Many bacterial species, such as the alphaproteobacterium Sinorhizobium meliloti, are characterized by open pangenomes and contain multipartite genomes consisting of a chromosome and other large-sized replicons, such as chromids, megaplasmids, and plasmids. The evolutionary forces in both functional and structural aspects that shape the pangenome of species with multipartite genomes are still poorly understood. Therefore, we sequenced the genomes of 10 new S. meliloti strains, analyzed with four publicly available additional genomic sequences. Results indicated that the three main replicons present in these strains (a chromosome, a chromid, and a megaplasmid) partly show replicon-specific behaviors related to strain differentiation. In particular, the pSymB chromid was shown to be a hot spot for positively selected genes, and, unexpectedly, genes resident in the pSymB chromid were also found to be more widespread in distant taxa than those located in the other replicons. Moreover, through the exploitation of a DNA proximity network, a series of conserved "DNA backbones" were found to shape the evolution of the genome structure, with the rest of the genome experiencing rearrangements. The presented data allow depicting a scenario where the pSymB chromid has a distinctive role in intraspecies differentiation and in evolution through positive selection, whereas the pSymA megaplasmid mostly contributes to structural fluidity and to the emergence of new functions, indicating a specific evolutionary role for each replicon in the pangenome evolution.

Sinorhizobium meliloti is a model system for the studies of symbiotic nitrogen fixation. An extensive polymorphism at the genetic and phenotypic level is present in natural populations of this species, especially in relation with symbiotic promotion of plant growth. AK83 and BL225C are two nodule-isolated strains with diverse symbiotic phenotypes; BL225C is more efficient in promoting growth of the Medicago sativa plants than strain AK83. In order to investigate the genetic determinants of the phenotypic diversification of S. meliloti strains AK83 and BL225C, we sequenced the complete genomes for these two strains.

In all domains of life, proper regulation of the cell cycle is critical to coordinate genome replication, segregation and cell division. In some groups of bacteria, e.g. Alphaproteobacteria, tight regulation of the cell cycle is also necessary for the morphological and functional differentiation of cells. Sinorhizobium meliloti is an alphaproteobacterium that forms an economically and ecologically important nitrogen-fixing symbiosis with specific legume hosts. During this symbiosis S. meliloti undergoes an elaborate cellular differentiation within host root cells. The differentiation of S. meliloti results in massive amplification of the genome, cell branching and/or elongation, and loss of reproductive capacity. In Caulobacter crescentus, cellular differentiation is tightly linked to the cell cycle via the activity of the master regulator CtrA, and recent research in S. meliloti suggests that CtrA might also be key to cellular differentiation during symbiosis. However, the regulatory circuit driving cell cycle progression in S. meliloti is not well characterized in both the free-living and symbiotic state. Here, we investigated the regulation and function of CtrA in S. meliloti. We demonstrated that depletion of CtrA cause cell elongation, branching and genome amplification, similar to that observed in nitrogen-fixing bacteroids. We also showed that the cell cycle regulated proteolytic degradation of CtrA is essential in S. meliloti, suggesting a possible mechanism of CtrA depletion in differentiated bacteroids. Using a combination of ChIP-Seq and gene expression microarray analysis we found that although S. meliloti CtrA regulates similar processes as C. crescentus CtrA, it does so through different target genes. For example, our data suggest that CtrA does not control the expression of the Fts complex to control the timing of cell division during the cell cycle, but instead it negatively regulates the septum-inhibiting Min system. Our findings provide valuable insight into how highly conserved genetic networks can evolve, possibly to fit the diverse lifestyles of different bacteria.

Bacterial genome evolution is characterized by gains, losses, and rearrangements of functional genetic segments. The extent to which large-scale genomic alterations influence genotype-phenotype relationships has not been investigated in a high-throughput manner. In the symbiotic soil bacterium Sinorhizobium meliloti, the genome is composed of a chromosome and two large extrachromosomal replicons (pSymA and pSymB, which together constitute 45% of the genome). Massively parallel transposon insertion sequencing (Tn-seq) was employed to evaluate the contributions of chromosomal genes to growth fitness in both the presence and absence of these extrachromosomal replicons. Ten percent of chromosomal genes from diverse functional categories are shown to genetically interact with pSymA and pSymB. These results demonstrate the pervasive robustness provided by the extrachromosomal replicons, which is further supported by constraint-based metabolic modeling. A comprehensive picture of core S. meliloti metabolism was generated through a Tn-seq-guided in silico metabolic network reconstruction, producing a core network encompassing 726 genes. This integrated approach facilitated functional assignments for previously uncharacterized genes, while also revealing that Tn-seq alone missed over a quarter of wild-type metabolism. This work highlights the many functional dependencies and epistatic relationships that may arise between bacterial replicons and across a genome, while also demonstrating how Tn-seq and metabolic modeling can be used together to yield insights not obtainable by either method alone.

