Background

Biological nitrogen fixation is carried out by a limited number of prokaryotes that all possess a nitrogenase enzyme complex that reduces molecular dinitrogen to ammonia. Nitrogen-fixing bacteria can be divided in two major groups: free-living nitrogen fixers or diazotrophs that directly assimilate ammonia for growth and symbiotic nitrogen fixers that pass ammonia to a eukaryotic host and indirectly profit from nitrogen fixation by occupying a particular ecological niche or by supporting the population through better feeding. In the latter group, the symbiosis between leguminous crop plants and rhizobia is of great importance for agriculture. The term "rhizobia" is used for bacteria that induce the formation of new organs, nodules, on the roots of a specific legume host. Inside the nodule, rhizobia are internalized in plant cells where they differentiate into nitrogen-fixing bacteroids [for a recent review on legume nodulation, see [1]].

Nitrogen-fixing nodules typically occur on roots; however, some members of the subfamilies Papilionoideae (Aeschenomyne sp., Sesbania sp., and Discolobium pulchellum) and Mimosoideae (Neptunia oleracea) form stem-located, aerial nodules [2]. These legumes grow in waterlogged soils of tropical regions and are characterized by dormant, stem-located adventitious root primordia that can develop into stem nodules upon inoculation with an appropriate microbial partner. Although stem and root nodulation are similar, in the latter the nodular vascular system is connected to that of the stem via the vascular bundles of the adventitious root primordium [3].

A particularly well-studied case of stem nodulation occurs in Sesbania rostrata Brem. upon inoculation with the microsymbiont Azorhizobium caulinodans [4]. S. rostrata, a fast-growing annual shrub from the Sahel region of West-Africa, carries numerous adventitious root primordia that protrude through the stem cortex and epidermis, creating a circular fissure, where bacteria can invade and proliferate [5]. The growth properties and the high rate of nitrogen fixation of stem-nodulated plants make S. rostrata well fit as green manure in rice cultivation and, possibly, as a pioneer plant for wetland improvement [6].

The bacterium, isolated from stem nodules [4] and originally designated Rhizobium sp. strain ORS571, was renamed Azorhizobium caulinodans inspired by the stem (cauli-)nodulating capacity and by the diazotrophic properties of the strain (azo-rhizobium). Its host range for effective nodulation is very narrow: although nodulation of several Sesbania sp. has been reported, nitrogen-fixing nodules are formed only on S. rostrata and S. punctata [7]. A. caulinodans also induces Fix nodules on Phaseolus vulgaris and Leucaena leucocephala [8]. Two features distinguish A. caulinodans from other rhizobia: its taxonomic position and its dual capacity for free-living and symbiotic nitrogen fixation. The latter is exceptional [9] and implies a regulatory mechanism to either assimilate the ammonia or donate it to the plant in the symbiotic interaction. The first taxonomic study of A. caulinodans strain ORS571 [10] showed that it belongs to the Rhodopseudomonas palustris rRNA branch of purple bacteria, but that it is quite distinct from both Rhodopseudomonas and Bradyrhizobium spp. Based on numerical analysis of phenotypes, protein patterns, and DNA-DNA and DNA-rRNA hybridizations, A. caulinodans was considered as a separate genus with Xanthobacter as closest relative [11]. Xanthobacter sp. are diazotrophic bacteria found in diverse soil habitats and in association with rice (Oryza sativa) roots [12, 13]. Comparison of 16S rRNA sequences indicated that X. flavus and A. caulinodans are strongly related [14].

Here, we present the genome sequence of the A. caulinodans strain ORS571 and discuss the annotation in function of the organism's biology with reference to comparative genomics. This information will stimulate the research on an organism that has real potential for novel applications in agriculture.

Results

Genome organization

Sequencing of the genome of A. caulinodans strain ORS571 (hereafter designated A. caulinodans) revealed a single circular chromosome of 5,369,772 base pairs [15]. Relevant genome features generated with the BLASTatlas tool [16] are presented in Figure 1 and can be viewed in detail as a web-based resource [17]. The putative origin of replication was predicted based on the position of a GC skew shift (Figure 1) [18] and coincided with the occurrence of a gene cluster typically associated with origins of replication in circular chromosomes of α-proteobacteria (Figure 2A) [19]. The specific distribution and orientation of the FtsK Orienting Polar Sequences (KOPS) motif 5'-GGGNAGGG-3', which is involved in loading the FtsK DNA translocase and directing it to the replication terminus in α-proteobacteria [20], confirmed the predicted location of the origin between AZC_4717 and AZC_0001 (Figure 2B).

