Phylogenomic analyses and reclassification of the Mesorhizobium complex: proposal for 9 novel genera and reclassification of 15 species.

BACKGROUD: The genus Mesorhizobium is shown by phylogenomics to be paraphyletic and forms part of a complex that includes the genera Aminobacter, Aquamicrobium, Pseudaminobacter and Tianweitania. The relationships for type strains belong to these genera need to be carefully re-evaluated. RESULTS: The relationships of Mesorhizobium complex are evaluated based on phylogenomic analyses and overall genome relatedness indices (OGRIs) of 61 type strains. According to the maximum likelihood phylogenetic tree based on concatenated sequences of 539 core proteins and the tree constructed using the bac120 bacterial marker set from Genome Taxonomy Database, 65 type strains were grouped into 9 clusters. Moreover, 10 subclusters were identified based on the OGRIs including average nucleotide identity (ANI), average amino acid identity (AAI) and core-proteome average amino acid identity (cAAI), with AAI and cAAI showing a clear intra- and inter-(sub)cluster gaps of 77.40-80.91% and 83.98-86.16%, respectively. Combined with the phylogenetic trees and OGRIs, the type strains were reclassified into 15 genera. This list includes five defined genera Mesorhizobium, Aquamicrobium, Pseudaminobacter, Aminobacterand Tianweitania, among which 40/41 Mesorhizobium species and one Aminobacter species are canonical legume microsymbionts. The other nine (sub)clusters are classified as novel genera. Cluster III, comprising symbiotic M. alhagi and M. camelthorni, is classified as Allomesorhizobium gen. nov. Cluster VI harbored a single symbiotic species M. albiziae and is classified as Neomesorhizobium gen. nov. The remaining seven non-symbiotic members were proposed as: Neoaquamicrobium gen. nov., Manganibacter gen. nov., Ollibium gen. nov., Terribium gen. nov., Kumtagia gen. nov., Borborobacter gen. nov., Aerobium gen. nov.. Furthermore, the genus Corticibacterium is restored and two species in Subcluster IX-1 are reclassified as the member of this genus. CONCLUSION: The Mesorhizobium complex are classified into 15 genera based on phylogenomic analyses and OGRIs of 65 type strains. This study resolved previously non-monophyletic genera in the Mesorhizobium complex.

Dynamic Transformation of Nano-MoS2 in a Soil-Plant System Empowers Its Multifunctionality on Soybean Growth.

Molybdenum disulfide (nano-MoS2) nanomaterials have shown great potential for biomedical and catalytic applications due to their unique enzyme-mimicking properties. However, their potential agricultural applications have been largely unexplored. A key factor prior to the application of nano-MoS2 in agriculture is understanding its behavior in a complex soil-plant system, particularly in terms of its transformation. Here, we investigate the distribution and transformation of two types of nano-MoS2 (MoS2 nanoparticles and MoS2 nanosheets) in a soil-soybean system through a combination of synchrotron radiation-based X-ray absorption near-edge spectroscopy (XANES) and single-particle inductively coupled plasma mass spectrometry (SP-ICP-MS). We found that MoS2 nanoparticles (NPs) transform dynamically in soil and plant tissues, releasing molybdenum (Mo) and sulfur (S) that can be incorporated gradually into the key enzymes involved in nitrogen metabolism and the antioxidant system, while the rest remain intact and act as nanozymes. Notably, there is 247.9 mg/kg of organic Mo in the nodule, while there is only 49.9 mg/kg of MoS2 NPs. This study demonstrates that it is the transformation that leads to the multifunctionality of MoS2, which can improve the biological nitrogen fixation (BNF) and growth. Therefore, MoS2 NPs enable a 30% increase in yield compared to the traditional molybdenum fertilizer (Na2MoO4). Excessive transformation of MoS2 nanosheets (NS) leads to the overaccumulation of Mo and sulfate in the plant, which damages the nodule function and yield. The study highlights the importance of understanding the transformation of nanomaterials for agricultural applications in future studies.

The convergent xenogeneic silencer MucR predisposes α-proteobacteria to integrate AT-rich symbiosis genes.

