRNA ligase CysS
557	11d02	primosomal replication factor Y PriA	608	26h03	glycyl tRNA-synthetase chain GlyQ
558	08g09	DNA gyrase sub. A (DNA topoisomerase II) GyrA	609	20a08	histidyl-tRNA synthetase HisS
559	14e01	DNA gyrase sub. A GyrA	610	21g03	leucyl-tRNA synthase LeuS
560	12b05	DNA gyrase sub. A GyrA	611	16h08	lysyl-tRNA synthetase LysS
561	23b07	DNA gyrase sub. B GyrB	612	19g05	phenylalanyl-tRNA synthetase chain PheS
562	29f12	DNA gyrase sub. B GyrB	613	25f01	seryl-tRNA synthetase SerS
563	12g08	replication prot. RepB	614	29d09	tryptophan- tRNA ligase TrprS
563a	02d09	overlaps clone 12g08	615	03g06	tryptophan- tRNA ligase TrprS
564	24g07	DNA polymerase I. PolA	616	10f04	tyrosyl-tRNA synthetase TyrS
564a	19a01	overlaps clone 24g07	617	22f04	valyl-tRNA synthetase ValS
565	01b07	excinuclease ABC sub. A (DNA repair prot.) UvrA
566	13d07	excinuclease ABC sub. A UvrA	617a	01f12	overlaps clone 22f04
567	21e12	excinuclease ABC sub. A UvrA	618	10b10	50S ribosomal prot. L2 RplB
568	25d05	excinuclease ABC sub. C UvrC	619	10e10	50S ribosomal prot. L4 RplD
569	18g04	excinuclease ABC sub. C UvrC	620	03h08	50S ribosomal prot. L7/ L12 RplL
570	02a02	excinuclease ABC sub. C UvrC	621	18e03	50S ribosomal prot. L9 RplI
571	18g06	transcription-repair coupling factor Mfd	622	12h01	50S ribosomal prot. L13 RplM
572	04d05	uracil-DNA glycosylase Ung	623	06a03	50S ribosomal prot. L14 RplN
573	07g08	uracil-DNA glycosylase Ung	623a	20e04	overlaps clone 06a03
574	17d05	type I restriction-modification enzyme M sub. HsdM	624	23h06	50S ribosomal prot. L17 RplQ
575	23g05	type I restriction-modification enzyme M sub. HsdM	625	06c05	50S ribosomal prot. L21 RplU
576	21f03	type I restriction enzyme S sub. HsdS	626	12b06	50S ribosomal prot. L22 RplV
			627	29f09	50S ribosomal prot. L33 RpmG
DNA segregation, recombination and transfer			628	17a03	30S ribosomal prot. S1 RpsA
577	10d01	integrase/recombinase	629	25d09	30S ribosomal prot. S1 RpsA
578	11a04	integrase/recombinase	630	13c09	30S ribosomal prot. S2 RpsB
579	14b05	integrase/recombinase (y4qK pNGR234a)	631	29h04	30S ribosomal prot. S3 RpsC
580	30a10	recombination prot. RecA	632	17h03	30S ribosomal prot. S5 RpsE
581	19f08	conjugal transfer prot. TraA	633	13c05	30S ribosomal prot. S12 RpsL
582	21d09	secretory prot. kinase sim. to TrbB pNGR234a	634	22f07	30S ribosomal prot. S15 RpsQ
582a	05d11	overlaps clone 21d09	635	02b05	30S ribosomal prot. S17 RpsQ
636	12a11	30S ribosomal prot. S21 RpsU	692	14c08	gluconate utilization system repressor; lacI family GntR
637	10f09	30S ribosomal prot. S21 RpsU	693	07e11	transcript. regulatory prot. NtaR; GntR family
638	15f06	ribosomal prot. L11 methylTase PrmA	694	08h12	hydrogen peroxide-inducible activator; lysR family OxyR
639	02c08	y4tL pNGR234a; hydrolase/ peptidase	695	08c08	ribitol operon repressor; lacI family
640	27e11	clp protease ATP binding sub.	696	14f06	transcript. repressor CytR; lacI family
641	06h11	ATP-dependent Clp protease binding sub. ClpA	697	24g11	transcript. repressor; LacI family
642	02e07	ATP-dependent Clp protease binding sub. ClpA	698	22e09	transcript. repressor; LacI family
643	22f12	ATP-dependent Clp protease binding sub. ClpA	699	28g11	transcript. repressor; LacI family
644	02f09	ATP-dependent protease regulatory ATPase sub. ClpB	700	22b04	transcript. repressor; LacI family
645	13a07	ATP-dependent protease regulatory ATPase sub. ClpB	701	17g03	catabolite control prot.; LacI family
646	26f08	ATP-dependent protease regulatory ATPase sub. ClpB	702	18b10	extragenic suppressor prot. SuhB
647	04a02	serine protease, heat shock HtrA like -prot.	703	22b12	extragenic suppressor prot. SuhB
648	10a05	N-carbamyl-L-amino acid amidohydrolase AmaB	704	25d07	transcript. regulator HexA; LysR family
649	18e01	N-carbamyl-L-amino acid amidohydrolase AmAB	704a	20a07	overlaps clone 25d07
650	26f09	peptide chain release factor 1 PrfA	705	07c07	transcript. regulator HexA; LysR family
651	12a02	peptide chain release factor 3 PrfC	706	10d07	transcript. regulator GstR; LysR family
652	14f04	O-sialoglycoprotein endopeptidase	707	18h01	transcript. regulator GstR; LysR family
653	15d11	periplasmic endopeptidase RmDEGP	708	11f05	transcript. regulator; LysR family
654	05a07	ATP-dependent RNA helicase HelO	708a	10d08	overlaps clone 11f05
