Multipartite bacterial genomes pose challenges for genome engineering and the establishment of additional replicons. We simplified the tripartite genome structure (3.65 Mbp chromosome, 1.35 Mbp megaplasmid pSymA, 1.68 Mbp chromid pSymB) of the nitrogen-fixing plant symbiont Sinorhizobium meliloti. Strains with bi- and monopartite genome configurations were generated by targeted replicon fusions. Our design preserved key genomic features such as replichore ratios, GC skew, KOPS, and coding sequence distribution. Under standard culture conditions, the growth rates of these strains and the wild type were nearly comparable, and the ability for symbiotic nitrogen fixation was maintained. Spatiotemporal replicon organization and segregation were maintained in the triple replicon fusion strain. Deletion of the replication initiator-encoding genes, including the oriVs of pSymA and pSymB from this strain, resulted in a monopartite genome with oriC as the sole origin of replication, a strongly unbalanced replichore ratio, slow growth, aberrant cellular localization of oriC, and deficiency in symbiosis. Suppressor mutation R436H in the cell cycle histidine kinase CckA and a 3.2 Mbp inversion, both individually, largely restored growth, but only the genomic rearrangement recovered the symbiotic capacity. These strains will facilitate the integration of secondary replicons in S. meliloti and thus be useful for genome engineering applications, such as generating hybrid genomes.

Host range specificity is a prominent feature of the legume-rhizobial symbiosis. Sinorhizobium meliloti and Sinorhizobium medicae are two closely related species that engage in root nodule symbiosis with legume plants of the Medicago genus, but certain Medicago species exhibit selectivity in their interactions with the two rhizobial species. We have identified a Medicago receptor-like kinase, which can discriminate between the two bacterial species, acting as a genetic barrier against infection by most S. medicae strains. Activation of this receptor-mediated nodulation restriction requires a bacterial gene that encodes a glycine-rich octapeptide repeat protein with distinct variants capable of distinguishing S. medicae from S. meliloti. This study sheds light on the coevolution of host plants and rhizobia, shaping symbiotic selectivity in their respective ecological niches.

The two-component regulatory system CenK-CenR has recently emerged as a regulator of cell envelope and cell division processes in the alpha-proteobacteria. In Sinorhizobium meliloti, CenK-CenR regulates the expression of SrlA, a thioredoxin-domain protein of unknown function. Deletion of srlA causes sensitivity to salt and oxidizing agents on solid growth medium. In this work, we report that the response regulator CenR, but not the histidine kinase CenK, is essential for cell viability in S. meliloti. We also demonstrate that phosphorylation of the target residue D55 is not required for viability, suggesting that the unphosphorylated transcription factor sufficiently regulates expression of one or more essential genes in the genome. Using transcription assays and phenotype testing we examine CenK-CenR-dependent activation of the srlA promoter and demonstrate its absolute dependence on phosphoryl-CenR for activity and that the CenR substitution D55E acts as a phosphomimetic that partially restores activity at the srlA promoter in the absence of phosphorylation by CenK. Finally, we report a mutational analysis of the CenR binding site in the srlA promoter required for transcriptional activation.

The species Rhizobium indigoferae and Sinorhizobium kummerowiae were isolated from legume nodules and the 16S rRNA sequences of their respective type strains, CCBAU 71042T and CCBAU 71714T, were highly divergent from those of the other species of the genera Rhizobium and Sinorhizobium, respectively. However, the 16S rRNA gene sequences obtained for strains CCBAU 71042T and CCBAU 71714T several years after description, were different from the original ones, showing 100 % similarity to the type strains of Rhizobium leguminosarum and Sinorhizobium meliloti, respectively. Phylogenetic analyses of two housekeeping genes, recA and atpD, confirmed the high phylogenetic closeness of strains CCBAU 71042T and CCBAU 71714T to the respective type strains of R. leguminosarum and S. meliloti. In the present work, we compared the genomes of the type strains of R. indigoferae and S. kummerowiae available in several culture collections with those of the respective type strains of R. leguminosarum and S. meliloti, some of them obtained in this study. The calculated average nucleotide identity-blast and digital DNA-DNA hybridization values in both cases were higher than those recommended for species differentiation, supporting the proposal for the reclassification of the type strains of R. indigoferae and S. kummerowiae into the species R. leguminosarum and S. meliloti, respectively.

