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.

Molybdenum Nanofertilizer Boosts Biological Nitrogen Fixation and Yield of Soybean through Delaying Nodule Senescence and Nutrition Enhancement.

Soybean (Glycine max) is a crop of global significance and has low reliance on N fertilizers due to its biological nitrogen fixation (BNF) capacity, which harvests ambient N2 as a critical ecosystem service. BNF can be severely compromised by abiotic stresses. Enhancing BNF is increasingly important not only to alleviate global food insecurity but also to reduce the environmental impact of agriculture by decreasing chemical fertilizer inputs. However, this has proven challenging using current genetic modification or bacterial nodulation methods. Here, we demonstrate that a single application of a low dose (10 mg/kg) of molybdenum disulfide nanoparticles (MoS2 NPs) can enhance soybean BNF and grain yield by 30%, compared with conventional molybdate fertilizer. Unlike molybdate, MoS2 NPs can more sustainably release Mo, which then is effectively incorporated as a cofactor for the synthesis of nitrogenase and molybdenum-based enzymes that subsequently enhance BNF. Sulfur is also released sustainably and incorporated into biomolecule synthesis, particularly in thiol-containing antioxidants. The superior antioxidant enzyme activity of MoS2 NPs, together with the thiol compounds, protect the nodules from reactive oxygen species (ROS) damage, delay nodule aging, and maintain the BNF function for a longer term. The multifunctional nature of MoS2 NPs makes them a highly effective strategy to enhance plant tolerance to abiotic stresses. Given that the physicochemical properties of nanomaterials can be readily modulated, material performance (e.g., ROS capturing capacity) can be further enhanced by several synthesis strategies. This study thus demonstrates that nanotechnology can be an efficient and sustainable approach to enhancing BNF and crop yield under abiotic stress and combating global food insecurity.

Adaptive Evolution of Rhizobial Symbiosis beyond Horizontal Gene Transfer: From Genome Innovation to Regulation Reconstruction.

There are ubiquitous variations in symbiotic performance of different rhizobial strains associated with the same legume host in agricultural practices. This is due to polymorphisms of symbiosis genes and/or largely unexplored variations in integration efficiency of symbiotic function. Here, we reviewed cumulative evidence on integration mechanisms of symbiosis genes. Experimental evolution, in concert with reverse genetic studies based on pangenomics, suggests that gain of the same circuit of key symbiosis genes through horizontal gene transfer is necessary but sometimes insufficient for bacteria to establish an effective symbiosis with legumes. An intact genomic background of the recipient may not support the proper expression or functioning of newly acquired key symbiosis genes. Further adaptive evolution, through genome innovation and reconstruction of regulation networks, may confer the recipient of nascent nodulation and nitrogen fixation ability. Other accessory genes, either co-transferred with key symbiosis genes or stochastically transferred, may provide the recipient with additional adaptability in ever-fluctuating host and soil niches. Successful integrations of these accessory genes with the rewired core network, regarding both symbiotic and edaphic fitness, can optimize symbiotic efficiency in various natural and agricultural ecosystems. This progress also sheds light on the development of elite rhizobial inoculants using synthetic biology procedures.

Bacteroid Development, Transcriptome, and Symbiotic Nitrogen-Fixing Comparison of Bradyrhizobium arachidis in Nodules of Peanut (Arachis hypogaea) and Medicinal Legume Sophora flavescens.