Figure 1
figure 1

Snapshot of the output generated after analysis of the A. caulinodans genome with the Genome Atlas tool. The output is accessible as a web-based resource [17] that can can be used as a tool to zoom in on specific regions of interest. Hits within the UNIPROT database, a comparison at the protein level between 14 α-proteobacteria and A. caulinodans, and the synteny between these genomes, the genome annotation, and structural features are represented. The origin and terminus of replication are indicated and the symbiotic region is boxed. From the outer to the inner circle: circle 1, protein hits in the UNIPROT database; circle 2, synteny between 15 α-proteobacterial genomes; circle 3, Agrobacterium tumefaciens C58; circle 4, Xanthobacter autotrophicus Py2; circle 5, Sinorhizobium meliloti 1021; circle 6, Bradyrhizobium japonicum USDA 110; circle 7, Rhizobium leguminosarum 3841; circle 8, Rhizobium etli CFN42; circles 9 and 10, Mesorhizobium loti strains BNC1 and MAFF303099, respectively; circle 11, Nitrobacter winogradskyi Nb225; circles 12, 13, 14, 15, and 16, Rhodopseudomonas palustris strains CGA009, HaA2, BisA53, BisB18, and BisB5, respectively; circle 17, intrinsic curvature; circle 18, stacking energy; circle 19, position preference; circle 20, genome annotation; circle 21, global repeats; circle 22, inverted repeats; circle 23, GC skew; circle 24, percent AT. The structural DNA parameters in circles 21 and 22 relate to the occurrence of repeats that might indicate inserted sequences, and circles 18 and 19 designate the accessibility and flexibility of the DNA as a measure for the capacity to interact with proteins.

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Figure 2
figure 2

Predicted position of the origin of replication. A. Conservation of a cluster of 12 genes located around the origin of replication in several α-proteobacteria: (1) Azorhizobium caulinodans, (2) Agrobacterium tumefaciens, Sinorhizobium meliloti, Rhizobium leguminosarum, and Rhizobium etli, (3) Mesorhizobium loti, Brucella abortus, and Brucella suis, (4) Bradyrhizobium japonicum and Rhodopseudomonas palustris, (5) Caulobacter crescentus, and (6) Xanthobacter autotrophicus. The putative proteins and the origin of replication are indicated. B. Cumulative distribution in forward and reverse orientation of the 8-base KOPS motif 5'-GGGNAGGG-3' in the genome of A. caulinodans. The orientation of this motif is strongly biased toward dif sites at the terminus of replication (Terminus).

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Although the overall GC content of the A. caulinodans genome is 67% and the average GC incidence at the third position of the codon (GC3) is 85%, the chromosome has many islands of varying size with different GC (Figure 3A) and GC3 contents (Figure 3B). In accordance with the overall high GC content, the codon usage is shifted toward GC-rich codons (Figure 4A) and, consequently, GC-coded amino acids are overrepresented (Figure 4B).

Figure 3
figure 3

Nucleotide composition of the A. caulinodans genome. The GC and GC3 contents for each open reading frame were calculated and positioned on the genome. Every gene was classified in one of four classes: orphans, genes of the A. caulinodans genome without homolog in other bacteria of the data set (44 genomes) (red squares); singletons, genes with one representative in A. caulinodans and homologs in the data set (green stars); phage- or integrase-related genes (yellow triangles); duplicated genes with more than one paralog in the A. caulinodans genome (blue diamonds). GC (A) and GC3 (B) distribution across the genome; GC (C) and GC3 (D) distribution across the symbiotic region. Circles (C and D) indicate the location of the three nod loci.

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Figure 4
figure 4

Codon (A) and amino acid (B) usage in the A. caulinodans genome (red) and the symbiotic region (blue).

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Combined computer prediction and similarity searches (Methods) revealed 4717 protein-encoding genes with an average coding density of one gene in every 1123 bp (89%). With the BLASTP program (Methods), the amino acid sequences were compared with the sequences in the nonredundant protein database at NCBI. A putative function could be assigned to 3588 genes (76.1%), 954 genes (20.2%) were similar to hypothetical genes, and the remaining 175 (3.7%) had no significant similarity to any registered gene (Figure 1; Table 1; Additional file 1).