Interactions between photosynthetic microalgae and bacteria impact the physiology of both partners, which influence the fitness and ecological trajectories of each partner in an environmental context-dependent manner. Thermal tolerance of Chlamydomonas reinhardtii can be enhanced through a mutualistic interaction with vitamin B12 (cobalamin)-producing Sinorhizobium meliloti. Here, we used label-free quantitative proteomics to reveal the metabolic networks altered by the interaction under normal and high temperatures. We created a scenario where the growth of Sinorhizobium requires carbon provided by Chlamydomonas for growth in co-cultures, and survival of Chlamydomonas under high temperatures relies on cobalamin and possibly other metabolites produced by Sinorhizobium. Differential abundance analysis identified proteins produced by each partner in co-cultures compared to mono-cultures at each temperature. Proteins involved in cobalamin production by Sinorhizobium increased in the presence of Chlamydomonas under elevated temperatures, whereas in Chlamydomonas, there was an increase in cobalamin-dependent methionine synthase and certain proteins associated with methylation reactions. Co-cultivation and heat stress strongly modulated the central metabolism of both partners as well as various transporters that could facilitate nutrient cross-utilization. Co-cultivation modulated expression of various components of two- or one-component signal transduction systems, transcriptional activators/regulators, or sigma factors, suggesting complex regulatory networks modulate the interaction in a temperature-dependent manner. Notably, heat and general stress-response and antioxidant proteins were upregulated in co-cultures, suggesting that the interaction is inherently stressful to each partner despite the benefits of mutualism. Our results shed insight into the metabolic tradeoffs required for mutualism and how metabolic networks are modulated by elevated temperature.
OBJECTIVE: Photosynthetic microalgae are key primary producers in aquatic ecosystems, playing an important role in the global carbon cycle. Nearly every alga lives in association with a diverse community of microorganisms that influence each other and their metabolic activities or survival. One chemical produced by bacteria that influence algae is vitamin B12, an enzyme cofactor used for a variety of metabolic functions. The alga Chlamydomonas reinhardtii benefits from vitamin B12 produced by Sinorhizobium meliloti by producing the amino acid methionine under high temperatures which are required for Chlamydomonas thermotolerance. Yet, our understanding of this interaction under normal and stressful temperatures is poor. Here, we used quantitative proteomics to identify differentially expressed proteins to reveal metabolic adjustments made by Chlamydomonas and Sinorhizobium that could facilitate this mutualism. These findings will enhance our understanding of how photosynthetic algae and their associated microbiomes will respond as global temperatures increase.

BACKGROUND: Alfalfa (Medicago sativa L.) experiences many negative effects under salinity stress, which may be mediated by recurrent selection. Salt-tolerant alfalfa may display unique adaptations in association with rhizobium under salt stress.
RESULTS: To elucidate inoculation effects on salt-tolerant alfalfa under salt stress, this study leveraged a salt-tolerant alfalfa population selected through two cycles of recurrent selection under high salt stress. After experiencing 120-day salt stress, mRNA was extracted from 8 random genotypes either grown in 0 or 8 dS/m salt stress with or without inoculation by Ensifer meliloti. Results showed 320 and 176 differentially expressed genes (DEGs) modulated in response to salinity stress or inoculation x salinity stress, respectively. Notable results in plants under 8 dS/m stress included upregulation of a key gene involved in the Target of Rapamycin (TOR) signaling pathway with a concomitant decrease in expression of the SNrK pathway. Inoculation of salt-stressed plants stimulated increased transcription of a sulfate-uptake gene as well as upregulation of the Lysine-27-trimethyltransferase (EZH2), Histone 3 (H3), and argonaute (AGO, a component of miRISC silencing complexes) genes related to epigenetic and post-transcriptional gene control.
CONCLUSIONS: Salt-tolerant alfalfa may benefit from improved activity of TOR and decreased activity of SNrK1 in salt stress, while inoculation by rhizobiumstimulates production of sulfate uptake- and other unique genes.

Legumes have evolved a nitrogen-fixing symbiotic interaction with rhizobia, and this association helps them to cope with the limited nitrogen conditions in soil. The compatible interaction between the host plant and rhizobia leads to the formation of root nodules, wherein internalization and transition of rhizobia into their symbiotic form, termed bacteroids, occur. Rhizobia in the nodules of the Inverted Repeat-Lacking Clade legumes, including Medicago truncatula, undergo terminal differentiation, resulting in elongated and endoreduplicated bacteroids. This transition of endocytosed rhizobia is mediated by a large gene family of host-produced nodule-specific cysteine-rich (NCR) peptides in M. truncatula. Few NCRs have been recently found to be essential for complete differentiation and persistence of bacteroids. Here, we show that a M. truncatula symbiotic mutant FN9285, defective in the complete transition of rhizobia, is deficient in a cluster of NCR genes. More specifically, we show that the loss of the duplicated genes NCR086 and NCR314 in the A17 genotype, found in a single copy in Medicago littoralis R108, is responsible for the ineffective symbiotic phenotype of FN9285. The NCR086 and NCR314 gene pair encodes the same mature peptide but their transcriptional activity varies considerably. Nevertheless, both genes can restore the effective symbiosis in FN9285 indicating that their complementation ability does not depend on the strength of their expression activity. The identification of the NCR086/NCR314 peptide, essential for complete bacteroid differentiation, has extended the list of peptides, from a gene family of several hundred members, that are essential for effective nitrogen-fixing symbiosis in M. truncatula.