The genome of about 10% of bacterial species is divided among two or more large chromosome-sized replicons. The contribution of each replicon to the microbial life cycle (for example, environmental adaptations and/or niche switching) remains unclear. Here we report a genome-scale metabolic model of the legume symbiont Sinorhizobium meliloti that is integrated with carbon utilization data for 1,500 genes with 192 carbon substrates. Growth of S. meliloti is modelled in three ecological niches (bulk soil, rhizosphere and nodule) with a focus on the role of each of its three replicons. We observe clear metabolic differences during growth in the tested ecological niches and an overall reprogramming following niche switching. In silico examination of the inferred fitness of gene deletion mutants suggests that secondary replicons evolved to fulfil a specialized function, particularly host-associated niche adaptation. Thus, genes on secondary replicons might potentially be manipulated to promote or suppress host interactions for biotechnological purposes.

In the symbiosis between rhizobia and legumes, host plants can form symbiotic root nodules with multiple rhizobial strains, potentially showing different symbiotic performances in nitrogen fixation. Here, we investigated the presence of mixed nodules, containing rhizobia with different degrees of mutualisms, and evaluate their relative fitness in the Sinorhizobium meliloti-Medicago sativa model symbiosis. We used three S. meliloti strains, the mutualist strains Rm1021 and BL225C and the non-mutualist AK83. We performed competition experiments involving both in vitro and in vivo symbiotic assays with M. sativa host plants. We show the occurrence of a high number (from 27 to 100%) of mixed nodules with no negative effect on both nitrogen fixation and plant growth. The estimation of the relative fitness as non-mutualist/mutualist ratios in single nodules shows that in some nodules the non-mutualist strain efficiently colonized root nodules along with the mutualist ones. In conclusion, we can support the hypothesis that in S. meliloti-M. sativa symbiosis mixed nodules are formed and allow non-mutualist or less-mutualist bacterial partners to be less or not sanctioned by the host plant, hence allowing a potential form of cheating behavior to be present in the nitrogen fixing symbiosis.

Integration of newly acquired genes into existing regulatory networks is necessary for successful horizontal gene transfer (HGT). Ten percent of bacterial species contain at least two DNA replicons over 300 kilobases in size, with the secondary replicons derived predominately through HGT. The Sinorhizobium meliloti genome is split between a 3.7 Mb chromosome, a 1.7 Mb chromid consisting largely of genes acquired through ancient HGT, and a 1.4 Mb megaplasmid consisting primarily of recently acquired genes. Here, RNA-sequencing is used to examine the transcriptional consequences of massive, synthetic genome reduction produced through the removal of the megaplasmid and/or the chromid. Removal of the pSymA megaplasmid influenced the transcription of only six genes. In contrast, removal of the chromid influenced expression of ∼8% of chromosomal genes and ∼4% of megaplasmid genes. This was mediated in part by the loss of the ETR DNA region whose presence on pSymB is due to a translocation from the chromosome. No obvious functional bias among the up-regulated genes was detected, although genes with putative homologs on the chromid were enriched. Down-regulated genes were enriched in motility and sensory transduction pathways. Four transcripts were examined further, and in each case the transcriptional change could be traced to loss of specific pSymB regions. In particularly, a chromosomal transporter was induced due to deletion of bdhA likely mediated through 3-hydroxybutyrate accumulation. These data provide new insights into the evolution of the multipartite bacterial genome, and more generally into the integration of horizontally acquired genes into the transcriptome.

Plant-associated bacteria exhibit a number of different strategies and specific genes allow bacteria to communicate and metabolically interact with plant tissues. Among the genes found in the genomes of plant-associated bacteria, the gene encoding the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase (acdS) is one of the most diffused. This gene is supposed to be involved in the cleaving of plant-produced ACC, the precursor of the plant stress-hormone ethylene toning down the plant response to infection. However, few reports are present on the actual role in rhizobia, one of the most investigated groups of plant-associated bacteria. In particular, still unclear is the origin and the role of acdS in symbiotic competitiveness and on the selective benefit it may confer to plant symbiotic rhizobia. Here we present a phylogenetic and functional analysis of acdS orthologs in the rhizobium model-species Sinorhizobium meliloti. Results showed that acdS orthologs present in S. meliloti pangenome have polyphyletic origin and likely spread through horizontal gene transfer, mediated by mobile genetic elements. When acdS ortholog from AK83 strain was cloned and assayed in S. meliloti 1021 (lacking acdS), no modulation of plant ethylene levels was detected, as well as no increase in fitness for nodule occupancy was found in the acdS-derivative strain compared to the parental one. Surprisingly, AcdS was shown to confer the ability to utilize formamide and some dipeptides as sole nitrogen source. Finally, acdS was shown to be negatively regulated by a putative leucine-responsive regulator (LrpL) located upstream to acdS sequence (acdR). acdS expression was induced by root exudates of both legumes and non-leguminous plants. We conclude that acdS in S. meliloti is not directly related to symbiotic interaction, but it could likely be involved in the rhizospheric colonization or in the endophytic behavior.