Table 1 Overview of the functional categories of proteins present in the A. caulinodans genome according to the classification of Riley [26].
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Three rRNA clusters are ordered as 5S-23S-16S (located between the protein-coding genes AZC_0613-AZC_0614, AZC_4195-AZC_4196, and AZC_4435-AZC_4436) and all have an insertion of a tRNA-Ile and a tRNA-Ala between the 16S and 23S genes. A total of 53 tRNA genes representing 44 tRNA species for all 20 amino acids were assigned by sequence similarity and computer prediction with the tRNAscan-SE program [21]. Most of the tRNA genes are dispersed on the genome and are probably transcribed as single units. Thirty out of 57 ribosomal protein genes occur in a cluster (AZC_2529-AZC_2559), whereas the others are scattered over the genome (Additional file 1).

Phylogeny and comparative genomics

For phylogenetic analysis (Methods), the genomes of A. caulinodans and of 44 α-proteobacteria were compared (Additional file 2). The data set was assembled based on the available complete genome sequences (closure date August 15, 2007) and ecological or phylogenetic relatedness. The resulting maximum-likelihood tree (Figure 5A) showed a great concordance with α-proteobacterial trees based on complete 16S rRNA genes [22] or sets of protein families [23]. Our analysis placed A. caulinodans closest to X. autotrophicus, Nitrobacter winogradskyi, Rhodopseudomonas palustris, and Bradyrhizobium japonicum, consistent with previous taxonomic studies [9, 10, 13]. With A. caulinodans as a reference genome, a graphical representation of the BLAST hits of the proteins encoded by the genomes of the 13 closest relatives was generated with the BLASTatlas tool (Figure 1) [16, 17].

Figure 5
figure 5

Maximum-likelihood tree and prevalence of duplicated, singleton, and orphan genes within 45 complete α-proteobacterial genome sequences. A. Unrooted maximum-likelihood tree based on 108 conserved protein sequences (for construction of the tree and references to the genomes, see Methods and Additional file 2, respectively). Bootstraps are 100, unless indicated otherwise. B. Percentual presence of the four gene categories: orphans (dark blue); singletons (cyan); phage- or integrase-related genes (red); and duplicated genes or paralogs (light blue). Genome sizes are indicated on the right.

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For a broader view of the gene relationships, the occurrence and organization of the proteins encoded by these 45 genomes were evaluated (Methods). Each gene of a total data set of 146,315 was classified in one of four groups: orphans, genes without homologs in other bacteria of the data set; singletons, genes with one representative in the genome and homologs in other genomes; phage or integrase-related genes; and duplicated genes or paralogs with more than one paralog in the genome. The distribution of each of these categories differed in the surveyed genomes (Figure 5B). Paralog representation ranged from 5% for the Neorickettsia sennetsu strain Miyayama (genome size 0.86 Mb) to 44% for Rhizobium leguminosarum bv. vicae (strain 3841) (genome size 7.79 Mb), whereas A. caulinodans had 36% paralogs (genome size 5.37 Mb). The data confirmed the observation that the number of paralogs strongly correlates with the genome size in a linear regression [24].

Altogether, these analyses demonstrate that currently X. autotrophicus is the closest sequenced relative of A. caulinodans. However, a comparison of the genomes with the ARTEMIS comparison tool [25] revealed a very low degree of synteny (Additional file 3). Although short sequence stretches are conserved, extensive rearrangements have taken place. The occurrence of four prophages and numerous transposases in the A. caulinodans genome suggests a high genome plasticity. In A. caulinodans, 1412 proteins have no counterpart in X. autotrophicus of which 544 (38%) are catalogued as unknown or hypothetical (Additional file 4). In the remaining group of functionally classified proteins, 46% have GC and GC3 contents different from the genome averages, suggesting recent acquisition.

Functional protein classes and metabolic pathways

The putative protein-encoding genes were ordered into 17 classes [26] (Table 1) and the metabolic potential of A. caulinodans was analyzed with the PathoLogic tool of the BioCyC/MetaCYC suite [27].