655	16f11	ATP-dependent RNA helicase HelO	709	05e08	transcript. regulator y4mQ pNGR234a; LysR family
656	21f01	translation elongation factor EF-Tu	709a	16c06	overlaps clone 05e08
657	29d04	translation elongation factor EF-Tu	710	07c01	LysR-type β-lactamase transcriptional regulator
658	23f05	translation elongation factor EF-G	711	26d11	LysR-type β-lactamase transcriptional regulator
659	28d12	translation elongation factor EF-Ts	712	26g10	transcriptional regulator; LysR family
660	23b05	ATP-dependent Lon protease	713	20b04	transcriptional regulator TrpI; LysR family
661	01h03	L-isoaspartyl protein carboxyl methylTase	714	21c12	transcriptional regulator TrpI; LysR family
662	26c02	aminoacyl-histidine dipeptidase PepD	715	21f05	sigma-54 dependent transcript. activator 4_Rme
Regulatory functions			716	22a01	transcriptional modulator MgpS
663	05e05	exoenzyme regulatory prot. AepA	717	22a05	transcriptional regulator ChvI
664	07e12	MucR transcriptional regulatory prot.; Ros/mucR family	717a	26f05	overlaps clone 22a05
664a	16e11	overlaps clone 07e12	718	22d06	Lrp-like transcript. regulatory4sM pNGR234a
665	29a08	SyrB (syrM repressor, sim.to y4aN, pNGR234a)	719	22g04	leucine-responsive regulatory prot.
666	07f06	sugar fermentation stimulation prot.	720	24d06	phosphoTase enzyme II, A PtsN, nitrogen regulation
667	08f09	two-component response regulator	721	25c01	cell division response regulator DivK
668	25g06	transcript. regulator; XylS/AraC family	722	27c07	response regulator PleD
669	15g01	transcript. regulator; XylS/AraC family	723	25d04	transcript. regulatory4tD pNGR234a ; AsnC family
670	09g10	transcript. regulator of NodD3, sim. to y4fK pNGR234a	724	28b08	LacI-GalR family of regulators, e.g. PckR
671	19c04	transcript. regulator of NodD3, sim. to y4fK pNGR234a	Other categories
672	11c01	transcript. regulator GlxA; XylS/AraC family	Adaptation to atypical conditions and protection
672a	17g08	overlaps clone 11c01	725	02b01	nodulation competitiveness prot. NfeD
673	10d05	DNA-binding prot., sim. to y4wC/y4aM pNGR234a	726	16b08	choline DHase (osmoregulation) BetA
674	06c07	adenylate cyclase CyaA	727	26e09	choline DHase (osmoregulation) BetA
675	10h04	adenylate cyclase CyaA	728	19c10	betaine aldehyde DHase (osmoregulation) BetB
675a	17b12	overlaps clone 10h04	729	06h05	choline sulfatase (osmoregulation) BetC
676	27b01	transcript. regulator of sensory transduction systems	729a	08c12	overlaps clone 06h05
676a	13f09	overlaps clone 27b01	730	09g04	choline sulfatase (osmoregulation) BetC
677	02e03	phosphate regulatory prot. PhoB	731	13g02	betaine-aldehyde DHase (osmoregulation)
678	01d05	phosphate regulatory prot. PhoU	732	04f08	acid tolerance ActA prot.
679	03f08	pyruvate Dhase complex repressor	733	08c02	copper resistance prot. precursor (detoxification)
680	03g07	regulatory prot. PcaR	734	10f07	catalase (detoxification)
681	05a10	catabolite control prot. A CcpA	735	29c08	epoxide hydrolase (detoxification)
682	08d09	acetate repressor prot.; IclR family	736	29e02	peroxidase / catalase (detoxification)
683	09b11	FixK regulator	736a	22g02	overlaps clone 29e02
684	28b10	GacA (FixJ-like) response regulator; LuxR/UhpA family	737	19f11	cytochrome P450 (detoxification)
685	14c12	two-component response regulator; LuxR family	738	24b03	nickel resistance prot. NreB
686	17g10	transcript. regulator y4qH pNGR234a, LuxR family	739	24d08	ice nucleation prot. (cold adaptation)
687	28c10	NifR3-like regulator	740	11f07	ice nucleation prot. (cold adaptation)
688	14d06	transcript. regulatory prot. (two-component system)	741	10a08	poly-β-hydroxybutyrate polymerase PhbC
689	14h07	transcript. activator prot.	742	24f07	poly-β-hydroxybutyrate polymerase PhbC
690	15c11	transcript. regulator	743	14g10	poly-β-hydroxybutyrate polymerase PhbC
691	20d01	transcript. regulator; GntR family	744	12a10	survival prot. SurE
691a	15f07	overlaps clone 20d01	745	28e06	biotin-regulated prot. BioS
746	15b05	6′-aminoglycoside (kanamycin 6′)-N-acetylTase AacA	793	28c01	bacteriophage P22 DNA packaging prot. GP2
747	19d11	arsenate reductase (arsenical pump modifier) ArsC	794	15a01	phage T7 internal virion prot. D
748	05a05	5′-hydroxystreptomycin biosynthesis prot. StrU
749	28e07	haloperoxidase	Miscellaneous
			795	27b07	siroheme synthetase-like prot. CysG