Bradyrhizobium arachidis strain CCBAU 051107 could differentiate into swollen and nonswollen bacteroids in determinate root nodules of peanut (Arachis hypogaea) and indeterminate nodules of Sophora flavescens, respectively, with different N2 fixation efficiencies. To reveal the mechanism of bacteroid differentiation and symbiosis efficiency in association with different hosts, morphologies, transcriptomes, and nitrogen fixation efficiencies of the root nodules induced by strain CCBAU 051107 on these two plants were compared. Our results indicated that the nitrogenase activity of peanut nodules was 3 times higher than that of S. flavescens nodules, demonstrating the effects of rhizobium-host interaction on symbiotic effectiveness. With transcriptome comparisons, genes involved in biological nitrogen fixation (BNF) and energy metabolism were upregulated, while those involved in DNA replication, bacterial chemotaxis, and flagellar assembly were significantly downregulated in both types of bacteroids compared with those in free-living cells. However, expression levels of genes involved in BNF, the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway, hydrogenase synthesis, poly-ß-hydroxybutyrate (PHB) degradation, and peptidoglycan biosynthesis were significantly greater in the swollen bacteroids of peanut than those in the nonswollen bacteroids of S. flavescens, while contrasting situations were found in expression of genes involved in urea degradation, PHB synthesis, and nitrogen assimilation. Especially higher expression of ureABEF and aspB genes in bacteroids of S. flavescens might imply that the BNF product and nitrogen transport pathway were different from those in peanut. Our study revealed the first differences in bacteroid differentiation and metabolism of these two hosts and will be helpful for us to explore higher-efficiency symbiosis between rhizobia and legumes. IMPORTANCE Rhizobial differentiation into bacteroids in leguminous nodules attracts scientists to investigate its different aspects. The development of bacteroids in the nodule of the important oil crop peanut was first investigated and compared to the status in the nodule of the extremely promiscuous medicinal legume Sophora flavescens by using just a single rhizobial strain of Bradyrhizobium arachidis, CCBAU 051107. This strain differentiates into swollen bacteroids in peanut nodules and nonswollen bacteroids in S. flavescens nodules. The N2-fixing efficiency of the peanut nodules is three times higher than that of S. flavescens. By comparing the transcriptomes of their bacteroids, we found that they have similar gene expression spectra, such as nitrogen fixation and motivity, but different spectra in terms of urease activity and peptidoglycan biosynthesis. Those altered levels of gene expression might be related to their functions and differentiation in respective nodules. Our studies provided novel insight into the rhizobial differentiation and metabolic alteration in different hosts.

Rhizobial migration toward roots mediated by FadL-ExoFQP modulation of extracellular long-chain AHLs.

Migration from rhizosphere to rhizoplane is a key selecting process in root microbiome assembly, but not fully understood. Rhizobiales members are overrepresented in the core root microbiome of terrestrial plants, and here we report a genome-wide transposon-sequencing of rhizoplane fitness genes of beneficial Sinorhizobium fredii on wild soybean, cultivated soybean, rice, and maize. There were few genes involved in broad-host-range rhizoplane colonization. The fadL mutant lacking a fatty acid transporter exhibited high colonization rates, while mutations in exoFQP (encoding membrane proteins directing exopolysaccharide polymerization and secretion), but not those in exo genes essential for exopolysaccharide biosynthesis, led to severely impaired colonization rates. This variation was not explainable by their rhizosphere and rhizoplane survivability, and associated biofilm and exopolysaccharide production, but consistent with their migration ability toward rhizoplane, and associated surface motility and the mixture of quorum-sensing AHLs (N-acylated-L-homoserine lactones). Genetics and physiology evidences suggested that FadL mediated long-chain AHL uptake while ExoF mediated the secretion of short-chain AHLs which negatively affected long-chain AHL biosynthesis. The fadL and exoF mutants had elevated and depleted extracellular long-chain AHLs, respectively. A synthetic mixture of long-chain AHLs mimicking that of the fadL mutant can improve rhizobial surface motility. When this AHL mixture was spotted into rhizosphere, the migration toward roots and rhizoplane colonization of S. fredii were enhanced in a diffusible way. This work adds novel parts managing extracellular AHLs, which modulate bacterial migration toward rhizoplane. The FadL-ExoFQP system is conserved in Alphaproteobacteria and may shape the "home life" of diverse keystone rhizobacteria.

Intracellular common gardens reveal niche differentiation in transposable element community during bacterial adaptive evolution.