Bacterial adaptation is largely shaped by horizontal gene transfer, xenogeneic silencing mediated by lineage-specific DNA bridgers (H-NS, Lsr2, MvaT and Rok), and various anti-silencing mechanisms. No xenogeneic silencing DNA bridger is known for α-proteobacteria, from which mitochondria evolved. By investigating α-proteobacterium Sinorhizobium fredii, a facultative legume microsymbiont, here we report the conserved zinc-finger bearing MucR as a novel xenogeneic silencing DNA bridger. Self-association mediated by its N-terminal domain (NTD) is required for DNA-MucR-DNA bridging complex formation, maximizing MucR stability, transcriptional silencing, and efficient symbiosis in legume nodules. Essential roles of NTD, CTD (C-terminal DNA-binding domain), or full-length MucR in symbiosis can be replaced by non-homologous NTD, CTD, or full-length protein of H-NS from γ-proteobacterium Escherichia coli, while NTD rather than CTD of Lsr2 from Gram-positive Mycobacterium tuberculosis can replace the corresponding domain of MucR in symbiosis. Chromatin immunoprecipitation sequencing reveals similar recruitment profiles of H-NS, MucR and various functional chimeric xenogeneic silencers across the multipartite genome of S. fredii, i.e. preferring AT-rich genomic islands and symbiosis plasmid with key symbiosis genes as shared targets. Collectively, the convergently evolved DNA bridger MucR predisposed α-proteobacteria to integrate AT-rich foreign DNA including symbiosis genes, horizontal transfer of which is strongly selected in nature.

Coordinated regulation of core and accessory genes in the multipartite genome of Sinorhizobium fredii.

Prokaryotes benefit from having accessory genes, but it is unclear how accessory genes can be linked with the core regulatory network when developing adaptations to new niches. Here we determined hierarchical core/accessory subsets in the multipartite pangenome (composed of genes from the chromosome, chromid and plasmids) of the soybean microsymbiont Sinorhizobium fredii by comparing twelve Sinorhizobium genomes. Transcriptomes of two S. fredii strains at mid-log and stationary growth phases and in symbiotic conditions were obtained. The average level of gene expression, variation of expression between different conditions, and gene connectivity within the co-expression network were positively correlated with the gene conservation level from strain-specific accessory genes to genus core. Condition-dependent transcriptomes exhibited adaptive transcriptional changes in pangenome subsets shared by the two strains, while strain-dependent transcriptomes were enriched with accessory genes on the chromid. Proportionally more chromid genes than plasmid genes were co-expressed with chromosomal genes, while plasmid genes had a higher within-replicon connectivity in expression than chromid ones. However, key nitrogen fixation genes on the symbiosis plasmid were characterized by high connectivity in both within- and between-replicon analyses. Among those genes with host-specific upregulation patterns, chromosomal znu and mdt operons, encoding a conserved high-affinity zinc transporter and an accessory multi-drug efflux system, respectively, were experimentally demonstrated to be involved in host-specific symbiotic adaptation. These findings highlight the importance of integrative regulation of hierarchical core/accessory components in the multipartite genome of bacteria during niche adaptation and in shaping the prokaryotic pangenome in the long run.

Coordinated Regulation of the Size and Number of Polyhydroxybutyrate Granules by Core and Accessory Phasins in the Facultative Microsymbiont Sinorhizobium fredii NGR234.

