We isolated and characterized nodA genes from photosynthetic and non-photosynthetic rhizobia nodulating the legume genus Aeschynomene, and found that the nodA sequence from photosynthetic stem-nodulating bacteria was phylogenetically distant from the other already described nodA genes. Characterization of the photosynthetic strain ORS285 common nod gene cluster (nodABC) showed, upstream of nodA, the presence of a new insertion sequence element belonging to the IS3 family and specific to a group of photosynthetic strains from Aeschynomene.
Rhizobia form nitrogen-fixing nodules on the roots of their leguminous host plants. Some species of the genus Aeschynomene have the unusual capacity to develop stem nodules, a property shared only with a very few species of the genera Sesbania, Neptunia and Discolobium. A number of stem isolates from Aeschynomene are of special interest because of their ability to develop a photosynthetic system , which is a rare trait in rhizobia. Three cross-inoculation groups among Aeschynomene species have been described . Group I never forms stem nodules and is nodulated by the typical, broad host range, non-photosynthetic Bradyrhizobium strains. The two other groups, II and III, develop stem nodulation. Group III is exclusively nodulated by photosynthetic rhizobia, which are highly specific to that group . Group II is intermediary as it is nodulated by both non-photosynthetic Bradyrhizobium, and with photosynthetic strains which are also able to nodulate Group III .
In the rhizobium–legume interaction, specificity is mainly controlled by Nod factors recognized by the host plant. All Nod factors are chitin oligomers mono N-acylated at the non-reducing end and diversely substituted at both ends of the molecules. These various substitutions, which confer plant-specificity, are encoded by host-specific nod genes. The synthesis of the N-acylated oligosaccharide core of the Nod factors is controlled by the nodABC genes, which are present in all rhizobia. The nodA gene, involved in the transfer of an acyl chain to the chitin oligosaccharide backbone Nod factor, has been shown to be a good nodulation marker .
In this study, we isolated and characterized the nodA genes of different photosynthetic and non-photosynthetic Aeschynomene rhizobia, and phylogenetically compared their sequence divergence. We found that the nodA gene sequence from photosynthetic bacteria was distant from that of the already described rhizobia and, therefore, we sequenced a 6.5-kb fragment containing the common nodulation gene nodABC. Here, we report the presence of a new insertion sequence element (IS) upstream of the nodA gene specific to Group II photosynthetic strains.
2Materials and methods
All the strains used are reported in Table 1. The strains were cultured on YEM medium  at 37°C. All strains were stored at −80°C on YEM medium adjusted to 20% (v/v) glycerol.
Table 1. Characteristics of the strains used in this work
2.2Polymerase chain reaction (PCR) amplification, DNA sequencing and sequence analysis
The nodA genes from Group I and II non-photosynthetic Bradyrhizobium strains were amplified using the degenerated primers nodAf.brad (5’-GTYCAGTGGAGSSTKCGCTGGG-3’)/nodAr.brad (5’-TCACARCTCKGGCCCGTTCCG-3’) whereas the nodA genes from the photosynthetic strains were amplified using the degenerated primers nodA1f (5’-TGCRGTGGAARNTRBVYTGGGAAA-3’)/nodAb1r (5'GGNCCGTCRTCRAASGTCARGTA-3’). These four degenerate primers were defined from conserved motifs of NodA sequences available from the databanks. The primers Isf (5’-AGCCCAGCGACCTATTTTAG-3’) and Isr (5’- GCCTCGATGAAAGCATTGTC-3’) which amplified a 822-bp fragment inside the IS identified in ORS285 were used to check for the presence of this IS element in the different Aeschynomene symbionts.
A touchdown PCR was performed for primer pairs nodAf.brad/nodAr.brad and nodA1f/nodAb1r (annealing temperature 60 to 50°C in 20 cycles) as previously described . For the primer pair Isf/Isr (annealing temperature of 55°C), the standard PCR method was used. PCR products were purified with a QIAquick Gel extraction Kit (Qiagen, France). The ABI Prism BigDye Terminator Cycle sequence Kit (Applied Biosystems, Foster City, CA, USA) was used to directly sequence the purified PCR with the primers used for the amplification. Sequencing reactions were analyzed on an Applied Biosystem model 310 DNA sequencer. An alignment was performed, using the program PILEUP , with a set of available NodA sequences. A phylogenetic tree was constructed by the neighbor-joining method , and a bootstrap confidence analysis was performed on 1000 replicates to determine the reliability of the tree topology so obtained .
2.3Construction and screening of a genomic library of Bradyrhizobium ORS285
Total DNA of Bradyrhizobium strain ORS285 was subjected to a partial digestion with Sau3AI and dephosphorylated by alkaline phosphatase treatment. Fragments were then ligated to SuperCos I XbaI/BamHI arms (Stratagene, La Jolla, CA, USA) as instructed by the manufacturer. Ligated DNA was packaged by using the Gigapack III Gold packaging extract (Stratagene). Screening was performed by PCR using the primers nodA.sp.285.f (5’-ACGCGCTCCCGTTCATGTCG-3’)/nodA.sp.285r (5’-GAAATACAAGCACCAACGGC-3’) which were designed from the nodA sequence of ORS285.
2.4Nucleotide sequence accession number
The DNA sequences of the common nod genes of ORS285 strain have been submitted to the GenBank database under accession number AF284858.