Associations between leguminous plants and symbiotic nitrogen-fixing rhizobia are a classic example of mutualism between a eukaryotic host and a specific group of prokaryotic microbes. Although this symbiosis is in part species specific, different rhizobial strains may colonize the same nodule. Some rhizobial strains are commonly known as better competitors than others, but detailed analyses that aim to predict rhizobial competitive abilities based on genomes are still scarce. Here, we performed a bacterial genome-wide association (GWAS) analysis to define the genomic determinants related to the competitive capabilities in the model rhizobial species Sinorhizobium meliloti. For this, 13 tester strains were green fluorescent protein (GFP) tagged and assayed versus 3 red fluorescent protein (RFP)-tagged reference competitor strains (Rm1021, AK83, and BL225C) in a Medicago sativa nodule occupancy test. Competition data and strain genomic sequences were employed to build a model for GWAS based on k-mers. Among the k-mers with the highest scores, 51 k-mers mapped on the genomes of four strains showing the highest competition phenotypes (>60% single strain nodule occupancy; GR4, KH35c, KH46, and SM11) versus BL225C. These k-mers were mainly located on the symbiosis-related megaplasmid pSymA, specifically on genes coding for transporters, proteins involved in the biosynthesis of cofactors, and proteins related to metabolism (e.g., fatty acids). The same analysis was performed considering the sum of single and mixed nodules obtained in the competition assays versus BL225C, retrieving k-mers mapped on the genes previously found and on vir genes. Therefore, the competition abilities seem to be linked to multiple genetic determinants and comprise several cellular components. IMPORTANCE Decoding the competitive pattern that occurs in the rhizosphere is challenging in the study of bacterial social interaction strategies. To date, the single-gene approach has mainly been used to uncover the bases of nodulation, but there is still a knowledge gap regarding the main features that a priori characterize rhizobial strains able to outcompete indigenous rhizobia. Therefore, tracking down which traits make different rhizobial strains able to win the competition for plant infection over other indigenous rhizobia will improve the strain selection process and, consequently, plant yield in sustainable agricultural production systems. We proved that a k-mer-based GWAS approach can efficiently identify the competition determinants of a panel of strains previously analyzed for their plant tissue occupancy using double fluorescent labeling. The reported strategy will be useful for detailed studies on the genomic aspects of the evolution of bacterial symbiosis and for an extensive evaluation of rhizobial inoculants.

Malic enzymes decarboxylate the tricarboxylic acid (TCA) cycle intermediate malate to the glycolytic end-product pyruvate and are well positioned to regulate metabolic flux in central carbon metabolism. Despite the wide distribution of these enzymes, their biological roles are unclear in part because the reaction catalyzed by these enzymes can be by-passed by other pathways. The N2-fixing alfalfa symbiont Sinorhizobium meliloti contains both a NAD(P)-malic enzyme (DME) and a separate NADP-malic enzyme (TME) and to help understand the role of these enzymes, we investigated growth, metabolomic, and transcriptional consequences resulting from loss of these enzymes in free-living cells.

Plant-associated bacterial communities caught the attention of several investigators which study the relationships between plants and soil and the potential application of selected bacterial species in crop improvement and protection. Medicago sativa L. is a legume crop of high economic importance as forage in temperate areas and one of the most popular model plants for investigations on the symbiosis with nitrogen fixing rhizobia (mainly belonging to the alphaproteobacterial species Sinorhizobium meliloti). However, despite its importance, no studies have been carried out looking at the total bacterial community associated with the plant. In this work we explored for the first time the total bacterial community associated with M. sativa plants grown in mesocosms conditions, looking at a wide taxonomic spectrum, from the class to the single species (S. meliloti) level.