Transposon-related functions			796	09h11	indoleacetamide hydrolase (auxin biosynthesis)
750	25f04	ATP-binding prot. y4bM/kI/tA pNGR234a NGRIS-3	797	16e08	2,4-dihydroxyhept-2-ene-1,7-dioic acid aldolase HpcH
751	26c12	ATP-binding prot. y4bL/kJ/tB pNGR234a NGRIS-3	798	18c10	NifS-like prot.
752	22b05	y4bA/pH pNGR234a NGRIS-4	799	12h07	serine/threonine prot. phosphatase
753	06e03	y4bA/pH pNGR234a NGRIS-4	800	29f06	y4vD pNGR234a, peroxiredoxin 2 family
753a	26e03	overlaps clone 06e03	801	22b03	y4wM pNGR234a, possible binding-prot
754	11c03	y4bA/pH pNGR234a NGRIS-4	802	14c03	MelA, melanin synthesis; 4HPPD family
755	29f05	y4bA/pH pNGR234a NGRIS-4	803	14a03	aldehyde DHase
756	12a08	y4bB/pI pNGR234a NGRIS-4	804	07d01	aldehyde DHase
757	07a07	y4bC/pJ pNGR234a NGRIS-4	805	02f01	aldehyde DHase
757a	20b11	overlaps clone 07a07	806	05g03	aldehyde DHase
758	11a02	y4bD/pK pNGR234a NGRIS-4	807	03e10	aldehyde DHase
759	23f01	y4ba/pH pNGR234a (NGRRS-1a left) NGRIS-4	808	07c02	aldehyde DHase
760	02e12	transposase y4jA/nE/sE pNGR234a NGRIS-5	809	12h02	betaine / aldehyde DHase
761	11d01	ISRm2011-2 transposase (IS630-Tc1 family)	810	09h07	betaine / aldehyde DHase
762	06d03	ISRm2011-2 transposase (IS630-Tc1 family)	811	29b08	oxidoRDase, sim. to various polyketide synthase
763	17c03	ISRm2011-2 transposase (IS630-Tc1 family)	812	14h02	molybdenum-containing aldehyde oxidoRDase
763a	28d04	overlaps clone 17c03	813	01e11	oxidoRDase (short-chain type DHase/ RDase)
764	03f02	transposase IS1380	814	06g07	oxidoRDase (short-chain type DHase/ RDase)
765	25h02	transposase IS1380	815	18d03	y4eL pNGR234a, short-chain type DHase/ RDase
765a	17f06	overlaps clone 25h02	816	06b08	short-chain DHase homolog
766	05h02	transposase IS1594	817	24c07	oxidoRDase
767	17h11	transposase IS200	818	16c05	oxidoRDase
768	26g05	ATP-binding prot. y4iQ/nD/sD pNGR234a NGRIS-5	819	12g04	oxidoRDase
769	17e12	IS1248b orf1; sim. to frag. fs4 pNGR234a NGRIS-9	820	07f05	NADH-dependent flavin oxidoRDase
770	13h02	IS869 orf1; sim. to frag. fs4 pNGR234a NGRIS-9	821	04e11	NADH-dependent flavin oxidoRDase
771	07h02	transposase y4sN pNGR234a NGRIS-9	822	18a09	2-hydroxyacid DHase
772	28h05	IS427 orf4; sim. to y4sN pNGR234a	822a	21c10	overlaps clone 18a09
773	18e04	transposase IS870.1	823	11b06	chlorophenol-4-monooxygenase component 1
774	19e10	RFRS9 25 kDa prot.	824	09b01	phenylacetic acid degradation prot.
774a	04e10	overlaps clone 19e10	825	12c05	phenylacetic acid degradation prot.
775	15d07	transposase y4bF pNGR234a	826	24g08	phenylacetic acid degradation prot.
776	18f10	transposase y4qJ pNGR234a	827	09b09	export prot.
776a	06d11	overlaps clone 18f10	828	16h05	potential multicopper oxidase
777	18c09	transposase y4qJ pNGR234a	829	24h11	2-hydroxyhepta-2,4-diene-1,7-dioate isomerase
777a	22a11	overlaps clone 18c09	830	26d12	2-hydroxyhepta-2,4-diene-1,7-dioate isomerase
778	17g01	IS110 family transposase y4uE pNGR234a	831	09a06	ferredoxin RDase (naphthalene conversion)
779	25e06	IS110 family transposase y4uE pNGR234a	832	11f11	Carboxymuconolactone decarboxylase
780	28g06	IS110 family transposase y4uE pNGR234a	833	07b07	biotin / pyruvate carboxylase
781	11d04	IS110 transposase/integrase (C-term)	834	22h07	GTP-binding prot
782	02e10	H- repeat associated prot.	835	22b09	L-sorbose DHase (GMC oxidoRDase family)
783	27d02	H- repeat associated prot.	836	10e01	L-sorbose DHase (GMC oxidoRDase family)
784	06e10	IS-related y4hQ	837	02d05	L-sorbose DHase FAD dependent
785	26h04	IS-related y4hQ	838	24c08	carbon monoxide DHase medium sub.
786	12b01	IS-related y4hP	839	22f10	D-arabino 3-hexulose 6-P formaldehyde lyase
787	12e04	IS-related y4qI	840	27e05	NADH-dependent DHase homolog
787a	11h07	overlaps clone 12e04	841	28b07	molybdenum-containing quinoline 2-oxidoRDase
788	30b11	IS-related y4qI	842	20e01	DHase sub. precursor
788a	05h12	overlaps clone 30b11	843	20g03	pterin-4a-carbinolamine DTase
789	04d03	IS-related y4gE	844	21d04	contains hemolysin-type calcium-binding domain
790	20b02	IS-related y4rI	845	16e10	sulfate-starvation induced prot
790a	25d08	overlaps clone 20b02
			Unknown proteins of: (primary accession number)
Phage-related functions			Escherichia coli
791	12h09	symbiosis island integrase (phage P4 family)	846	15g10	P77388
791a	12h08	overlaps clone 12h09	847	02a07	P32683
792	17b04	bacteriophage P22 DNA packaging prot. GP2	848	02a09	P33362