The distribution and abundance of transposable elements across the tree of life have significantly shaped the evolution of cellular organisms, but the underlying mechanisms shaping these ecological patterns remain elusive. Here we establish a "common garden" approach to study causal ecological interactions between a xenogeneic conditional lethal sacB gene and the community of transposable insertion sequences (ISs) in a multipartite prokaryote genome. Xenogeneic sacB of low, medium, or high GC content was individually inserted into three replicons of a model bacterium Sinorhizobium fredii, and exhibited replicon- and GC-dependent variation in genetic stability. This variation was largely attributable to multidimensional niche differentiation for IS community members. The transposition efficiency of major active ISs depended on the nucleoid-associated xenogeneic silencer MucR. Experimentally eliminating insertion activity of specific ISs by deleting MucR strongly demonstrated a dominant role of niche differentiation among ISs. This intracellular common garden approach in the experimental evolution context allows not only for evaluating genetic stability of natural and synthetic xenogeneic genes of different sequence signatures in host cells but also for tracking and testing causal relationships in unifying ecological principles in genome ecology.

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.

The PTSNtr-KdpDE-KdpFABC Pathway Contributes to Low Potassium Stress Adaptation and Competitive Nodulation of Sinorhizobium fredii.

The rhizobium-legume symbiosis is essential for sustainable agriculture by reducing nitrogen fertilizer input, but its efficiency varies under fluctuating soil conditions and resources. The nitrogen-related phosphotransferase system (PTSNtr) consisting of PtsP, PtsO, and PtsN is required for optimal nodulation and nitrogen fixation efficiency of the broad-host-range Sinorhizobium fredii CCBAU45436 associated with diverse legumes, though the underlying mechanisms remain elusive. This work characterizes the PtsN-KdpDE-KdpFABC pathway that contributes to low potassium adaptation and competitive nodulation of CCBAU45436. Among three PtsN, PtsN1 is the major functional homolog. The unphosphorylated PtsN1 binds the sensory kinase KdpD through a non-canonical interaction with the GAF domain of KdpD, while the region covering HisKA-HATPase domains mediates the interaction of KdpD with the response regulator KdpE. KdpE directly activates the kdpFABC operon encoding the conserved high-affinity potassium uptake system. Disruption of this signaling pathway leads to reduced nodule number, nodule occupancy, and low potassium adaptation ability, but without notable effects on rhizoplane colonization. The induction of key nodulation genes NIN and ENOD40 in host roots during early symbiotic interactions is impaired when inoculating the kdpBC mutant that shows delayed nodulation. The nodulation defect of the kdpBC mutant can be rescued by supplying replete potassium. Potassium is actively consumed by both prokaryotes and eukaryotes, and components of the PTSNtr-KdpDE-KdpFABC pathway are widely conserved in bacteria, highlighting the global importance of this pathway in bacteria-host interactions. IMPORTANCE In all ecological niches, potassium is actively consumed by diverse prokaryotes and their interacting eukaryote hosts. It is only just emerging that potassium is a key player in host-pathogen interactions, and the role of potassium in mutualistic interactions remains largely unknown. This work is focused on the mutualistic symbiosis between rhizobia and legumes. We report that the nitrogen-related phosphotransferase system PTSNtr, the two-component system KdpDE, and the high-affinity potassium uptake system KdpFABC constitute a pathway that is important for low potassium adaptation and optimal nodulation of rhizobia. Given the widely conserved PTSNtr, KdpDE, and KdpFABC in bacteria and increasing knowledge on microbiome for various niches, the PTSNtr-KdpDE-KdpFABC pathway can be globally important in the biosphere.

The zinc-finger bearing xenogeneic silencer MucR in α-proteobacteria balances adaptation and regulatory integrity.