The exact roles of various granule-associated proteins (GAPs) of polyhydroxybutyrate (PHB) are poorly investigated, particularly for bacteria associated with plants. In this study, four structural GAPs, named phasins PhaP1 to PhaP4, were identified and demonstrated as true phasins colocalized with PHB granules in Sinorhizobium fredii NGR234, a facultative microsymbiont of Vigna unguiculata and many other legumes. The conserved PhaP2 dominated in regulation of granule size under both free-living and symbiotic conditions. PhaP1, another conserved phasin, made a higher contribution than accessory phasins PhaP4 and PhaP3 to PHB biosynthesis at stationary phase. PhaP3, with limited phyletic distribution on the symbiosis plasmid of Sinorhizobium, was more important than PhaP1 in regulating PHB biosynthesis in V. unguiculata nodules. Under the test conditions, no significant symbiotic defects were observed for mutants lacking individual or multiple phaP genes. The mutant lacking two PHB synthases showed impaired symbiotic performance, while mutations in individual PHB synthases or a PHB depolymerase yielded no symbiotic defects. This phenomenon is not related to either the number or size of PHB granules in test mutants within nodules. Distinct metabolic profiles and cocktail pools of GAPs of different phaP mutants imply that core and accessory phasins can be differentially involved in regulating other cellular processes in the facultative microsymbiont S. fredii NGR234.IMPORTANCE Polyhydroxybutyrate (PHB) granules are a store of carbon and energy in bacteria and archaea and play an important role in stress adaptation. Recent studies have highlighted distinct roles of several granule-associated proteins (GAPs) in regulating the size, number, and localization of PHB granules in free-living bacteria, though our knowledge of the role of GAPs in bacteria associated with plants is still limited. Here we report distinct roles of core and accessory phasins associated with PHB granules of Sinorhizobium fredii NGR234, a broad-host-range microsymbiont of diverse legumes. Core phasins PhaP2 and PhaP1 are conserved major phasins in free-living cells. PhaP2 and accessory phasin PhaP3, encoded by an auxiliary gene on the symbiosis plasmid, are major phasins in nitrogen-fixing bacteroids in cowpea nodules. GAPs and metabolic profiles can vary in different phaP mutants. Contrasting symbiotic performances between mutants lacking PHB synthases, depolymerase, or phasins were revealed.

Minimal standards for the description of new genera and species of rhizobia and agrobacteria.

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.

NsrA, a Predicted ß-Barrel Outer Membrane Protein Involved in Plant Signal Perception and the Control of Secondary Infection in Sinorhizobium meliloti.

An ongoing signal exchange fine-tunes the symbiotic interactions between rhizobia and legumes, ensuring the establishment and maintenance of mutualism. In a recently identified regulatory loop, endosymbiotic Sinorhizobium meliloti exerts negative feedback on root infection in response to unknown plant cues. Upon signal perception, three bacterial adenylate cyclases (ACs) of the inner membrane, namely, CyaD1, CyaD2, and CyaK, synthesize the second messenger cAMP, which, together with the cAMP-dependent Clr transcriptional activator, activates the expression of genes involved in root infection control. The pathway that links signal perception at the surface of the cell to cytoplasmic cAMP production by ACs was thus far unknown. Here we first show that CyaK is the cognate AC for the plant signal, called signal 1, that was observed previously in mature nodule and shoot extracts. We also show that inactivation of the gene immediately upstream of cyaK, nsrA (smb20775), which encodes a ß-barrel protein of the outer membrane, abolished signal 1 perception ex planta, whereas nsrA overexpression increased signal 1 responsiveness. Inactivation of the nsrA gene abolished all Clr-dependent gene expression in nodules and led to a marked hyperinfection phenotype on plants, similar to that of a cyaD1 cyaD2 cyaK triple mutant. We suggest that the NsrA protein acts as the (co)receptor for two signal molecules, signal 1 and a hypothetical signal 1', in mature and young nodules that cooperate in controlling secondary infection in S. meliloti-Medicago symbiosis. The predicted topology and domain composition of the NsrA protein hint at a mechanism of transmembrane signaling.IMPORTANCE Symbiotic interactions, especially mutualistic ones, rely on a continuous signal exchange between the symbionts. Here we report advances regarding a recently discovered signal transduction pathway that fine-tunes the symbiotic interaction between S. meliloti and its Medicago host plant. We have identified an outer membrane protein of S. meliloti, called NsrA, that transduces Medicago plant signals to adenylate cyclases in the inner membrane, thereby triggering a cAMP signaling cascade that controls infection. Besides their relevance for the rhizobium-legume symbiosis, these findings shed light on the mechanisms of signal perception and transduction by adenylate cyclases and transmembrane signaling in bacteria.

Evidence for Phosphate Starvation of Rhizobia without Terminal Differentiation in Legume Nodules.