792a	06h08	overlaps clone 17b04	849	03e01	P45528
850	03h01	P76631	908	28a02	P96267
851	05g07	P76481	909	08e12	Q50709
852	06h04	P75774	910	22g11	Q11157
853	07f09	P37007	911	10f08	O69646
854	07h06	Q46890	912	11a08	O07756
855	08b02	P21498	913	15c10	O06378
856	08b06	BAA14942	914	16f03	P95223
857	08d12	P39333	915	18e10	P96914
858	13e12	P76641	916	21g05	P72043
858a	09c08	overlaps clone 13e12	917	25f02	P95223
859	10a03	P45568	918	29h03	O53720
860	10d02	P37675
861	10d04	P42901	Bacillus subtilis
862	10f12	P76481	919	16d09	O34932
863	12c11	P45475	919a	02a06	overlaps clone 16d09
864	12g02	AAC75037	920	05a01	P94437
865	15e04	P52049	920a	05g06	overlaps clone 05a01
866	15f04	P77748	921	06c04	P94937
867	16f04	BAA31826	922	06e01	AAB72069
868	17c04	P23522	923	04h03	O32272
869	18g09	AAA83544	924	07f08	AAB35255
870	18h06	AAC74284	925	08a08	BAA06611
871	18h11	P77368	926	17e06	BAA23396
872	19h07	P77165	927	19c11	P54724
873	20a06	P37619	928	11d08	P54178
874	22d02	AAC74824	929	11b08	P39640
875	22e07	AAC44004	929a	08g04	overlaps clone 11b08
876	22f08	P33362	930	11f08	P96683
877	25f09	P76397	931	09c03	P42966
878	28e04	P76397	932	17f04	O34398
879	25f10	P77470	933	24b07	P35155
880	26e05	AAC74522	934	18c06	O05220
881	27a05	AAC75727	935	24d11	Q07835
882	27e02	P39829	936	13a01	O07618
883	09c12	P37053	937	25a06	P37508
884	28f07	AAC75038	938	21a05	Q45584
885	17d02	P77391	938a	12b10	overlaps clone 21a05
886	24f10	P76235
887	28f08	P76235	Synechocystis sp.
888	28a06	P39338	939	04a01	BAA17151
889	30e08	P08367	940	06c03	BAA17443
890	27c11	P77165	941	04h11	BAA18318
891	02b12	P75844	942	09g07	BAA18319
892	24h06	P77334	943	13c07	BAA18330
893	22h02	P46854	944	08e11	BAA16904
893a	21d08	overlaps clone 22h02	945	08f05	BAA17017
			946	10e06	BAA17950
Mycobacterium tuberculosis			947	10f05	BAA16766
894	02h04	O05841	948	15h10	BAA18186
895	02h08	O06320	949	25e03	AAB41278
			950	26c01	P72872
895a	08h06	overlaps clone 02h08	950a	11h09	overlaps clone 26c01
896	03d06	O05865	951	29a07	BAA10710
897	04f10	O53858	952	01c06	BAA10835
898	04h01	O06804
899	07e03	P71838	Haemophilus influenzae
900	19b02	Q10846	953	01c01	P44250
901	19f01	P96814	954	01e07	P31777
902	20c01	O50466	955	06b02	Q57151
903	24h02	O07220	956	13a08	P44093
904	25a05	O06235	957	25h09	P44886
905	25b06	P71984	958	01h02	P44540
906	25h05	O53203	959	22c12	P44543
907	27h05	Q10849	960	19a07	Q57184
Agrobacterium sp.			1005	07d10	Q58322
961	03a07	AAB91569	1006	05a02	Q57883
962	05f01	AAB67296	1006a	29e09	overlaps clone 05a02
963	28b11	AAB67297	1006b	17d09	overlaps clone 05a02
964	06b11	AAB51512	1007	06f06	Q46063
965	19e06	AAC17194	1008	07b09	AAB50572
966	09h12	AAB67297	1009	02h03	AAB50572
967	17e09	P70791	1009a	12e05	overlaps clone 02h03
968	16h09	P70795	1010	04e08	AAC46053
969	01g12	P70795	1011	28h08	AAC46056
970	22b02	P70795	1012	19f10	AAA96787
			1013	10g08	BAA29686
Rhizobia			1014	02g03	BAA29099
971	17f01	P55362	1014a	13b10	overlaps clone 02g03
972	30f11	P55362	1015	11g07	AAC82835
973	16c11	P55388	1016	17e05	P46378
974	30e04	P55424	1017	17h12	AAB51777
975	27b10	P55480	1018	18a11	CAA55879
976	10b09	P55552	1019	18h10	AAB66497
977	12g11	P55552	1020	19f09	AAB85316
978	29e06	P55552	1021	19f12	O52867
979	11d10	P55590	1021a	23b03	overlaps clone 19f12
980	07c10	P55694	1022	21a11	AAB38705
981	20h08	P55706	1023	21d12	AAB09035
982	02g08	P25893	1024	12d11	AAD03878
983	11f12	P49305	1025	21a04	AAC44077
984	14b08	AAB63673	1026	22f06	AAD03845
985	16b12	AAA74241	1027	16d06	AAD03912
986	16f09	AAA74241	1028	22g06	P70734
987	16c07	AAB4153	1029	22h06	AAC46243
988	09g03	Q52991	1030	05d03	AAC06984
989	07h12	P25893	1030a	06d06	overlaps clone 05d03
990	22h03	AAA88525	1031	24b01	AAC07457
991	25c11	Q52967	1032	30a07	AAC06721
992	25g03	AAB81416	1033	01a12	P38102
993	20a04	CAA11961	1034	24d02	P55176
994	30e03	CAB01954	1035	28e05	P29938
995	20g09	AAC64871	1036	09c10	AAC44553
996	04f06	AAB17515	1037	26a12	AAB89525
997	22h04	AAB17515	1038	27f04	AAC34291
998	19e07	AAB17514	1039	29c07	C36925
999	05f08	AAA96138	1040	29d11	Q49092
1000	24e08	O69244	1041	11d06	Q15595
			1041a	07a02	overlaps clone 11d06
Other organisms			1041b	23g10	overlaps clone 11d06
1001	19a11	AAC16153	1042	06h07	P40896
1002	28a01	AAC16139	1043	24d09	P34227
1003	22d01	P30790	1043a	15f12	overlaps clone 24d09
1004	03g04	Q06373	1043b	18f08	overlaps clone 15f12