Foreign AT-rich genes drive bacterial adaptation to new niches while challenging the existing regulation network. Here we report that MucR, a conserved regulator in α-proteobacteria, balances adaptation and regulatory integrity in Sinorhizobium fredii, a facultative microsymbiont of legumes. Chromatin immunoprecipitation sequencing coupled with transcriptomic data reveal that average transcription levels of both target and non-target genes, under free-living and symbiotic conditions, increase with their conservation levels. Targets involved in environmental adaptation and symbiosis belong to genus or species core and can be repressed or activated by MucR in a condition-dependent manner, implying regulatory integrations. However, most targets are enriched in strain-specific genes of lower expression levels and higher AT%. Within each conservation levels, targets have higher AT% and average transcription levels than non-target genes and can be further up-regulated in the mucR mutant. This is consistent with higher AT% of spacers between -35 and -10 elements of promoters for target genes, which enhances transcription. The MucR recruitment level linearly increases with AT% and the number of a flexible pattern (with periodic repeats of Ts) of target sequences. Collectively, MucR directly represses AT-rich foreign genes with predisposed high transcription potential while progressive erosions of its target sites facilitate regulatory integrations of foreign genes.

Global Transcriptional Repression of Diguanylate Cyclases by MucR1 Is Essential for Sinorhizobium-Soybean Symbiosis.

The ubiquitous bacterial second messenger c-di-GMP is intensively studied in pathogens but less so in mutualistic bacteria. Here, we report a genome-wide investigation of functional diguanylate cyclases (DGCs) synthesizing c-di-GMP from two molecules of GTP in Sinorhizobium fredii CCBAU45436, a facultative microsymbiont fixing nitrogen in nodules of diverse legumes, including soybean. Among 25 proteins harboring a putative GGDEF domain catalyzing the biosynthesis of c-di-GMP, eight functional DGCs were identified by heterogenous expression in Escherichia coli in a Congo red binding assay. This screening result was further verified by in vitro enzymatic assay with purified full proteins or the GGDEF domains from representative functional and nonfunctional DGCs. In the same in vitro assay, a functional EAL domain catalyzing the degradation of c-di-GMP into pGpG was identified in a protein that has an inactive GGDEF domain but with an active phosphodiesterase (PDE) function. The identified functional DGCs generally exhibited low transcription levels in soybean nodules compared to free-living cultures, as revealed in transcriptomes. An engineered upregulation of a functional DGC in nodules led to a significant increase of c-di-GMP level and symbiotic defects, which were not observed when a functional EAL domain was upregulated at the same level. Further transcriptional analysis and gel shift assay demonstrated that these functional DGCs were all transcriptionally repressed in nodules by a global pleiotropic regulator, MucR1, that is essential in Sinorhizobium-soybean symbiosis. These findings shed novel insights onto the systematic regulation of c-di-GMP biosynthesis in mutualistic symbiosis. IMPORTANCE The ubiquitous second messenger c-di-GMP is well-known for its role in biofilm formation and host adaptation of pathogens, whereas it is less investigated in mutualistic symbioses. Here, we reveal a cocktail of eight functional diguanylate cyclases (DGCs) catalyzing the biosynthesis of c-di-GMP in a broad-host-range Sinorhizobium that can establish nitrogen-fixing nodules on soybean and many other legumes. These functional DGCs are generally transcribed at low levels in soybean nodules compared to free-living conditions. The engineered nodule-specific upregulation of DGC can elevate the c-di-GMP level and cause symbiotic defects, while the upregulation of a phosphodiesterase that quenches c-di-GMP has no detectable symbiotic defects. Moreover, eight functional DGCs located on two different replicons are all directly repressed in nodules by a global silencer, MucR1, that is essential for Sinorhizobium-soybean symbiosis. These findings represent a novel mechanism of a strategic regulation of the c-di-GMP biosynthesis arsenal in prokaryote-eukaryote interactions.

Comparative Analysis of Core and Accessory Genes in Coexpression Network.

