Genetic snapshots of the <it>Rhizobium</it> species NGR234 genome

gb-2000-1-6-research0014 GBJ Research Genetic snapshots of the <it>Rhizobium</it> species NGR234 genome Viprey Virginie Rosenthal André Broughton William J broughtw@sc2a.unige.ch Perret Xavier

Laboratoire de Biologie Moléculaire des Plantes Supérieures, Université de Genève, chemin de l'Impératrice, 1292 Chambésy, Genève, Switzerland

Institut für Molekulare Biotechnologie, Abteilung Genomanalyze, Beutenbergstrasse, 07745 Jena, Germany

Current address: John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK

Genome Biology 1465-6906 2000 1 6 research0014.1 research0014.17 http://genomebiology.com/2000/1/6/research/0014 10.1186/gb-2000-1-6-research0014 11178268 17 4 2000 25 10 2000 31 10 2000 4 12 2000 2000 GenomeBiology.com

Skimming' the genome of Rhizobium sp. NGR234, a nitrogen-fixing symbiont of many legumes, 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.

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 112 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.

Genome studies Microbiology and parasitology Genetics

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

(0.1%)

tRNA

(0.1%)

repeated sequences / intergenic elements only

(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 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

(1.9)

(3.9)

Transport/binding proteins and lipoproteins

184

(20.0)

381

(16.0)

Sensors (signal transduction)

(2.3)

(1.6)

Membrane bioenergetics (electron transport and ATP synthase)

(5.3)

(3.3)

Surface polysaccharides biosynthesis and export

(2.7)

(0.7)

Sporulation

(0.1)

139

(5.8)

Germination/transformation

(1.8)

Mobility and chemotaxis

(2.8)

(2.3)

Cell division

(0.5)

(0.9)

Protein secretion

(1.4)

(0.8)

Chaperones/heat-shock proteins

(1.3)

(0.6)

Cell death

(0.9)

(0.2)

Intermediary metabolism

Carbohydrates and related molecules

(7.5)

261

(11.0)

Amino acids and related molecules

(9.9)

202

(8.5)

Nucleotides and nucleic acids

(1.2)

(3.5)

Lipids

(2.1)

(3.2)

Cofactors/prosthetic groups

(4.0)

(4.2)

Phosphate

(0.3)

(0.4)

Opine-like compounds

(0.9)

(0.1)

Sulphur

(0.2)

(0.3)

Information pathways

DNA replication, restriction, modification and repair

(2.8)

(2.6)

DNA segregation, recombination and transfer

(1.1)

RNA synthesis and modification

(2.1)

Protein synthesis and modification

(6.8)

123

(5.2)

Regulatory functions

(7.4)

213

(8.9)

Other categories

Adaptation to atypical conditions and protection

(2.9)

147

(6.2)

Transposon-related functions

(5.5)

(0.4)

Phage-related functions

(0.5)

(3.5)

Miscellaneous

(5.6)

(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

26d04

sugar transp. ATP-binding prot.

Cell wall

26h11

sugar transp. ATP-binding prot.

01d07

N-acetylmuramoyl-L-alanine amidase

05b03

sugar transp. system permease prot.

06g04

N-acetylglucosamine-1-phosphate uridyl Tase

30b08

sugar transp. system permease prot.

26b06

UDP-N-acetylenolpyruvoylglucosamine RDase MurB

26a07

sugar ABC transp., ATP-binding prot.

28f02

UDP-N-acetylmuramate-alanine ligase MurC

14e06

xylose transp. permease prot.

22b10

UDP-N-acetylmuramoylalanine-D-glutamate ligase MurD

03f04

xylose transp. permease prot.

29g07

UDP-N-acetylmuramyl-tripeptide synthetase MurE

19a04

maltose binding prot.

08h05

UDP-N-acetylmuramyl-tripeptide synthetase MurE

11c09

membrane bound sugar transp. prot.

12f10

outer membrane prot.

18a12

sugar transp. ATP-binding prot.

17a08

outer membrane prot. Omp28

28e01

sugar transp. ATP-binding prot.

21h07

group 1 outer membrane prot. OMP1 precursor60

30f09

sugar transp. prot.