Abbreviations: No, number; prot., protein; sim., similar; sub, subunit; transcript., transcriptional; transp., transport; ATase, aminotransferase; CoA, coenzyme A; DHase, dehydrogenase; DTase, dehydratase; RDase, reductase; Sase, synthase; Tase, transferase; TGase, transglycosylase.

As in other bacterial genomes, such as that of Escherichia coli [30], the largest functional class represents transport and binding proteins (see Tables 2 and 3). A number of essential genes, including those required for replication, transcription and translation as well as those linked to primary metabolism, were also found. As expected of a soil-borne prokaryote, many loci (18%) involved in carbon and nitrogen metabolism were identified (encoding enzymes for the assimilation of nitrate/ammonia, the tri-carboxylic acid cycle, or transporters of dicarboxylic acids, and so on). In B. subtilis, 19% of the protein-coding genes are devoted to the metabolism of carbohydrates, amino acids and related molecules (Table 2). This is in contrast to microorganisms such as Haemophilus influenzae and M. genitalium that are not able to grow on many nitrogen and carbon sources (only 10% of their predicted genes code for such metabolic functions [31]). Interestingly, homologs of various chaperones such as GroES/GroEL, DnaJ, and other small heat-shock proteins (sHsps), were identified (Table 3, clones 308 to 318). The presence of multiple sHsps is not common in prokaryotes, but was shown to be widespread in rhizobia [32].

