Tunnel-like infection thread (IT) structures support root colonization by symbiotic nitrogen-fixing rhizobia bacteria in most legume species. These tip-grown structures are key to directing rhizobia from root hairs to developing nodules, where they are hosted to fix nitrogen. Rhizobia likely progress inside ITs by combining growth and motility by modes not yet defined. Here, we tackled this question by combining mathematical modeling, live cell imaging, and bacterial mutant phenotyping in Medicago truncatula. Modeling the motion of fluorescently-labeled Sinorhizobium meliloti inside root hair IT compartments estimated slow movement (2-6 μm h-1), compatible with passive rather than active motility. Consistent with this model, flagella-less S. meliloti mutants were impaired in active swimming motility in vitro, yet could colonize host roots and nodules in planta. By contrast, mutation in the rhizobactin 1021 siderophore rhbE biosynthesis gene affected surface motility in vitro and host root and nodule colonization. This mutation also promoted the formation of branched ITs in root hairs, which ultimately resulted in impaired nodule development and infection. In line with the slow motion of S. meliloti inside ITs estimated by modeling, our findings suggest that rhizobia favor flagella-independent surface translocation to reach developing nodules in M. truncatula.

Soil salinity poses a major challenge to the legume-rhizobia symbiosis development, thereby affecting sustainable agriculture. Selecting NaCl-tolerant strains and enhancing the native strains' fitness under salt stress are essential steps for the restoration of marginal areas. In this work, 49 Sinorhizobium meliloti strains, the rhizobial species forming symbiotic nitrogen-fixing associations with alfalfa-including 21 de novo-sequenced field isolates-were subjected to a thorough invitro screening for salt tolerance at progressively higher NaCl concentrations. Field isolates showed genome-based geographical clustering but contrasting salt tolerance abilities. Indeed, genome-wide association (GWA) analysis on the strains' whole-genome sequencing data indicated several loci associated with the variability in salt tolerance. Candidate genes were involved in various processes including cell wall organisation, LPS biosynthesis, quorum sensing, and carbohydrate transport and metabolism. The relationship with carbohydrate metabolism was further confirmed by Phenotype Microarray analysis which indicated salt-tolerant strains having enhanced capacity in carbon source usage. These findings reveal synergistic pathways underlying salt tolerance and suggest candidate traits (e.g., quorum sensing, carbohydrate synthesis and modification) for developing bioinoculants to enhance legume performance in saline soils.

Microbial community structure is driven, in part, by the metabolic interdependencies of resident microbes. Thus, manipulating specific metabolic interactions represents one attractive way to both understand how microbial communities perform complex functions and alter them for therapeutic or environmental effects. However, it is not yet possible to control the availability of those metabolites produced by some members of the community that are required by others. Here, we report the development of a metabolite ‘quenching’ strategy that disrupts a specific metabolic interaction involving corrinoids, the vitamin B12 family of cofactors, by applying a high-affinity corrinoid-binding protein, BtuG, to bacteria engaged corrinoid cross-feeding. Using a model coculture composed of Sinorhizobium meliloti, a bacterium that produces a corrinoid (cobalamin), and an Escherichia coli strain engineered to be corrinoid-dependent, we demonstrate corrinoid quenching by sequestration of extracellular corrinoid and show that BtuG specifically blocks corrinoid-dependent growth. We use this tool to calculate the amount of cobalamin released by S. meliloti cells and find that the cobalamin release rate is dependent on the growth phase of the producer, increasing to a maximum of approximately 40 cobalamin molecules per minute per cell in late exponential phase. This work establishes a strategy to selectively block microbial interactions that may be more broadly applied to dissecting community structure and function. We expect that applying high-affinity ‘molecular sponges’ to quench nutrient sharing will allow for the identification of key nutrients that structure microbial communities and empower precision microbiome manipulation strategies.

Global copper response of the soil bacterial predator Myxococcus xanthus and its contribution to antibiotic cross-resistance.