In the understanding of the molecular interaction between plants and their microbiome, a key point is to identify simplified models of the microbiome including relevant bacterial and fungal partners which could also be effective in plant growth promotion. Here, as proof-of-concept, we aim to identify the possible molecular interactions between symbiotic nitrogen-fixing rhizobia and soil fungi (Trichoderma spp.), hence shed light on synergistic roles rhizospheric fungi could have in the biology of symbiotic nitrogen fixation bacteria. We selected 4 strains of the model rhizobium Sinorhizobium meliloti and 4 Trichoderma species (T. velutinum, T. tomentosum, T. gamsii and T. harzianum). In an experimental scheme of 4 ×4 strains x species combinations, we investigated the rhizobia physiological and transcriptomic responses elicited by fungal spent media, as well as spent media effects on rhizobia-host legume plant (alfalfa, Medicago sativa L.) symbiosis. Fungal spent media had large effects on rhizobia, specific for each fungal species and rhizobial strains combination, indicating a generalized rhizobia genotype x fungal genotype interaction, including synergistic, neutral and antagonistic effects on alfalfa symbiotic phenotypes. Differential expression of a high number of genes was shown in rhizobia strains with up to 25% of total genes differentially expressed upon treatment of cultures with fungal spent media. Percentages over total genes and type of genes differentially expressed changed according to both fungal species and rhizobial strain. To support the hypothesis of a relevant rhizobia genotype x fungal genotype interaction, a nested Likelihood Ratio Test indicated that the model considering the fungus-rhizobium interaction explained 23.4% of differentially expressed genes. Our results provide insights into molecular interactions involving nitrogen-fixing rhizobia and rhizospheric fungi, highlighting the panoply of genes and genotypic interactions (fungus, rhizobium, host plant) which may concur to plant symbiosis.

Sensory adaptation in bacterial chemotaxis is mediated by posttranslational modifications of methyl-accepting chemotaxis proteins (MCPs). In Escherichia coli, the adaptation proteins CheR and CheB tether to a conserved C-terminal receptor pentapeptide. Here,we investigated the function of the pentapeptide motif (N/D)WE(E/N)F in Sinorhizobium meliloti chemotaxis. Isothermal titration calorimetry revealed stronger affinity of the pentapeptides to CheR and activated CheB relative to unmodified CheB. Strains with mutations of the conserved tryptophan in one or all four MCP pentapeptides resulted in a significant decrease or loss of chemotaxis to glycine betaine, lysine, and acetate, chemoattractants sensed by pentapeptide-bearing McpX and pentapeptide-lacking McpU and McpV, respectively. Importantly, we discovered that the pentapeptide mediates chemotaxis when fused to the C-terminus of pentapeptide-lacking chemoreceptors via a flexible linker. We propose that adaptational assistance and a threshold number of available sites enable the efficient docking of adaptation proteins to the chemosensory array. Altogether, these results demonstrate that S. meliloti effectively utilizes a pentapeptide-dependent adaptation system with a minimal number of tethering units to assist pentapeptide-lacking chemoreceptors and hypothesize that the higher abundance of CheR and CheB in S. meliloti compared to E. coli allows for ample recruitment of adaptation proteins to the chemosensory array.

The symbiovar mediterranense of Sinorhizobium meliloti was initially found in Phaseolus vulgaris nodules in Tunisia and in an eastern location of Lanzarote (Canary Islands). Here we show that the symbiovar mediterranense of S. meliloti also nodulates P. vulgaris in two western locations of this Island. The analyses of the symbiotic nodA and nodC genes reveal the complexity of the symbiovar mediterranense which encompasses strains belonging to several phylogenetic lineages and clusters. The comparison of the nodA and nodC phylogenies showed that the nodC was the most resolutive phylogenetic marker for the delineation of Sinorhizobium symbiovars. Considering that the similarity of this gene within several symbiovars, particularly mediterranense, is around 95 %, the cut-off value for their differentiation should be lower. Considering that a nodC gene cut-off similarity value of around 92 % is accepted for the genus Bradyrhizobium and that the symbiovar concept is identical in all rhizobial genera, we propose to apply this value for symbiovars delineation within all these genera. Therefore, using this cut-off value for the nodC gene analysis of Sinorhizobium symbiovars, we propose to merge the symbiovars aegeanense and fredii into the single symbiovar fredii and to define four novel symbiovars with the names asiaense, culleni, sudanense and tunisiaense.