Obviously, the ability of rhizobia to respond to plant compounds that stimulate their growth contributes to successful colonization of the root [33] and absence of vitamins often limits the growth or rhizobia. Furthermore, the ability to either take up or synthesize vitamins is thought to be an essential characteristic of rhizobia [33]. For these reasons, it is not surprizing that several ANU265 sequences matched genes for biotin and thiamine utilization, such as that coding for a homolog of bioS (clone 745), a biotin-regulated locus of R. meliloti [34]. In R. meliloti, bioS is part of an operon which includes the surE and IppB/nlpD genes that are also found in ANU265 (clones 744 and 183). Homologs of thiamine biosynthetic genes thiCG of R. etli (clones 512 and 513) were also found. Miranda-Rios et al. [35] reported a direct correlation between the expression of thiC and the production of the symbiotic terminal oxidase cbb3, which is required for bacteroid respiration under conditions of low oxygen.

Putative symbiotic genes include loci involved in exopolysaccharide (EPS) biosynthesis and/or export, which are encoded by pNGR234b [10], as well as genes involved in the elaboration of acidic capsular polysaccharides (K-anti-gens), lipopolysaccharides and cyclic β-glucans (Table 3, clones 245 to 270). A sequence homologous to fixN of R. meliloti was also identified (clones 208 and 209). The chromosomal fixNOPQ locus encodes an oxidase complex that is probably active during nitrogen fixation. Although sequences of the regulatory fixK genes [3] were identified (clone 683), no significant match to the oxygen-responsive system encoded by fixLJ was found. Members of other symbiotic two-component regulatory systems were detected in ANU265, however, including homologs of the sensor histi-dine kinase exoS (clone 200) and the response regulator chvI (clone 717). Both are necessary for regulating production of succinoglycans that are important in R. meliloti-Medicago sativa symbioses [36]. Similarly, the nwsA locus (clone 202) encodes a putative sensor kinase that is involved in the expression of nodulation genes in Bradyrhizobium strains [37].

