To achieve effective symbiosis with legume, rhizobia should fine-tune their background regulation network in addition to activating key genes involved in nodulation (nod) and nitrogen fixation (nif). Here, we report that an ancestral zinc finger regulator, MucR1, other than its paralog, MucR2, carrying a frameshift mutation, is essential for supporting nitrogen fixation of Sinorhizobium fredii CCBAU45436 within soybean nodules. In contrast to the chromosomal mucR1, mucR2 is located on symbiosis plasmid, indicating its horizontal transfer potential. A MucR2 homolog lacking the frameshift mutation, such as the one from S. fredii NGR234, can complement phenotypic defects of the mucR1 mutant of CCBAU45436. RNA-seq analysis revealed that the MucR1 regulon of CCBAU45436 within nodules exhibits significant difference compared with that of free-living cells. MucR1 is required for active expression of transporters for phosphate, zinc, and elements essential for nitrogenase activity (iron, molybdenum, and sulfur) in nodules but is dispensable for transcription of key genes (nif/fix) involved in nitrogen fixation. Further reverse genetics suggests that S. fredii uses high-affinity transporters to meet the demand for zinc and phosphate within nodules. These findings, together with the horizontal transfer potential of the mucR homolog, imply an intriguing evolutionary role of this ancestral regulator in supporting nitrogen fixation.

In order to investigate the genetic differentiation of Sinorhizobium strains nodulating Glycine max and related microevolutionary mechanisms, three housekeeping genes (SMc00019, truA, and thrA) and 16 symbiosis-related genes on the chromosome (7 genes), pSymA (6 genes), and pSymB (3 genes) were analyzed. Five distinct species were identified among the test strains by calculating the average nucleotide identity (ANI) of SMc00019-truA-thrA: Sinorhizobium fredii, Sinorhizobium sojae, Sinorhizobium sp. I, Sinorhizobium sp. II, and Sinorhizobium sp. III. These species assignments were also supported by population genetics and phylogenetic analyses of housekeeping genes and symbiosis-related genes on the chromosome and pSymB. Different levels of genetic differentiation were observed among these species or different replicons. S. sojae was the most divergent from the other test species and was characterized by its low intraspecies diversity and limited geographic distribution. Intergenic recombination dominated the evolution of 19 genes from different replicons. Intraspecies recombination happened frequently in housekeeping genes and symbiosis-related genes on the chromosome and pSymB, whereas pSymA genes showed a clear pattern of lateral-transfer events between different species. Moreover, pSymA genes were characterized by a lower level of polymorphism and recombination than those on the chromosome and pSymB. Taken together, genes from different replicons of rhizobia might be involved in the establishment of symbiosis with legumes, but these symbiosis-related genes might have evolved differently according to their corresponding replicons.

Receiving nodulation and nitrogen fixation genes does not guarantee rhizobia an effective symbiosis with legumes. Here, variations in gene content were determined for three Sinorhizobium species showing contrasting symbiotic efficiency on soybeans. A nitrate-reduction gene cluster absent in S. sojae was found to be essential for symbiotic adaptations of S. fredii and S. sp. III. In S. fredii, the deletion mutation of the nap (nitrate reductase), instead of nir (nitrite reductase) and nor (nitric oxide reductase), led to defects in nitrogen-fixation (Fix ). By contrast, none of these core nitrate-reduction genes were required for the symbiosis of S. sp. III. However, within the same gene cluster, the deletion of hemN1 (encoding oxygen-independent coproporphyrinogen III oxidase) in both S. fredii and S. sp. III led to the formation of nitrogen-fixing (Fix ) but ineffective (Eff ) nodules. These Fix /Eff nodules were characterized by significantly lower enzyme activity of glutamine synthetase indicating rhizobial modulation of nitrogen-assimilation by plants. A distant homologue of HemN1 from S. sojae can complement this defect in S. fredii and S. sp. III, but exhibited a more pleotropic role in symbiosis establishment. These findings highlighted the lineage-dependent optimization of symbiotic functions in different rhizobial species associated with the same host.