These analyses revealed the presence of many regulatory genes (8%) and several RNA polymerase σ factors, among which two household σ70 factors (AZC_3643 and AZC_4253), two σ54 factors (AZC_2924 and AZC_3925; see below), and five σ factors of the extracytoplasmic subclass (AZC_0389, AZC_1202, AZC_2427, AZC_2453, and AZC_3238), implying responsiveness to many environmental triggers. As A. caulinodans is a motile bacterium, a large gene cluster is present (AZC_0615-AZC_0666) for the formation of a type-III flagellum. A significant number of chemotaxis genes predicts the capacity to respond to a wide array of molecules (Additional file 5). While no complete quorum sensing system could be detected, the presence of no less than five LuxR-type response regulators suggests that A. caulinodans has the potential to listen in on acyl-homoserine lactone-mediated communication in its surroundings.

A variety of encoded proteins might offer protection against toxic compounds in the environment (Additional file 6). Examples are two cytochrome P450 monooxygenases and pathways to degrade or modify plant-derived molecules, such as protocatechuate, and xenobiotics, such as cyanate, 1,4-dichlorobenzene, octane, and gallate. Several multidrug efflux pumps, antibiotic-modifying enzymes, and heavy metal translocation systems probably confer resistance to deleterious compounds. The production of the siderophores enterobactin and aerobactin might guarantee iron acquisition from the surroundings.

The surface of bacteria is important for recognition, attachment, and colonization during the interaction with a host. Exopolysaccharides and lipopolysaccharides are involved in nodulation as protective compounds against defense molecules generated by the plant and as communication signals [28–30]. Other functions could relate to surface structures, important for interaction with the host (Additional file 7), e.g. putative adhesion proteins, antigens, and 29 genes that code for proteins with GGDEF/EAL domains. The latter typically play a role in the transition from a motile planktonic form to a sessile biofilm by controlling the formation and degradation of the secondary messenger cyclic di-GMP [31]. Hormones also play an important role in plant-microbe interactions. Both a structural (AZC_0267) and a regulatory gene (AZC_0266) mediating degradation of the ethylene precursor 1-aminocyclopropane-1-carboxylate, are present in the genome.

Over 15% of the genes are dedicated to "transport and binding", of which more than 50% belong to the ATP-binding-cassette (ABC) transporter class. With 118 complete systems (consisting of a solute-binding protein, a permease, and an ABC component for the uptake systems, or an ABC component and a permease for the export systems), and numerous orphan subunits scattered over the genome, the transporter complement of A. caulinodans equals that of many other soil bacteria. These high-affinity transport systems are dedicated to the uptake of peptides, amino acids, sugars, polyamines, siderophores, nitrate/sulfonate/bicarbonate, or C4-dicarboxylate and many unknown substrates (Additional file 8). Accordingly, catabolic pathways are predicted for compounds, such as amino acids (including citrulline and ornithine), glucuronate, galactonate, galactarate, gluconate, quinate, L-idonate, creatinine, and 4-hydroxymandelate. Sugars, such as glucose, fructose, sucrose, ribose, xylose, xylulose, and lactose are not metabolized by A. caulinodans; instead, dicarboxylic acids are used as primary carbon source [10], as reflected by the presence of multiple C4-dicarboxylic acid transport systems. The occurrence of 16 putative alcohol dehydrogenase genes suggests that ethanol could be a major carbon source under flooded conditions. A. caulinodans is also capable of oxidizing hydrogen, an obligatory by-product of the nitrogenase, and the required hup, hyp, and hox A genes are located in a large gene cluster (AZC_0594-AZC_0613) [32]. Encoded energy metabolism pathways include glycolysis, Entner-Doudoroff, and TCA cycle. The absence of a gene encoding phosphofructokinase indicates the lack of a functional Emden-Meyerhof pathway.

Nitrogen fixation and related functions

Table 2 lists the genomic position of A. caulinodans genes related to free-living and symbiotic nitrogen fixation. These genes code for known functions, such as formation of the nitrogenase, assembly and stabilization of the complex, synthesis of the MoFe cofactor and the FeS clusters, electron transport, ammonium assimilation, and regulation of gene expression by nitrogen and oxygen, but also for proteins whose exact role await experimental confirmation. Several nif genes occur in more than one copy and are scattered over the genome as solitary loci or clusters of varying size with GC and GC3 contents matching the averages of the genome (Additional file 1). The NifH phylogeny was congruent with the phylogenetic relationships based on 16S rRNA [33] or on core protein families [Figure 5A]. The same holds true for the other genes listed in Table 2 (data not shown).