The rhizosphere and rhizoplane are nutrient-rich but selective environments for the root microbiome. Here, we deciphered a posttranscriptional network regulated by the homologous trans-small RNAs (sRNAs) AbcR1 and AbcR2, which rewire the metabolism of the nitrogen-fixing α-rhizobium Sinorhizobium meliloti during preinfection stages of symbiosis with its legume host alfalfa. The LysR-type regulator LsrB, which transduces the cell redox state, is indispensable for AbcR1 expression in actively dividing bacteria, whereas the stress-induced transcription of AbcR2 depends on the alternative σ factor RpoH1. MS2 affinity purification coupled with RNA sequencing unveiled exceptionally large and overlapping AbcR1/2 mRNA interactomes, jointly representing ⁓6% of the S. meliloti protein-coding genes. Most mRNAs encode transport/metabolic proteins whose translation is silenced by base pairing to two distinct anti-Shine Dalgarno motifs that function independently in both sRNAs. A metabolic model-aided analysis of the targetomes predicted changes in AbcR1/2 expression driven by shifts in carbon/nitrogen sources, which were confirmed experimentally. Low AbcR1/2 levels in some defined media anticipated overexpression growth phenotypes linked to the silencing of specific mRNAs. As a proof of principle, we confirmed AbcR1/2-mediated downregulation of the l-amino acid AapQ permease. AbcR1/2 interactomes are well represented in rhizosphere-related S. meliloti transcriptomic signatures. Remarkably, a lack of AbcR1 specifically compromised the ability of S. meliloti to colonize the root rhizoplane. The AbcR1 regulon likely ranks the utilization of available substrates to optimize metabolism, thus conferring on S. meliloti an advantage for efficient rhizosphere/rhizoplane colonization. AbcR1 regulation is predicted to be conserved in related α-rhizobia, which opens unprecedented possibilities for engineering highly competitive biofertilizers. IMPORTANCE Nitrogen-fixing root nodule symbioses between rhizobia and legume plants provide more than half of the combined nitrogen incorporated annually into terrestrial ecosystems, rendering plant growth independent of environmentally unfriendly chemical fertilizers. The success of symbiosis depends primarily on the capacity of rhizobia to establish competitive populations in soil and rhizosphere environments. Here, we provide insights into the regulation and architecture of an extensive RNA posttranscriptional network that fine-tunes the metabolism of the alfalfa symbiont S. meliloti, thereby enhancing the ability of this beneficial bacterium to colonize nutrient-rich but extremely selective niches, such as the rhizosphere of its host plant. This pervasive RNA regulation of metabolism is a major adaptive mechanism, predicted to operate in diverse rhizobial species. Because RNA regulation relies on modifiable base-pairing interactions, our findings open unexplored avenues for engineering the legumes rhizobiome within sustainable agricultural practices.

Reconstruction of the regulatory network is an important step in understanding how organisms control the expression of gene products and therefore phenotypes. Recent studies have pointed out the importance of regulatory network plasticity in bacterial adaptation and evolution. The evolution of such networks within and outside the species boundary is however still obscure. Sinorhizobium meliloti is an ideal species for such study, having three large replicons, many genomes available and a significant knowledge of its transcription factors (TF). Each replicon has a specific functional and evolutionary mark; which might also emerge from the analysis of their regulatory signatures. Here we have studied the plasticity of the regulatory network within and outside the S. meliloti species, looking for the presence of 41 TFs binding motifs in 51 strains and 5 related rhizobial species. We have detected a preference of several TFs for one of the three replicons, and the function of regulated genes was found to be in accordance with the overall replicon functional signature: house-keeping functions for the chromosome, metabolism for the chromid, symbiosis for the megaplasmid. This therefore suggests a replicon-specific wiring of the regulatory network in the S. meliloti species. At the same time a significant part of the predicted regulatory network is shared between the chromosome and the chromid, thus adding an additional layer by which the chromid integrates itself in the core genome. Furthermore, the regulatory network distance was found to be correlated with both promoter regions and accessory genome evolution inside the species, indicating that both pangenome compartments are involved in the regulatory network evolution. We also observed that genes which are not included in the species regulatory network are more likely to belong to the accessory genome, indicating that regulatory interactions should also be considered to predict gene conservation in bacterial pangenomes.