Phosphate homeostasis is tightly modulated in all organisms, including bacteria, which harbor both high- and low-affinity transporters acting under conditions of fluctuating phosphate levels. It was thought that nitrogen-fixing rhizobia, named bacteroids, inhabiting root nodules of legumes are not phosphate limited. Here, we show that the high-affinity phosphate transporter PstSCAB, rather than the low-affinity phosphate transporter Pit, is essential for effective nitrogen fixation of Sinorhizobium fredii in soybean nodules. Symbiotic and growth defects of the pst mutant can be effectively restored by knocking out PhoB, the transcriptional repressor of pit. The pst homologs of representative rhizobia were actively transcribed in bacteroids without terminal differentiation in nodules of diverse legumes (soybean, pigeonpea, cowpea, common bean, and Sophora flavescens) but exhibited a basal expression level in terminally differentiated bacteroids (alfalfa, pea, and peanut). Rhizobium leguminosarum bv. viciae Rlv3841 undergoes characteristic nonterminal and terminal differentiations in nodules of S. flavescens and pea, respectively. The pst mutant of Rlv3841 showed impaired adaptation to the nodule environment of S. flavescens but was indistinguishable from the wild-type strain in pea nodules. Taken together, root nodule rhizobia can be either phosphate limited or nonlimited regarding the rhizobial differentiation fate, which is a host-dependent feature.

Nonspecific Symbiosis Between Sophora flavescens and Different Rhizobia.

We explored the genetic basis of the promiscuous symbiosis of Sophora flavescens with diverse rhizobia. To determine the impact of Nod factors (NFs) on the symbiosis of S. flavescens, nodulation-related gene mutants of representative rhizobial strains were generated. Strains with mutations in common nodulation genes (nodC, nodM, and nodE) failed to nodulate S. flavescens, indicating that the promiscuous nodulation of this plant is strictly dependent on the basic NF structure. Mutations of the NF decoration genes nodH, nodS, nodZ, and noeI did not affect the nodulation of S. flavescens, but these mutations affected the nitrogen-fixation efficiency of nodules. Wild-type Bradyrhizobium diazoefficiens USDA110 cannot nodulate S. flavescens, but we obtained 14 Tn5 mutants of B. diazoefficiens that nodulated S. flavescens. This suggested that the mutations had disrupted a negative regulator that prevents nodulation of S. flavescens, leading to nonspecific nodulation. For Ensifer fredii CCBAU 45436 mutants, the minimal NF structure was sufficient for nodulation of soybean and S. flavescens. In summary, the mechanism of promiscuous symbiosis of S. flavescens with rhizobia might be related to its nonspecific recognition of NF structures, and the host specificity of rhizobia may also be controlled by currently unknown nodulation-related genes.

The nitrate-reduction gene cluster components exert lineage-dependent contributions to optimization of Sinorhizobium symbiosis with soybeans.

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.

MucR Is Required for Transcriptional Activation of Conserved Ion Transporters to Support Nitrogen Fixation of Sinorhizobium fredii in Soybean Nodules.

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.

Genetic Analysis Reveals the Essential Role of Nitrogen Phosphotransferase System Components in Sinorhizobium fredii CCBAU 45436 Symbioses with Soybean and Pigeonpea Plants.

The nitrogen phosphotransferase system (PTS(Ntr)) consists of EI(Ntr), NPr, and EIIA(Ntr). The active phosphate moiety derived from phosphoenolpyruvate is transferred through EI(Ntr) and NPr to EIIA(Ntr). Sinorhizobium fredii can establish a nitrogen-fixing symbiosis with the legume crops soybean (as determinate nodules) and pigeonpea (as indeterminate nodules). In this study, S. fredii strains with mutations in ptsP and ptsO (encoding EI(Ntr) and NPr, respectively) formed ineffective nodules on soybeans, while a strain with a ptsN mutation (encoding EIIA(Ntr)) was not defective in symbiosis with soybeans. Notable reductions in the numbers of bacteroids within each symbiosome and of poly-ß-hydroxybutyrate granules in bacteroids were observed in nodules infected by the ptsP or ptsO mutant strains but not in those infected with the ptsN mutant strain. However, these defects of the ptsP and ptsO mutant strains were recovered in ptsP ptsN and ptsO ptsN double-mutant strains, implying a negative role of unphosphorylated EIIA(Ntr) in symbiosis. Moreover, the symbiotic defect of the ptsP mutant was also recovered by expressing EI(Ntr) with or without the GAF domain, indicating that the putative glutamine-sensing domain GAF is dispensable in symbiotic interactions. The critical role of PTS(Ntr) in symbiosis was also observed when related PTS(Ntr) mutant strains of S. fredii were inoculated on pigeonpea plants. Furthermore, nodule occupancy and carbon utilization tests suggested that multiple outputs could be derived from components of PTS(Ntr) in addition to the negative role of unphosphorylated EIIA(Ntr).

