The rhizobium–legume symbiosis has been widely studied as the model of mutualistic evolution and the essential component of sustainable agriculture. Extensive genetic and recent genomic studies have led to the hypothesis that many distinct strategies, regardless of rhizobial phylogeny, contributed to the varied rhizobium–legume symbiosis. We sequenced 26 genomes ofSinorhizobiumandBradyrhizobiumnodulating soybean to test this hypothesis. TheBradyrhizobiumcore genome is disproportionally enriched in lipid and secondary metabolism, whereas several gene clusters known to be involved in osmoprotection and adaptation to alkaline pH are specific to theSinorhizobiumcore genome. These features are consistent with biogeographic patterns of these bacteria. Surprisingly, no genes are specifically shared by these soybean microsymbionts compared with other legume microsymbionts. On the other hand, phyletic patterns of 561 known symbiosis genes of rhizobia reflected the species phylogeny of these soybean microsymbionts and other rhizobia. Similar analyses with 887 known functional genes or the whole pan genome of rhizobia revealed that only the phyletic distribution of functional genes was consistent with the species tree of rhizobia. Further evolutionary genetics revealed that recombination dominated the evolution of core genome. Taken together, our results suggested that faithfully vertical genes were rare compared with those with history of recombination including lateral gene transfer, although rhizobial adaptations to symbiotic interactions and other environmental conditions extensively recruited lineage-specific shell genes under direct or indirect control through the speciation process.

Herein the members of the Subcommittee on Taxonomy of Rhizobia and Agrobacteria of the International Committee on Systematics of Prokaryotes review recent developments in rhizobial and agrobacterial taxonomy and propose updated minimal standards for the description of new species (and genera) in these groups. The essential requirements (minimal standards) for description of a new species are (1) a genome sequence of at least the proposed type strain and (2) evidence for differentiation from other species based on genome sequence comparisons. It is also recommended that (3) genetic variation within the species is documented with sequence data from several clearly different strains and (4) phenotypic features are described, and their variation documented with data from a relevant set of representative strains. Furthermore, it is encouraged that information is provided on (5) nodulation or pathogenicity phenotypes, as appropriate, with relevant gene sequences. These guidelines supplement the current rules of general bacterial taxonomy, which require (6) a name that conforms to the International Code of Nomenclature of Prokaryotes, (7) validation of the name by publication either directly in the International Journal of Systematic and Evolutionary Microbiology or in a validation list when published elsewhere, and (8) deposition of the type strain in two international culture collections in separate countries.

As the putative center of origin for soybean and the second largest region of soybean production in China, the North China Plain covers temperate and subtropical regions with diverse soil characteristics. However, the soybean rhizobia in this plain have not been sufficiently studied. To investigate the biodiversity and biogeography of soybean rhizobia in this plain, a total of 309 isolates of symbiotic bacteria from the soybean nodules collected from 16 sampling sites were studied by molecular characterization. These isolates were classified into 10 genospecies belonging to the genera Sinorhizobium and Bradyrhizobium , including four novel groups, with S. fredii (68.28%) as the dominant group. The phylogeny of symbiotic genes nodC and nifH defined four lineages among the isolates associated with Sinorhizobium fredii , Bradyrhizobium elkanii , B. japonicum , and B. yuanmingense , demonstrating the different origins of symbiotic genes and their coevolution with the chromosome. The possible lateral transfer of symbiotic genes was detected in several cases. The association between soil factors (available N, P, and K and pH) and the distribution of genospecies suggest clear biogeographic patterns: Sinorhizobium spp. were superdominant in sampling sites with alkaline-saline soils, while Bradyrhizobium spp. were more abundant in neutral soils. This study clarified the biodiversity and biogeography of soybean rhizobia in the North China Plain.