Table 2 Nitrogen fixation-related genes
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The transcriptional activator NifA (AZC_1049) acts together with a σ54 factor RpoN (AZC_3925) to control the nif/fix gene expression [34]. Nitrogen regulation of nifA expression is under control of the NtrBC (AZC_3086-AZC_3087) and NtrYX (AZC_3083-AZC_3084) two-component systems [35, 36] that respond to the intracellular and extracellular nitrogen status, respectively. The expression of these two loci depends on a hypothetical σ54 factor RpoF [34], which presumably corresponds to AZC_2924. Oxygen control of nif A expression is mediated by FixLJ (AZC_4654 and AZC_4655) [37], and the transcription factor FixK (AZC_4653) [38]. The nif A gene is further controlled at the transcriptional level by a LysR-type regulator [39] and at the translational level by the nrf A gene product (AZC_3080) [40]. FixK also activates transcription of the cyt NOQP operon (AZC_4523-AZC_4526), encoding the high-affinity terminal oxidase cytochrome cbb3 that is induced under microaerobiosis [41, 42]. Mutants in cyt NOQP still fix nitrogen under free-living conditions, suggesting the occurrence of another terminal oxidase [41, 43]. The survey of the genome excluded the presence of a second cytochrome cbb3 complex, but revealed two cytochrome bd complexes (AZC_1353-AZC_1354 and AZC_3759-AZC_3760).

A symbiosis region

A region of 87.6 kb, delimited by a Gly-tRNA (position 4346061) and an integrase (AZC_3882) and flanked by direct repeats (Figure 6), is characterized by an overall lower GC (Figure 3C) and GC3 contents (Figure 3D) than the genome averages, and a different preferential codon usage (Figure 4A). No less than 18 putative transposases and three integrases are present, suggesting a complex history of horizontal gene transfer events. The region contains the three nod loci that are involved in the synthesis and secretion of the lipochitooligosaccharide Nod factors (NFs) [44], but also genes related to chemotaxis, amino acid uptake, and a putative type-IV secretion system (Additional file 1).

Figure 6
figure 6

Schematic representation of the symbiosis region. Genes described in the text are indicated by arrows; the others are not individually represented, but their number is specified in the pentagons. The symbiotic region is flanked by tRNA-Gly (triangles) and interspersed by multiple transposases and integrases (blue lines). Genes in the nod A operon are nod ABCSUIJZnoe CHOP and genes in the trb operon are trb BCDEJLFGI.

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The three nod loci are not adjacent and have a GC content lower than that of the surrounding sequences (Figure 3C). The shifts in GC content correspond to the location of repeated elements that are flanked by insertion sequences or tRNAs (Figures 1 and 6). The constitutively expressed nod D gene (AZC_3792) [45, 46] codes for a LysR-type regulator that activates transcription of the two other flavonoid-inducible nod loci. The inducible operon nod ABCSUIJZnoe CHOP (AZC_3818-AZC_3807) [47–49] encodes most of the enzymatic machinery for NF backbone synthesis, decoration, and secretion. The biochemical role of these proteins has been extensively described, except for the last four open reading frames noe CHOP that are involved in NF arabinosylation and are still under study. Based on similarity with proteins involved in arabinosylation of the cell wall in Mycobacterium tuberculosis, noe C (AZC_3810), noe H (AZC_3809), and noe O (AZC_3808) might encode the synthesis of a D-arabinose precursor [50–52]. The third locus encodes the inducible nol K gene responsible for GDP-fucose synthesis for NF decoration (AZC_3850) [53, 54].

The symbiosis region also contains two conjugation-related gene clusters with GC and GC3 contents comparable to the genome averages. The cluster AZC_3844-AZC_3826 – flanked by two transposases – consists of repA and genes encoding conjugal transfer, partition, and plasmid stabilization proteins (Additional file 1). In the cluster AZC_3856–3877, flanked by a transposase and an integrase, genes are found that are homologous to the trb BCDEJLFGI genes, a type-IV secretion system involved in conjugative transfer of the tumor-inducing plasmid in Agrobacterium tumefaciens [55] (Figure 6).