Construction and pilot screening of a signature-tagged mutant library of Sinorhizobium fredii.

Sinorhizobium fredii is well known for its ability to establish symbiosis with diverse legumes such as Glycine max (soybean, determinate nodules) and Cajanus cajan (pigeon pea, indeterminate nodules). In order to make screening of S. fredii genes related to symbiosis cost-effective, we constructed a large Tn5 insertion mutant library of S. fredii CCBAU45436 using the signature-tagged mutagenesis (STM) technique. This STM library contains a total of 25,500 independent mutants distributed in 17 sublibraries tagged by corresponding distinct DNA bar-code sequences. After the pilot screening of 255 mutants in 15 batches, Tag85-4, Tag4-17, Tag4-11 and Tag10-13 were found to have attenuated competitiveness (0-30 % in nodule occupation) compared to the wild-type strain when inoculated on soybean. Further characterization of these mutants suggests that Tag4-11 (a pyrC mutant) and Tag10-13 (a nrdJ mutant) are defective in establishing symbiosis with soybean. The pyrC mutant induced uninfected pseudonodules while the nrdJ mutant formed significantly more nodules containing bacteroids with poor persistence ability. When these two mutants were tested on pigeon pea, host-specific symbiotic defects were found. These results demonstrated the STM library as a valuable resource for identifying S. fredii genes relevant to symbiosis.

Rhizobial Diversity and Nodulation Characteristics of the Extremely Promiscuous Legume Sophora flavescens.

In present study, we report our extensive survey on the diversity and biogeography of rhizobia associated with Sophora flavescens, a sophocarpidine (matrine)-containing medicinal legume. We additionally investigated the cross nodulation, infection pattern, light and electron microscopies of root nodule sections of S. flavescens infected by various rhizobia. Seventeen genospecies of rhizobia belonging to five genera with seven types of symbiotic nodC genes were found to nodulate S. flavescens in natural soils. In the cross-nodulation tests, most representative rhizobia in class α-Proteobacteria, whose host plants belong to different cross-nodulation groups, form effective indeterminate nodules, while representative rhizobia in class ß-Proteobacteria form ineffective nodules on S. flavescens. Highly host-specific biovars of Rhizobium leguminosarum (bv. trifolii and bv. viciae) and Rhizobium etli bv. phaseoli could establish symbioses with S. flavescens, providing further evidence that S. flavescens is an extremely promiscuous legume and it does not have strict selectivity on either the symbiotic genes or the species-determining housekeeping genes of rhizobia. Root-hair infection is found as the pattern that rhizobia have gained entry into the curled root hairs. Electron microscopies of ultra-thin sections of S. flavescens root nodules formed by different rhizobia show that the bacteroids are regular or irregular rod shape and nonswollen types. Some bacteroids contain poly-ß-hydroxybutyrate (PHB), while others do not, indicating the synthesis of PHB in bacteroids is rhizobia-dependent. The extremely promiscuous symbiosis between S. flavescens and different rhizobia provide us a basis for future studies aimed at understanding the molecular interactions of rhizobia and legumes.

Rhizobium anhuiense sp. nov., isolated from effective nodules of Vicia faba and Pisum sativum.