18f12

penicillin-binding prot. 1B

01c10

galactoside transp. system permease prot.

19h11

penicillin-binding prot. 1A

19g07

galactoside transp. ATP-binding prot.

29f10

D-alanyl-D-alanine carboxypeptidase

21e06

branched-chain amino acid transp.

19d01

monofunctional biosynthetic peptidoglycan TGase MtgA

29a02

amino-acid ABC transp. permease prot.

28f09

lysozyme M1 precursor Acm

27b03

amino-acid ABC transp. permease prot.

03f07

acriflavine resistance prot. E precursor AcrE

05c04

ABC transp. permease prot.

16a

09h01

overlaps clone 03f07

07b05

amino-acid ABC transp. ATP-binding

Transport/ binding proteins and lipoproteins

19h08

amino-acid ABC transp. ATP-binding

22a03

sugar-binding transp. ATP-binding prot.

06a09

amino-acid ABC transp. ATP-binding

08h08

ABC transp. ATP-binding prot.

11d03

glutamate/ aspartate transp. system permease prot.

29c10

sugar ABC transp., permease prot.

21d10

high-affinity branched-chain amino acid transp.

03f05

inner membrane prot.of trehalose/ maltose transp.

75a

24a05

overlaps clone 21d10

02h10

transp. permease prot.

02c01

amino acid ABC transp.

21a

09f05

overlaps clone 02h10

08f08

branched-chain amino acid transp. prot.

11c04

ABC transp. permease prot.

10e04

branched-chain amino acid transp. prot.

12d03

inner membrane ABC transp.

17b01

branched-chain amino acid transp. prot.

18h08

sugar ABC transp. ATP-binding prot.

08g08

branched-chain amino acid transp. prot.

21b03

ATP-binding transp. prot.

04g02

branched-chain amino acid transp. permease prot.

01h04

ATP-binding transp. prot.

28h02

high-affinity branched-chain amino acid transp.

21b11

ATP-binding transp. prot.

82a

08a04

overlaps clone 28h02

26g01

maltose/ maltodextrin transp. ATP-binding prot.

82b

11g01

overlaps clone 28h02

18g11

sugar ABC transp. ATP-binding prot.

16c12

periplasmic dipeptide transp. prot. precursor

24e03

ABC transp. ATP-binding prot.

03h07

dipeptide ABC transp.

02c09

ABC transp. ATP-binding prot.

10d11

peptide ABC transp. permease prot.

31a

12f05

overlaps clone 02c09

15e08

ABC transp. ATP-binding prot.

28g12

ABC transp. ATP-binding prot.

04a06

peptide ATP-bind. transp.

32a

01a11

overlaps clone 28g12

09a09

peptide ABC transp. permease prot.

32b

09h06

overlaps clone 28g12

12c06

peptide ABC transp. permease prot.

01a10

ribose transp. ATP-binding prot.

03f10

ABC transp.

06h03

D-ribose-binding periplasmic prot. precursor

22c01

ABC transp. ATP-binding prot.

14e04

sugar transp. system permease prot.

19b12

ABC transp. ATP-binding prot.

18d02

sugar transp. system permease prot.

23g09

peptide ABC transp. ATP-binding prot.

04e03

sugar transp. system permease prot.

18e05

ABC transp. ATP-binding prot.

14f09

sugar transp. system permease prot.

14d12

oligopeptide ABC transp.

08g05

sugar transp. system permease prot.

19c05

oligopeptide binding prot.

06b09

sugar transp. system permease prot.

21g02

peptide ABC transp.

19e12

sugar transp. system permease prot.

22h09

dipeptide transp. ATP-binding prot.

16f12

membrane-spanning permease

25c02

oligopeptide transp. ATP-binding prot.

26d10

sugar transp. system permease prot.

99a

23c05

overlaps clone 99

23b06

sugar transp. system permease prot.

100

27b09

oligopeptide ABC transp. permease prot.

27h12

ABC transp. integral membrane prot.

101

27c09

oligopeptide-binding prot. precursor

22d10

ribose ABC transp. permease prot.

102

30a09

oligopeptide transp. ATP-binding prot

25a11

sugar transp.

103

03b02

ABC transp., y4wM pNGR234a

21a06

sugar transp. ATP-binding prot.