It has been postulated that genes responsible for the synthesis (mos) and catabolism (moc) of rhizopines confer a competitive advantage on their host rhizobia [38]. Rhizopines are synthesized in nodules of M. sativa inoculated with R. meliloti strain L5-30, and can be used as growth substrates by certain rhizobia. Although mos and moc genes were thought to be limited to R. meliloti strains [39], homologs of mocABC, and mosA genes were also found in ANU265 (clones 543 to 549). Propagation of rhizobia in the soil, and hence their symbiotic efficiency, probably also depends on their tolerance to osmotic changes. It is thus notable that homologs of the R. meliloti betABC genes, which are involved in the osmoregulatory choline-glycine betaine pathway [40], were also found (clones 726 to 730).

Other putative symbiotic loci include homologs of the phbC and prsDE genes of R. meliloti, which encode a poly-3-hydroxybutyrate synthase [41] and a type I secretion system [42] (clones 741 to 743, and 298 to 301, respectively). Interestingly, PrsD and PrsE of R. meliloti are involved in the secretion of enzymes that modify succinoglycans [43], whereas a similar type I secretion system seems to be responsible for the export of the nodulation-signaling protein NodO in R. leguminosarum bv. viciae [44,45]. Although the role of these prsDE homologs in NGR234 is not clear, it is possible that more than one type of protein secretion system has a symbiotic role in this bacterium [46].