Prokaryotes harbor a various proportion of accessory genes in their genomes. The integration of accessory functions with the core regulation network is critical for environmental adaptation, particularly considering a theoretically unlimited number of niches on the earth for microorganisms. Comparative genomics can reveal a co-occurrence pattern between a subset of accessory genes (or variations in core genes) and an adaptation trait, while comparative transcriptomics can further uncover whether a coordinated regulation of gene expression is involved. In this chapter, we introduce a protocol for weighted gene coexpression network construction by using well-developed open source tools, and a further application of such a network in comparative analysis of bacterial core and accessory genes.

Differentially expressed microRNAs that target functional genes in mature soybean nodules.

MicroRNAs (miRNAs) are important regulators of biological functions in plants. To find out what roles miRNAs play in regulating symbiotic nitrogen fixation (SNF) in soybean [Glycine max (L.) Merr.], we identified high-confidence differentially expressed (DE) miRNAs from uninoculated roots (UR), rhizobium-inoculated roots (IR), and nodules (NODs) of soybean by robust small RNA sequencing (sRNA-seq). Based on their predicted target messenger RNAs (mRNAs), the expression profiles of some of these DE miRNAs could be linked to nodule functions. In particular, several miRNAs associated with nutrient transportation genes were differentially expressed in IRs and mature NODs. MiR399b, specifically, was highly induced in IRs and NODs, as well as by inorganic phosphate (Pi) starvation. In composite soybean plants overexpressing miR399b, PHOSPHATE2 (PHO2), a known target of miR399b that inhibits the activities of high-affinity Pi transporters, was strongly repressed. In addition, the overexpression of miR399b in the roots of transgenic composite plants significantly improved whole-plant Pi and ureide concentrations and the overall growth in terms of leaf node numbers and whole-plant dry weight. Our findings suggest that the induction of miR399b in NODs could enhance nitrogen fixation and soybean growth, possibly via improving Pi uptake to achieve a better Pi-nitrogen balance to promote SNF in nodules.

Lineage-Specific Rewiring of Core Pathways Predating Innovation of Legume Nodules Shapes Symbiotic Efficiency.

The interkingdom coevolution innovated the rhizobium-legume symbiosis. The application of this nitrogen-fixing system in sustainable agriculture is usually impeded by incompatible interactions between partners. However, the progressive evolution of rhizobium-legume compatibility remains elusive. In this work, deletions of rhcV encoding a structural component of the type three secretion system allow related Sinorhizobium strains to nodulate a previously incompatible soybean cultivar (Glycine max). These rhcV mutants show low to medium to high symbiotic efficiency on the same cultivated soybean while being indistinguishable on wild soybean plants (Glycine soja). The dual pantranscriptomics reveals nodule-specific activation of core symbiosis genes of Sinorhizobium and Glycine genes associated with genome duplication events along the chronogram. Unexpectedly, symbiotic efficiency is in line with lineage-dependent transcriptional profiles of core pathways which predate the diversification of Fabaceae and Sinorhizobium. This is supported by further physiological and biochemical experiments. Particularly, low-efficiency nodules show disordered antioxidant activity and low-energy status, which restrict nitrogen fixation activity. Collectively, the ancient core pathways play a crucial role in optimizing the function of later-evolved mutualistic arsenals in the rhizobium-legume coevolution.IMPORTANCE Significant roles of complex extracellular microbiota in environmental adaptation of eukaryotes in ever-changing circumstances have been revealed. Given the intracellular infection ability, facultative endosymbionts can be considered pioneers within complex extracellular microbiota and are ideal organisms for understanding the early stage of interkingdom adaptation. This work reveals that the later innovation of key symbiotic arsenals and the lineage-specific network rewiring in ancient core pathways, predating the divergence of legumes and rhizobia, underline the progressive evolution of rhizobium-legume compatibility. This insight not only is significant for improving the application benefits of rhizobial inoculants in sustainable agriculture but also advances our general understanding of the interkingdom coevolution which is theoretically explored by all host-microbiota interactions.