104

03g02

ABC transp., y4wM pNGR234a

21b02

sugar transp. ATP-binding prot.

105

07c06

ABC transp., y4wM pNGR234a

24d10

galactoside transp. ATP-binding prot. MglA

106

30e02

ATP-binding prot.

24e10

lactose transp. system permease prot. LacF

107

05c03

ATP-binding prot.

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. a PntA

155

29b02

ferric siderophore receptor

204

02h05

pyridine nucleotide transhydrogenase sub. a PntA

155a

18g03

overlaps clone 29b02

205

09d11

pyridine nucleotide transhydrogenase sub. a PntA

156

14e12

potassium uptake prot. Kup

206

20h04

pyridine nucleotide transhydrogenase sub. ß PntB

157

14f07

phosphoenolpyruvate-prot. phosphoTase

207

25g07

pyridine nucleotide transhydrogenase sub. ß 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

ß-(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 d, 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 a-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 ?-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-ß-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

a-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

a-ketoglutarate DHase

388

29h05

fumarate hydratase

330a

24d12

Overlaps clone 01c12

389

30b10

mannitol 2-DHase

331

16g02

acetoin:DCPIP oxidoRDase a

390

05d12

isocitrate lyase

332

02e09

acetoin:DCPIP oxidoRDase ß

Metabolism of amino acids and related molecules

333

02e11

succinyl-coA synthetase ß chain

391

27a11

a-isopropylmalate synthase LeuA

334

03e11

ribulose-bisphosphate carboxylase, large sub.

392

27d07

a-isopropylmalate synthase LeuA

335

03h09

citrate synthase

393

14d11

a-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 a-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

ß-glucosidase (cellulose degradation)

412

05g02

3-isopropylmalate DTase large sub. LeuC

356

07a05

ß-glucosidase (cellulose degradation)

413

05g05

class III pyridoxal-phosphate-dependent ATase

357

18b12

ß-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 a-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 a 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 a 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 ß-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 a SoxA

512

05b08

thiamine biosynthesis prot. ThiC

461

25e04

sarcosine oxidase a SoxA

513

03g09

thiamine biosynthesis prot. ThiG

462

01e05

sarcosine oxidase ß SoxB

514

05b12

S-adenosylmethionine: 2-demethylmenaquinonemethylTase

463

03c10

sarcosine oxidase d 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

?-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, ß 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 a sub. RpoA

Metabolism of phosphate

588

17b03

ribonuclease HII RnhB

541

04f07

inorganic pyrophosphatase Ppa

589

02a01

RNA polymerase ß sub RpoB

541a

29c05

overlaps clone 04f07

590

03e09

RNA polymerase ß sub RpoB

542

25h01

phosphonate utilization PhnJ

591

06d05

RNA polymerase ß sub RpoB

Metabolism of rhizopine

592

22h12

RNA polymerase ß sub RpoB

543

05a11

MocA oxidoreductase

593

28d10

RNA polymerase ß sub RpoB

544

07d03

MocB rhizopine-binding prot. precursor

594

16b02

RNA polymerase ß ’ 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 a sub. DnaE

605a

20h09

overlaps clone 01b01

554

28f04

DNA polymerase e 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 ß-lactamase transcriptional regulator

658

23f05

translation elongation factor EF-G

711

26d11

LysR-type ß-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-ß-hydroxybutyrate polymerase PhbC

689

14h07

transcript. activator prot.

742

24f07

poly-ß-hydroxybutyrate polymerase PhbC

690

15c11

transcript. regulator

743

14g10

poly-ß-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 ß-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 DH5aF'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 DH5aF'IQ [48,52], and transformants were plated on 5-bromo-4-chloro-indoyl-ß-D-galactoside (X-Gal) and isopropyl-ß-thiogalactopyranoside (IPTG)-containing petri dishes [48]. Fresh 1 ml cultures of E. coli DH5aF'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].

Acknowledgements

We thank S. Brenner, C. Freiberg, S. Taudien and D. Gerber for their help with many aspects of this work. Financial assistance was provided by the Fonds National Suisse de la Recherche Scientifique (Grant No. 31-45921.95) and the Université de Genève.

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