The genus Bradyrhizobium has been considered to be a taxonomically difficult group. In this study, phylogenetics and evolutionary genetics analyses were used to investigate divergence levels among Bradyrhizobium strains nodulating soybeans in China. Eleven genospecies were identified by sequence analysis of three phylogenetic and taxonomic markers (SMc00019, thrA, and truA). This was also supported by analyses of eight genes outside the symbiosis island ("off-island" genes; SMc00019, thrA, truA, fabB, glyA, phyR, exoN, and hsfA). However, seven genes inside the symbiosis island ("island" genes; nifA, nifH, nodC, nodV, fixA, trpD, and rhcC2) showed contrasting lower levels of nucleotide diversity and recombination rates than did off-island genes. Island genes had significantly incongruent gene phylogenies compared to the species tree. Four phylogenetic clusters were observed in island genes, and the epidemic cluster IV (harbored by Bradyrhizobium japonicum, Bradyrhizobium diazoefficiens, Bradyrhizobium huanghuaihaiense, Bradyrhizobium liaoningense, Bradyrhizobium daqingense, Bradyrhizobium sp. I, Bradyrhizobium sp. III, and Bradyrhizobium sp. IV) was not found in Bradyrhizobium yuanmingense, Bradyrhizobium sp. II, or Bradyrhizobium elkanii. The gene flow level of island genes among genospecies is discussed in the context of the divergence level of off-island genes. Soybeans (Glycine max L.) were first domesticated in China and then introduced into different parts of the planet (1), now with an annual harvest area of 100 million hectares around the world (FAO, 2011). Ninety percent of their production comes from the United States, Brazil, Argentina, China, and India (FAO, 2007(FAO, to 2011. One of the key features of soybean is its ability to form symbiotic nitrogen-fixing nodules with diverse rhizobial species (2, 3), implying its important role in sustainable agriculture. It has been recurrently reported that Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense, and Sinorhizobium fredii could nodulate soybeans (2-5). Recently, Bradyrhizobium huanghuaihaiense, Bradyrhizobium daqingense, Sinorhizobium sojae, and several unnamed species were also found to be effective microsymbionts of soybeans (2, 3, 6-8). Strain USDA110 represents a widely distributed type formerly known as B. japonicum Ia, but it has recently been proposed as a member of the new species Bradyrhizobium diazoefficiens (9).Recent studies not only suggested differences in the biogeographic distribution of rhizobial species nodulating soybeans but also demonstrated a biased selection of rhizobial species by different genotypes of soybeans (2,4,10,11). Consistent with these findings, comparative genomics of rhizobia revealed that the phyletic distribution of rhizobial functional genes involved in environmental adaptations and symbiotic interactions generally agrees with the phylogeny of rhizobial species (7). Therefore, it is important to distinguish different rhizobial species and even subdivis...

Aim Most culture‐independent studies of bacterial biogeography have been at genus or higher taxonomic levels, although many important processes mediated by bacteria are at the strain or species level, such as the competitive nodulation of rhizobia on legumes. Here, at the intra‐species level, we characterized the structural variation in rhizobial populations in soybean rhizosphere under field conditions across 32 sampling sites in three eco‐regions. Location North‐east (Heilongjiang), north (Hebei–Shandong) and south (Jiangxi) China. Methods The intra‐species diversity of rhizobia in soybean rhizospheres was investigated by rpoB pyrosequencing. Soybean nodule isolates were characterized using repetitive extragenic palindromic PCR, restriction fragment length polymorphism of 16S‐23S intergenic sequences and rpoB PCR sequencing. Patterns and processes of biogeography of identified rhizobia were studied. Results Significant biogeographical patterns of rhizosphere rhizobia at the species level were found, and the abundant species in the rhizosphere were generally the dominant microsymbionts in soybean nodules. The distribution of rhizobial species in the rhizosphere across eco‐regions correlated with combined effects of spatial, vegetative, edaphic and climatic variables. Gene flow and genetic differentiation analyses revealed that there was no dispersal limitation among geographical populations of the same rhizobial species, and drift or subsequent diversification might be involved in shaping the biogeographical pattern in addition to environmental factors. Main conclusions These results constitute the first report of the existence of rhizobial biogeography in legume rhizospheres at the species level, contrasting with most studies, which focused on nodule isolates. The method developed in this study could be used to uncover, at high resolution, the diversity of local rhizobial populations in soils. This information is crucial for choosing suitable strains or species as inoculants for a specific legume host under local field conditions. Moreover, a similar strategy could be used to study other bacteria with important ecological functions.