Defining the Rhizobium leguminosarum Species Complex.

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.

Rhizobiales-Specific RirA Represses a Naturally "Synthetic" Foreign Siderophore Gene Cluster To Maintain Sinorhizobium-Legume Mutualism.

Iron homeostasis is strictly regulated in cellular organisms. The Rhizobiales order enriched with symbiotic and pathogenic bacteria has evolved a lineage-specific regulator, RirA, responding to iron fluctuations. However, the regulatory role of RirA in bacterium-host interactions remains largely unknown. Here, we report that RirA is essential for mutualistic interactions of Sinorhizobium fredii with its legume hosts by repressing a gene cluster directing biosynthesis and transport of petrobactin siderophore. Genes encoding an inner membrane ABC transporter (fat) and the biosynthetic machinery (asb) of petrobactin siderophore are sporadically distributed in Gram-positive and Gram-negative bacteria. An outer membrane siderophore receptor gene (fprA) was naturally assembled with asb and fat, forming a long polycistron in S. fredii. An indigenous regulation cascade harboring an inner membrane protease (RseP), a sigma factor (FecI), and its anti-sigma protein (FecR) were involved in direct activation of the fprA-asb-fat polycistron. Operons harboring fecI and fprA-asb-fat, and those encoding the indigenous TonB-ExbB-ExbD complex delivering energy to the outer membrane transport activity, were directly repressed by RirA under iron-replete conditions. The rirA deletion led to upregulation of these operons and iron overload in nodules, impaired intracellular persistence, and symbiotic nitrogen fixation of rhizobia. Mutualistic defects of the rirA mutant can be rescued by blocking activities of this naturally "synthetic" circuit for siderophore biosynthesis and transport. These findings not only are significant for understanding iron homeostasis of mutualistic interactions but also provide insights into assembly and integration of foreign machineries for biosynthesis and transport of siderophores, horizontal transfer of which is selected in microbiota. IMPORTANCE Iron is a public good explored by both eukaryotes and prokaryotes. The abundant ferric form is insoluble under neutral and basic pH conditions, and many bacteria secrete siderophores forming soluble ferric siderophore complexes, which can be then taken up by specific receptors and transporters. Siderophore biosynthesis and uptake machineries can be horizontally transferred among bacteria in nature. Despite increasing attention on the importance of siderophores in host-microbiota interactions, the regulatory integration process of transferred siderophore biosynthesis and transport genes is poorly understood in an evolutionary context. By focusing on the mutualistic rhizobium-legume symbiosis, here, we report how a naturally synthetic foreign siderophore gene cluster was integrated with the rhizobial indigenous regulation cascade, which is essential for maintaining mutualistic interactions.

Ancestral zinc-finger bearing protein MucR in alpha-proteobacteria: A novel xenogeneic silencer?

The MucR/Ros family protein is conserved in alpha-proteobacteria and characterized by its zinc-finger motif that has been proposed as the ancestral domain from which the eukaryotic C2H2 zinc-finger structure evolved. In the past decades, accumulated evidences have revealed MucR as a pleiotropic transcriptional regulator that integrating multiple functions such as virulence, symbiosis, cell cycle and various physiological processes. Scattered reports indicate that MucR mainly acts as a repressor, through oligomerization and binding to multiple sites of AT-rich target promoters. The N-terminal region and zinc-finger bearing C-terminal region of MucR mediate oligomerization and DNA-binding, respectively. These features are convergent to those of xenogeneic silencers such as H-NS, MvaT, Lsr2 and Rok, which are mainly found in other lineages. Phylogenetic analysis of MucR homologs suggests an ancestral origin of MucR in alpha- and delta-proteobacteria. Multiple independent duplication and lateral gene transfer events contribute to the diversity and phyletic distribution of MucR. Finally, we posed questions which remain unexplored regarding the putative roles of MucR as a xenogeneic silencer and a general manager in balancing adaptation and regulatory integration in the pangenome context.