Sustainable agricultural production is frequently related with the use of agrochemicals, particularly pesticides, which are effective in combating plant infections and pests. Herbicides, a type of pesticide used to control weeds, can have a variety of effects on the growth of beneficial microbial populations in soil. The purpose of this paper was to investigate the effects of three pesticides on the growth of Rhizobium sp. strains. In this experiment, the growth of three bacterial strains (Rhizobium (Sinorhizobium) meliloti RM1, Rhizobium trifolii RT2, and RT3) was measured in the presence of three herbicides (Lumax 537.5, Agrodimark, and Siran 40 SC), which were added after impregnation of paper discs plated on Muller Hinton Agar. In this experiment, the recommended and doubled doses of each herbicide were employed. Resistance, susceptibility - increased exposure, and susceptibility - standard dosage were assessed following inhibition zone diameter measurement. Our findings demonstrated that the diameter of the inhibitory zone varies with herbicide type, dose, and rhizobial species. The findings revealed that treatments with increasing herbicide concentrations produced a larger inhibition zone diameter. The useof Lumax 537.5 SE led to higher inhibition zone diameter compared with other herbicides. In general, Rhizobium trifolii was more sensitive to Lumax 537.5 SE and Agrodimark than Rhizobium (Sinorhizobium) meliloti. This work may have scientific and applicative relevance for plant farmers, improving their knowledge of soil microbiology and pesticide application.

Elucidation of the complex mechanisms of action of antimicrobial peptides (AMPs) is critical for improving their efficacy. A major challenge in AMP research is distinguishing AMP effects resulting from various protein interactions from those caused by membrane disruption. Moreover, since AMPs often act in multiple cellular compartments, it is challenging to pinpoint where their distinct activities occur. Nodule-specific cysteine-rich (NCR) peptides secreted by some legumes, including NCR247, have evolved from AMPs to regulate differentiation of their nitrogen-fixing bacterial partner during symbiosis as well as to exert antimicrobial actions. At sub-lethal concentrations, NCR247 exhibits strikingly pleiotropic effects on Sinorhizobium meliloti. We used the L- and D-enantiomeric forms of NCR247 to distinguish between phenotypes resulting from stereospecific, protein-targeted interactions and those caused by non-specific interactions such as membrane disruption. In addition, we utilized an S. meliloti strain lacking BacA, a transporter that imports NCR peptides into the cytoplasm. The bacterial protein BacA, plays critical symbiotic roles by possibly reducing periplasmic peptide accumulation and fine-tuning symbiotic signaling. Use of the BacA-deficient strain made it possible to distinguish between phenotypes resulting from peptide interactions in the periplasm and those occurring in the cytoplasm. At high concentrations, both L- and D-NCR247 permeabilize bacterial membranes, consistent with nonspecific cationic AMP activity. In the cytoplasm, both NCR247 enantiomers sequester heme and trigger iron starvation in a chirality-independent but BacA-dependent manner. However, only L-NCR247 activates bacterial two-component systems via stereospecific periplasmic interactions. By combining stereochemistry and genetics, this work disentangles the spatial and molecular complexity of NCR247 action. This approach provides critical mechanistic insights into how host peptides with pleiotropic functions modulate bacterial physiology.