We provide here a comparative genome analysis of 31 strains within the genus Paenibacillus including 11 new genomic sequences of N₂-fixing strains. The heterogeneity of the 31 genomes (15 N₂-fixing and 16 non-N₂-fixing Paenibacillus strains) was reflected in the large size of the shell genome, which makes up approximately 65.2% of the genes in pan genome. Large numbers of transposable elements might be related to the heterogeneity. We discovered that a minimal and compact nif cluster comprising nine genes nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV encoding Mo-nitrogenase is conserved in the 15 N₂-fixing strains. The nif cluster is under control of a σ⁷⁰-depedent promoter and possesses a GlnR/TnrA-binding site in the promoter. Suf system encoding [Fe–S] cluster is highly conserved in N₂-fixing and non-N₂-fixing strains. Furthermore, we demonstrate that the nif cluster enabled Escherichia coli JM109 to fix nitrogen. Phylogeny of the concatenated NifHDK sequences indicates that Paenibacillus and Frankia are sister groups. Phylogeny of the concatenated 275 single-copy core genes suggests that the ancestral Paenibacillus did not fix nitrogen. The N₂-fixing Paenibacillus strains were generated by acquiring the nif cluster via horizontal gene transfer (HGT) from a source related to Frankia. During the history of evolution, the nif cluster was lost, producing some non-N₂-fixing strains, and vnf encoding V-nitrogenase or anf encoding Fe-nitrogenase was acquired, causing further diversification of some strains. In addition, some N₂-fixing strains have additional nif and nif-like genes which may result from gene duplications. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes. This study not only reveals the organization and distribution of nitrogen fixation genes in Paenibacillus, but also provides insight into the complex evolutionary history of nitrogen fixation. We sequenced the genomes of 11 N₂-fixing Paenibacillus strains and demonstrated the genomic diversity of the genus Paenibacillus by comparing these strains to each other and to 20 other strains (4 N₂-fixing and 16 non-N₂-fixing strains) that were sequenced previously. Phylogenetic analysis of the concatenated 275 single-copy core genes suggests that ancestral Paenibacillus did not fix nitrogen and the N₂-fixing strains fall into two sub-groups, which were likely originated from a N₂-fixing common ancestor. A minimal and compact nif cluster comprising nine nif genes encoding Mo-nitrogenase is highly conserved in the 15 N₂-fixing strains. Variations in the nif cluster and in the chromosomal regions surrounding the nif cluster between two sub-groups imply at least two independent acquisitions with insertion of distinct nif cluster variants in different genomic sites of Paenibacillus in early evolutionary history. Phylogeny of the concatenated NifHDK sequences suggests that Paenibacillus and Frankia are sister groups. The nif cluster, a functional unit for nitrogen fixation, was lost, producing some non-N₂-fixing strains. There were recent events of acquisition of vnf and anf genes, causing further diversification of some strains. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes.

Bacteria currently included in Rhizobium leguminosarum are too diverse to be considered a single species, so we can refer to this as a species complex (the Rlc). We have found 429 publicly available genome sequences that fall within the Rlc and these show that the Rlc is a distinct entity, well separated from other species in the genus. Its sister taxon is R. anhuiense. We constructed a phylogeny based on concatenated sequences of 120 universal (core) genes, and calculated pairwise average nucleotide identity (ANI) between all genomes. From these analyses, we concluded that the Rlc includes 18 distinct genospecies, plus 7 unique strains that are not placed in these genospecies. Each genospecies is separated by a distinct gap in ANI values, usually at approximately 96% ANI, implying that it is a ‘natural’ unit. Five of the genospecies include the type strains of named species: R. laguerreae, R. sophorae, R. ruizarguesonis, “R. indicum” and R. leguminosarum itself. The 16S ribosomal RNA sequence is remarkably diverse within the Rlc, but does not distinguish the genospecies. Partial sequences of housekeeping genes, which have frequently been used to characterize isolate collections, can mostly be assigned unambiguously to a genospecies, but alleles within a genospecies do not always form a clade, so single genes are not a reliable guide to the true phylogeny of the strains. We conclude that access to a large number of genome sequences is a powerful tool for characterizing the diversity of bacteria, and that taxonomic conclusions should be based on all available genome sequences, not just those of type strains.

Six strains were isolated from root nodules of Vicia faba grown in Nanchang, Yifeng, Taihu, Huaibei, Bengbu and Lujiang, in the middle and lower reaches of the Yangtze River. According to phylogenetic analyses of 16S rRNA gene, atpD and recA sequences, these strains belong to the genus Rhizobium, with Rhizobium etli and Rhizobium leguminosarum as the closest related species. CCBAU 33202^T, a representative of these novel isolates, showed sequence similarity to its closest relatives R. etli CFN 42^T and R. leguminosarum USDA 2370^T of 99.5 and 99.1 % for the 16S rRNA gene, 91.9 and 91.9 % for atpD and 90.3 and 93.2 % for recA. The strains from this study could also be differentiated from R. etli CFN 42^T and R. leguminosarum USDA 2370^T by 16S–IGS RFLP and SDS-PAGE of whole-cell proteins, fatty acid profiles and several phenotypic characteristics. DNA–DNA hybridization yielded relatedness of 19 and 14–43 %, respectively, with R. etli CFN 42^T and strains representing different biovars of R. leguminosarum. All data obtained in this study showed that these V. faba isolates belong to a novel species, for which the name Rhizobium fabae sp. nov. is proposed. The type strain, CCBAU 33202^T (=LMG 23997^T =JCM 14381^T), was isolated from Nanchang.