Many bacteria carry two or more chromosome-like replicons. This occurs in pathogens such as Vibrio cholerea and Brucella abortis as well as in many N2-fixing plant symbionts including all isolates of the alfalfa root-nodule bacteria Sinorhizobium meliloti. Understanding the evolution and role of this multipartite genome organization will provide significant insight into these important organisms; yet this knowledge remains incomplete, in part, because technical challenges of large-scale genome manipulations have limited experimental analyses. The distinct evolutionary histories and characteristics of the three replicons that constitute the S. meliloti genome (the chromosome (3.65 Mb), pSymA megaplasmid (1.35 Mb), and pSymB chromid (1.68 Mb)) makes this a good model to examine this topic. We transferred essential genes from pSymB into the chromosome, and constructed strains that lack pSymB as well as both pSymA and pSymB. This is the largest reduction (45.4%, 3.04 megabases, 2866 genes) of a prokaryotic genome to date and the first removal of an essential chromid. Strikingly, strains lacking pSymA and pSymB (ΔpSymAB) lost the ability to utilize 55 of 74 carbon sources and various sources of nitrogen, phosphorous and sulfur, yet the ΔpSymAB strain grew well in minimal salts media and in sterile soil. This suggests that the core chromosome is sufficient for growth in a bulk soil environment and that the pSymA and pSymB replicons carry genes with more specialized functions such as growth in the rhizosphere and interaction with the plant. These experimental data support a generalized evolutionary model, in which non-chromosomal replicons primarily carry genes with more specialized functions. These large secondary replicons increase the organism's niche range, which offsets their metabolic burden on the cell (e.g. pSymA). Subsequent co-evolution with the chromosome then leads to the formation of a chromid through the acquisition of functions core to all niches (e.g. pSymB).

The mutualistic association between leguminous plants and endosymbiotic rhizobial bacteria is a paradigmatic example of a symbiosis driven by metabolic exchanges. Here, we report the reconstruction and modelling of a genome-scale metabolic network of Medicago truncatula (plant) nodulated by Sinorhizobium meliloti (bacterium). The reconstructed nodule tissue contains five spatially distinct developmental zones and encompasses the metabolism of both the plant and the bacterium. Flux balance analysis (FBA) suggests that the metabolic costs associated with symbiotic nitrogen fixation are primarily related to supporting nitrogenase activity, and increasing N2-fixation efficiency is associated with diminishing returns in terms of plant growth. Our analyses support that differentiating bacteroids have access to sugars as major carbon sources, ammonium is the main nitrogen export product of N2-fixing bacteria, and N2 fixation depends on proton transfer from the plant cytoplasm to the bacteria through acidification of the peribacteroid space. We expect that our model, called 'Virtual Nodule Environment' (ViNE), will contribute to a better understanding of the functioning of legume nodules, and may guide experimental studies and engineering of symbiotic nitrogen fixation.

Rhizobia are ecologically important, facultative plant-symbiotic microbes. In nature, there is a large variability in the association of rhizobial strains and host plants of the same species. Here, we evaluated whether plant and rhizobial genotypes influence the initial transcriptional response of rhizobium following perception of a host plant. RNA sequencing of the model rhizobium Sinorhizobium meliloti exposed to root exudates or luteolin (an inducer of nod genes, involved in the early steps of symbiotic interaction) was performed on a combination of three S. meliloti strains and three alfalfa varieties as host plants. The response to root exudates involved hundreds of changes in the rhizobium transcriptome. Of the differentially expressed genes, 35% were influenced by the strain genotype, 16% were influenced by the plant genotype, and 29% were influenced by strain-by-host plant genotype interactions. We also examined the response of a hybrid S. meliloti strain in which the symbiotic megaplasmid (∼20% of the genome) was mobilized between two of the above-mentioned strains. Dozens of genes were upregulated in the hybrid strain, indicative of nonadditive variation in the transcriptome. In conclusion, this study demonstrated that transcriptional responses of rhizobia upon perception of legumes are influenced by the genotypes of both symbiotic partners and their interaction, suggesting a wide spectrum of genetic determinants involved in the phenotypic variation of plant-rhizobium symbiosis.IMPORTANCE A sustainable way for meeting the need of an increased global food demand should be based on a holobiont perspective, viewing crop plants as intimately associated with their microbiome, which helps improve plant nutrition, tolerance to pests, and adverse climate conditions. However, the genetic repertoire needed for efficient association with plants by the microbial symbionts is still poorly understood. The rhizobia are an exemplary model of facultative plant symbiotic microbes. Here, we evaluated whether genotype-by-genotype interactions could be identified in the initial transcriptional response of rhizobium perception of a host plant. We performed an RNA sequencing study to analyze the transcriptomes of different rhizobial strains elicited by root exudates of three alfalfa varieties as a proxy of an early step of the symbiotic interaction. The results indicated strain- and plant variety-dependent variability in the observed transcriptional changes, providing fundamentally novel insights into the genetic basis of rhizobium-plant interactions. Our results provide genetic insights and perspective to aid in the exploitation of natural rhizobium variation for improvement of legume growth in agricultural ecosystems.