The genome annotation indicates the presence of a few additional nodulation-related genes outside of the symbiosis region (Additional file 1). Two response regulators (AZC_1361 and AZC_2281) homologous to nod W genes of Bradyrhizobium japonicum and part of a two-component signal transduction system might be involved in the response to host-exuded flavonoids [56]. A nod T-related gene (AZC_3288) [57] might act as the outer-membrane component in NF secretion together with the inner-membrane NodIJ proteins. None of these four potential nodulation genes has a different GC or GC3 content, in contrast to the nod genes of the symbiosis region.

Discussion

Azorhizobium caulinodans is a member of the α-proteobacteria, a group with diverse genome architectures. Several plant-associated representatives, such as Agrobacterium and Sinorhizobium, have quite considerable genomes and large circular or linear plasmids. In contrast, A. caulinodans has a single circular chromosome of 5.37 Mb and no auxiliary replicons. The GC content and the coding density are in range with other rhizobia and soil bacteria. A. caulinodans is a motile, nitrogen-fixing, hydrogen-oxidizing, aerobic bacterium with a preference for organic acids as carbon source. This lifestyle is reflected in the metabolic pathways and in clusters for flagellum synthesis, motility, and chemotaxis. A high number of genes are dedicated to transport and regulation, indicating that a wide range of substrates can be taken up, but that the pathways are tightly regulated to limit the metabolic burden. Besides the well-described role of surface polysaccharides during plant-microbe interactions, the genome of A. caulinodans encodes functions that might be involved in biofilm formation, possibly facilitating the interaction with a host. Ongoing functional analysis will undoubtedly reveal new players in the ecology of the dual lifestyle of A. caulinodans [58, 59].

Genome analysis combined with phylogenetic studies has shed new light on bacterial evolution and taxonomy. Core functions can be identified that are highly conserved between related groups, but that may acquire individual characteristics through accessory genes [60]. Analysis of a family of core proteins and 16S rDNA sequence comparison revealed that the closest relative of A. caulinodans is Xanthobacter autotrophicus. Xanthobacter sp. are free-living nitrogen fixers and the nif and fix genes can thus be considered part of the core functions of the Azorhizobium-Xanthobacter group. The major difference in the lifestyle of both organisms is that A. caulinodans has acquired the ability to establish a symbiosis with S. rostrata.

The nodulation capacities are related to the presence of a symbiosis region with distinct GC and GC3 contents and codon usage. The association with tRNA loci, which presumably act as targets for the integration of foreign DNA, and multiple transposons suggest a high plasticity of this region, as reflected in its composition. The symbiosis region contains three subclusters related to nodulation, nod ABCSUIJZnoe CHOP, nod D, and nol K that are flanked by sequences suggestive of independent horizontal acquisition. The repeated elements could be the relics of insertion elements that once played a role in the evolution of the A. caulinodans nodulation genes that have all the characteristics of archetypal accessory genes.

To study the evolution of nod genes, A. caulinodans forms an interesting case. The azorhizobial nod genes are only distantly related to their counterparts in other rhizobia. Phylogenetic comparisons demonstrated that the nod A and nod C genes from rhizobia that nodulate temperate legumes (e.g. S. meliloti, R. leguminosarum bv. viciae and bv. trifolii, and R. galegae) are grouped together and the genes from rhizobia that nodulate tropical legumes (e.g. B. japonicum, B. elkanii, R. loti, R. tropici, and R. etli) form a second cluster [61, 62]. However, the nod A, nod B, and nod C genes of A. caulinodans belong neither to the tropical nor the temperate clusters [62, 63]. Also, the genetic distance between the nod SU genes of A. caulinodans and their counterparts in other rhizobia is much greater than the mutual genetic distance between the nod SU genes of these rhizobia [8]. The organization of the nod ABCSUIJ genes in A. caulinodans resembles the situation in B. japonicum, but the upstream and downstream regions are different [64, 65].

At present we do not know the origin of the symbiotic genes of A. caulinodans. The Rhizobiaceae, which have been historically considered a true family in phylogenetic terms, now seem a rather diverse group of bacteria, including Methylobacterium, Ralstonia, and Burkholderia that share variant, relatively recently acquired, symbiotic gene clusters. Possibly, the A. caulinodans nod genes are derived from unexplored rhizobia or even from obligate endophytes. Undoubtedly, the recent and ongoing explosion in meta-genomic projects will provide more insight into the origin of the nodulation functions.