Conclusions

Random sequencing of ANU265 followed by homology searches of public databases resulted in the identification of 1,130 putative protein-coding sequences, of which 922 (41%) could be classified into functional groups. Comparison of these data with those derived from the complete sequence of the B. subtilis genome showed a similar distribution of putative coding sequences, except perhaps for functions related to transposable elements (Table 2). In fact, the genome of ANU265 carries more putative transposases and other IS-related functions (5.5% of all identified genes, and 2.2% of all shotgun sequences) than that of B. subtilis. Nevertheless, in proportion to their size, the chromosome and megaplasmid of NGR234 carry fewer IS sequences than pNGR234a. Furthermore, hybridization data indicate that the density of known transposable elements is higher in pNGR234b than on the chromosome (order of IS accretion is: pNGR234a > pNGR234b > chromosome) [11]. This suggests that IS elements preferentially accumulate on plasmids, possibly because they are less likely to disrupt essential functions. In contrast, the many RIME elements present in NGR234 are clearly more abundant on the chromosome and megaplasmid than on pNGR234a. Together, the distinct G+C contents and structural features of the symbiotic plasmid, megaplasmid and chromosome suggest that different evolutionary constraints and histories contributed to shape these three replicons.

'Skimming' the genome of Rhizobium sp. NGR234 has given new insights into the evolution of its replicons and the integration of symbiotic functions in the genome of a soil bacterium. It also reinforced the assumption, which originated from host-range extension experiments [12,47], that pNGR234a carries most of the symbiotic genes. Although few nod, nif and fix homologs were found amongst the random clones, it is likely that additional chromosome- and megaplasmid-encoded functions contribute to successful symbioses between NGR234 and its many host plants. In this respect, transcriptional analyses using shotgun sequences as hybridization templates [11] will help identify such new symbiotic loci.

Materials and methods

Microbiological techniques

Rhizobium strain ANU265 [19], a strain of Rhizobium sp. NGR234 [7] cured of pNGR234a, was grown in Rhizobium minimal medium supplemented with succinate (RMM) [47]. Escherichia coli was grown on SOC or in TY [48]. Subclones in M13mp18 vectors [49] were grown in E. coli strain DH5α F'IQ [50].

Preparation of the random genomic library and M13 templates

Genomic DNA of Rhizobium strain ANU265 was prepared as in Perret and Broughton [51]. ANU265 genomic DNA (15 μg) was sheared by sonication and incubated for 10 min at 30°C with 30 units of mung bean nuclease. The resulting digest was extracted with phenol/chloroform (1:1) and precipitated with ethanol. Fragments ranging in size from 900 to 1,500 bp were purified from agarose gels and ligated into SmaI-digested M13mp18 vector DNA. Ligation mixtures were electroporated into E. coli strain DH5αF'IQ [48,52], and transformants were plated on 5-bromo-4-chloro-indoyl-β-D-galactoside (X-Gal) and isopropyl-β-thiogalactopyranoside (IPTG)-containing petri dishes [48]. Fresh 1 ml cultures of E. coli DH5αF'IQ were infected with phages from randomly selected white plaques, and grown for 6 h at 37°C in TY medium. Phages were precipitated from 600 μl of the culture supernatant by adding 150 μl 2.5 M NaCl/20% polyethylene glycol (PEG-8,000) (20 min at 25°C). Afterwards, they were centrifuged for 20 min at 3,000g at 25°C, and resuspended in 20 μl Triton-TE extraction buffer (0.5% Triton X-100; 10 mM Tris-HCl, 1 mM EDTA pH 8.0). Following 10 min incubation at 80°C and ethanol precipitation, single-stranded phage DNA was recovered in 50 μl H₂O.

Sequence analysis

Dye-terminator cycle sequencing of individual M13 sub-clones, gel electrophoresis and sequence editing was performed as described by Freiberg et al. [53]. Shotgun sequences were checked for redundancy using the XGAP program [54] and for significant homologies with BLASTX-BLASTN software [55] using nonredundant databases at NCBI [25].