3Results and discussion
The nodA genes of both photosynthetic and non-photosynthetic Aeschynomene nodulating rhizobia were first isolated by PCR. By using the degenerated primers nodAf.brad/nodAr.brad, products of the expected size were obtained from all tested non-photosynthetic Bradyrhizobium strains isolated from either Group I (e.g. ORS301, ORS302, ORS304, ORS309) or Group II (e.g. ORS336). In contrast, all photosynthetic strains nodulating Aeschynomene Group II (e.g. ORS285, ORS287, ORS364) gave a positive amplification signal when using the degenerate primers nodA1f/nodAb1r. In spite of testing numerous primers defined from the consensus region of either NodA, NodB or NodC, we did not succeed in isolating the common nod genes from Group III photosynthetic strains. These results indicate a great divergence of the symbiotic genes as compared with the other rhizobia, which could be related to the extreme narrow host-specificity of the Group III photosynthetic strains.
Sequence analysis revealed that the nodA gene sequences from Groups I and II non-photosynthetic Bradyrhizobium-tested strains are polymorphs with a percentage of similarity ranging from 73.78 to 96.62% at the DNA level. In contrast, the nodA gene sequences of the Group II photosynthetic strains were found to be 100% identical, demonstrating that this symbiotic gene is very well conserved between these photosynthetic Bradyrhizobium. Phylogenetic NodA sequence analysis between the different rhizobia revealed that the nodA gene from the Group II photosynthetic strains constituted a new branch, related but distinct from the other Bradyrhizobium nodA gene (Fig. 1). Sequence similarity of NodA from those photosynthetic strains with other NodA Bradyrhizobium species was low (63%). These data are in full agreement with previous phylogenetic studies, which showed that photosynthetic Aeschynomene strains form a homogeneous and distinct cluster , for which a new species should be proposed. Conversely, NodA sequences from the non-photosynthetic Group I and II strains intertwined with the other species of Bradyrhizobium (Fig. 1), and the sequence similarity for this genus was high, ranging from 80 to 88%, thus confirming their taxonomic position within the Bradyrhizobium japonicum and Bradyrhizobium elkanii branches .
The high level of divergence of the NodA sequence of photosynthetic strains led us to characterize their complete common nodABC genes. Two specific primers nodA.sp.285.f/nodA.sp.285r based on nodA gene sequence were designed for the PCR screening of a DNA genomic library for strain ORS285 used as a model for Group II photosynthetic Bradyrhizobium. A positive clone (pSTM75) which contained an insert of approximately 35 kb was used to characterize the common nodulation genes.
A 6.5-kb region in the inserted DNA fragment of the pSTM75 cosmid, showing a positive hybridization signal to the nodA gene used as probe, was sequenced and analyzed, as shown in Fig. 2. This nucleotide sequence had four open reading frames (ORFs) encoding proteins with similarity to known Nod proteins (Fig. 2). Based on sequence similarities, three of these ORFs located from position 3992 to 6407 were assigned to nodA, nodB and nodC genes which presented the same classical organization as already found for other rhizobia. The nodB sequence showed 62 to 61% homology with the other Bradyrhizobium nodB genes available in databanks, and the nodC sequence, 63% homology with that of Rhizobium sp. strain N33 and 53% homology with the nodC gene of Bradyrhizobium sp. strain SNU001. The fourth ORF encoding a putative Nod protein was located from position 1746 to 617 and showed 55% amino acid homology with the nolL gene from Rhizobium etli (Fig. 2). The nolL gene has been shown to be responsible for the acetylation of the fucosyl residue in the nodulation factor , which indicates that Nod factors of photosynthetic strains are fucosylated, as already found for all Bradyrhizobium studied so far [11–12]. Indeed, we have detected by PCR in strain ORS285 (data not shown) a sequence homologous to the nodZ gene which encodes a fucosyl transferase.
Interestingly, we did not find a nodD regulatory gene upstream of nodA, as usually described for most rhizobia. Instead, we found in the 2-kb region located between nodA and nolL, an unexpected putative insertion element showing homology to IS sequences belonging to the IS3 family . The analysis of this IS sequence revealed the presence of two ORFs, ORFA and ORFB (Fig. 2). The ORFA, starting with an ATG, encoded a putative protein of 88 amino acids and showed a strong homology to a putative transposase identified in Yersinia pestis (59% of identity), the ORFA of ISD, an insertion element found in Desulfovibrio vulgaris (50% of identity) , the ORFA of IS1222 from Enterobacter agglomerans (48% of identity) , and also other members of the IS3 family. The ORFB, for which the proposed start codon overlapped the stop codon of ORFA, encoded a putative protein of 244 amino acids and also showed homology to the same ISs of the IS3 family. The greatest homologies found were 41% identity with the Yersinia pestis putative transposase, and 38 and 32% identity with the ORFBs of IS1222  and ISD1, respectively.
The distribution of this IS element among Aeschynomene rhizobia was investigated by PCR. All of the photosynthetic strains from Group II, and only those, gave an amplified fragment of the expected size, indicating that this IS element is specific to this group of photosynthetic Bradyrhizobium. IS elements have been identified as mobile DNA elements in the genome of a wide range of bacterial genera and species . They have been postulated to play an important role in the evolution and adaptation of bacteria. They have also been identified inside or flanking the symbiotic gene [16–18] where they could play an important role in the generation of nodulation polymorphism and consequently in the evolution of different symbiotic phenotypes . Therefore, we can hypothesize that the transposition events within nodulation genes could have played a role, either by gene rearrangement or gene transfer, in the adaptation of photosynthetic bacteria to Aeschynomene stem-nodulation.
This work was supported in part by CNRS (Dynamique de la Biodiversité et Environnement). C.C. is indebted to IRD for a doctoral grant.