Conclusion

Extension of symbiotic nitrogen fixation to non-legume cereal plants is a challenging long-standing goal. Especially, there is a growing interest in nitrogen-fixing organisms that could establish an endophytic, beneficial relation with important crops, such as rice and wheat (Triticum aestivum). Interestingly in this context, the occurrence of A. caulinodans has been reported in intercellular infection pockets located in the cortex of roots of Arabidopsis thaliana and wheat [66]. In fields where S. rostrata and rice are grown as rotation crops, A. caulinodans seems to survive very well in the rhizosphere of the rice plants and in the soil [67]. Moreover, the bacterium invades emerging lateral roots of rice, and rice seedlings inoculated with A. caulinodans have a high nitrogenase activity [68]. Finally, A. caulinodans fixes nitrogen under relatively high oxygen tension as a free-living organism, invades the host via cracks, and establishes intercellular colonies. Altogether, these features might be advantageous for primary infection of nitrogen-starved root systems and highlight the potential of A. caulinodans as a candidate model organism. The genome sequence data provide new opportunities for exploring the regulatory aspects of Azorhizobium nitrogen fixation and the essential features that implement the ability for endosymbiosis.

Methods

DNA sequencing

The nucleotide sequence of the entire genome of A. caulinodans ORS571 was determined by the whole-genome shotgun strategy method. For shotgun cloning, DNA fragments of 2 to 3 kb were cloned into the Hin cII site of pUC118. For gap closing, the pCC1Fos vector (Epicentre, Madison, WI, USA) was used, and approximately 35-kb clones were prepared. The accumulated sequence files were assembled with the Phrap program [69]. A total of 71,424 random sequence files corresponding to approximately 7.7 genome equivalents were assembled to generate draft sequences. Finishing was carried out by visual editing of the sequences, followed by gap closing, and additional sequencing to obtain sequence data with a Phred score of 20 or higher [70, 71]. The integrity of the final genome sequence was assessed by comparing the insert length of each fosmid clone with the computed distance between the end sequences of the clones. The end sequence data facilitated gap closure as well as accurate reconstruction of the entire genome. The final gaps in the sequences were filled by the primer walking method. A lower threshold of acceptability for the generation of consensus sequences was set at a Phred score of 20 for each base. The nucleotide sequence is available in the DDBJ/EMBL/GenBank databases under the accession number AP009384.

Structural and functional annotation

Coding regions were assigned by a combination of computer prediction and similarity search. Briefly, the protein-coding regions were predicted with the Glimmer 2.02 program [72] and all regions equal to or longer than 90 bp were translated into amino acid sequences that were subjected to similarity searches against the nonredundant protein database at NCBI with the BLASTP program [73]. In parallel, the entire genomic sequence was compared with those in the nonredundant protein database with the BLASTX program [73] to identify genes that had escaped prediction and/or were smaller than 90 bp, especially in the predicted intergenic regions. For predicted genes without sequence similarity to known genes, only those equal to or longer than 150 bp were considered as candidates. Functions were assigned to the predicted genes based on sequence similarity of their deduced products to that of genes of known function. For genes that encode proteins of 100 amino acid residues or more, an E-value of 10-20 was considered significant, whereas a higher E-value was significant for genes encoding smaller proteins (E-value treshold of 10-10). Genes for structural RNAs were assigned by similarity search against the in-house structural RNA database that had been generated based on the GenBank data. tRNA-encoding regions were predicted by the tRNAscan-SE 1.21 program [21] in combination with the similarity search.

MetaCyc analysis [27, 74] detected 229 metabolic pathways, containing 1037 reaction steps. To assess the presence or absence of a metabolic pathway and to decrease the likelihood of being misled by the many enzymes that are shared among multiple pathways, the analysis was emphasized on the presence of enzymes that are unique to a pathway.

Construction of a phylogenetic tree

The Maximum-likelihood tree was based on 108 core proteins of 45 α-proteobacteria [23] whose sequence data and annotation files were available and downloaded from the NCBI Microbial Genome Resource database [75]. The set of core genes was determined by an all-against-all BLAST at protein level. Best reciprocal hits were selected, taking into account a cut-off value defined as 20% similarity and an overlap of at least 150 amino acids. Only proteins present in all 45 genomes as single copy were considered as "core proteins" and used to construct the phylogenetic tree. The total alignment contained 32,327 amino acids. The tree was constructed with the Phyml program [76] and a WAG substitution model [77] and 100 bootstrap replicates were run. Unless indicated otherwise, bootstraps are 100 (Figure 5A).