Four rhizobia-like strains, isolated from root nodules of Pisum sativum and Vicia faba grown in Anhui and Jiangxi Provinces of China, were grouped into the genus Rhizobium but were distinct from all recognized species of the genus Rhizobium by phylogenetic analysis of 16S rRNA and housekeeping genes. The combined sequences of the housekeeping genes atpD, recA and glnII for strain CCBAU 23252(T) showed 86.9 to 95% similarity to those of known species of the genus Rhizobium. All four strains had nodC and nifH genes and could form effective nodules with Pisum sativum and Vicia faba, and ineffective nodules with Phaseolus vulgaris, but did not nodulate Glycine max, Arachis hypogaea, Medicago sativa, Trifolium repens or Lablab purpureus in cross-nodulation tests. Fatty acid composition, DNA-DNA relatedness and a series of phenotypic tests also separated these strains from members of closely related species. Based on all the evidence, we propose a novel species, Rhizobium anhuiense sp. nov., and designate CCBAU 23252(T) ( = CGMCC 1.12621(T) = LMG 27729(T)) as the type strain. This strain was isolated from a root nodule of Vicia faba and has a DNA G+C content of 61.1 mol% (Tm).

Plant-activated bacterial receptor adenylate cyclases modulate epidermal infection in the Sinorhizobium meliloti-Medicago symbiosis.

Legumes and soil bacteria called rhizobia have coevolved a facultative nitrogen-fixing symbiosis. Establishment of the symbiosis requires bacterial entry via root hair infection threads and, in parallel, organogenesis of nodules that subsequently are invaded by bacteria. Tight control of nodulation and infection is required to maintain the mutualistic character of the interaction. Available evidence supports a passive bacterial role in nodulation and infection after the microsymbiont has triggered the symbiotic plant developmental program. Here we identify in Sinorhizobium meliloti, the Medicago symbiont, a cAMP-signaling regulatory cascade consisting of three receptor-like adenylate cyclases, a Crp-like regulator, and a target gene of unknown function. The cascade is activated specifically by a plant signal during nodule organogenesis. Cascade inactivation results in a hyperinfection phenotype consisting of abortive epidermal infection events uncoupled from nodulation. These findings show that, in response to a plant signal, rhizobia play an active role in the control of infection. We suggest that rhizobia may modulate the plant's susceptibility to infection. This regulatory loop likely aims at optimizing legume infection.

Comparative genomics of rhizobia nodulating soybean suggests extensive recruitment of lineage-specific genes in adaptations.

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 of Sinorhizobium and Bradyrhizobium nodulating soybean to test this hypothesis. The Bradyrhizobium core 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 the Sinorhizobium core 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.

Replicon-dependent differentiation of symbiosis-related genes in Sinorhizobium strains nodulating Glycine max.

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.

Genetic divergence of bradyrhizobium strains nodulating soybeans as revealed by multilocus sequence analysis of genes inside and outside the symbiosis island.

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.

Cofitness network connectivity determines a fuzzy essential zone in open bacterial pangenome.

Most in silico evolutionary studies commonly assumed that core genes are essential for cellular function, while accessory genes are dispensable, particularly in nutrient-rich environments. However, this assumption is seldom tested genetically within the pangenome context. In this study, we conducted a robust pangenomic Tn-seq analysis of fitness genes in a nutrient-rich medium for Sinorhizobium strains with a canonical open pangenome. To evaluate the robustness of fitness category assignment, Tn-seq data for three independent mutant libraries per strain were analyzed by three methods, which indicates that the Hidden Markov Model (HMM)-based method is most robust to variations between mutant libraries and not sensitive to data size, outperforming the Bayesian and Monte Carlo simulation-based methods. Consequently, the HMM method was used to classify the fitness category. Fitness genes, categorized as essential (ES), advantage (GA), and disadvantage (GD) genes for growth, are enriched in core genes, while nonessential genes (NE) are over-represented in accessory genes. Accessory ES/GA genes showed a lower fitness effect than core ES/GA genes. Connectivity degrees in the cofitness network decrease in the order of ES, GD, and GA/NE. In addition to accessory genes, 1599 out of 3284 core genes display differential essentiality across test strains. Within the pangenome core, both shared quasi-essential (ES and GA) and strain-dependent fitness genes are enriched in similar functional categories. Our analysis demonstrates a considerable fuzzy essential zone determined by cofitness connectivity degrees in Sinorhizobium pangenome and highlights the power of the cofitness network in understanding the genetic basis of ever-increasing prokaryotic pangenome data.