Biomolecular condensates are non-membrane-bound assemblies of proteins and nucleic acids that facilitate specific cellular processes. Like eukaryotic P-bodies, the recently discovered bacterial ribonucleoprotein bodies (BR-bodies) organize the mRNA decay machinery in α-proteobacteria; however, the similarities in molecular and cellular functions across species have been poorly explored. Here, we examine the functions of BR-bodies in the nitrogen-fixing endosymbiont Sinorhizobium meliloti, which colonizes the roots of compatible legume plants. Similar to Caulobacter crescentus, assembly of BR-bodies into visible foci in S. meliloti cells requires the C-terminal intrinsically disordered region (IDR) of RNase E in vivo and in vitro, and foci fusion is readily observed in vivo, suggesting that they are liquid-like condensates that form via mRNA sequestration. Using Rif-seq to measure mRNA lifetimes, we found a global slowdown in mRNA decay in a mutant deficient in BR-bodies, indicating that compartmentalization of the degradation machinery promotes efficient mRNA turnover across α-proteobacteria. Although BR-bodies are constitutively present during exponential growth, the abundance of BR-bodies increases upon cell stress, whereby they promote resistance to environmental stresses. Finally, we show that BR-bodies enhance competitive fitness during Medicago truncatula root colonization and appear to be required for effective symbiosis, as mutants without BR-bodies failed to promote robust plant growth on nitrogen-free medium. These results suggest that BR-bodies provide a fitness advantage for bacteria during host colonization, perhaps by enabling better resistance against the host immune response.IMPORTANCEAlthough eukaryotes often organize their biochemical pathways in membrane-bound organelles, bacteria generally lack such subcellular structures. Instead, membraneless compartments called biomolecular condensates have recently been found in bacteria to organize and enhance biochemical activities. Bacterial ribonucleoprotein bodies (BR-bodies), as one of the most characterized bacterial biomolecular condensates identified to date, assemble the mRNA decay machinery via the intrinsically disordered regions (IDRs) of proteins. However, the implications of such assemblies are unclear. Using a plant-associated symbiont, we show that the absence of BR-bodies results in slower mRNA decay, sensitivity to environmental stresses, and ineffective symbiosis, suggesting that BR-bodies play critical roles in regulating biochemical pathways and promoting fitness during host colonization.

This study presents the complete assembly and analysis of the mitochondrial genome (mitogenome) of Medicago lupulina L. var. vulgaris Koch, cultivar-population VIK32, line MlS-1, which forms an effective symbiosis not only with arbuscular mycorrhiza but also with the root nodule bacteria Sinorhizobium meliloti. The assembly, generated using a hybrid sequencing approach, revealed sequences of putative horizontal origin. These include a highly conserved open reading frame (ORF), orf279, encoding a protein structurally homologous to maturase K, yet bearing remote similarity to bacterial reverse transcriptases and CRISPR-associated proteins. We also identified sequences homologous to mitovirus RNA-dependent RNA polymerases and a fragment of the chloroplast 23S ribosomal RNA (rRNA), suggesting historical gene transfers from viruses and plastids. This work establishes a foundation for investigating the role of mitochondrial genome variation in key plant’s phenotypic traits, such as the enhanced responsiveness to arbuscular mycorrhiza observed in this agronomically valuable line.

Phosphate-solubilizing bacteria (PSB), as a green alternative to chemical phosphorus fertilizers, can enhance phosphorus use efficiency in crops through an environmentally friendly manner, thereby increasing crop yields. To address the limitations in the application and promotion of phosphate-solubilizing bacterial inoculants caused by the constraints in strain resources and viable counts, a potential phosphate-solubilizing bacterium, designated H9, was isolated from farmland in the suburban area of Luoyang. After six days of cultivation, the maximum soluble phosphorus content in the fermentation broth reached 71.17 mg·L-1. Based on morphological characteristics and 16S rDNA sequencing, the strain H9 was identified as Sinorhizobium meliloti. We further conducted high-density fermentation optimization to determine the optimal culture conditions. The results showed that at the condition of lactose 48.32 g·L-1, yeast extract 11.32 g·L-1, NaCl 3.29 g·L-1, an initial pH of 7.0, a working volume of 20 mL, inoculum size of 3%, and incubation at 34 ℃ with shaking at 180 r·min-1, the viable density of H9 reached (1.491±0.05)×1010 CFU·mL-1. Collectively, these findings suggested that S. meliloti H9 is a promising microbial resource for the development of high-quality phosphate-solubilizing biofertilizers and offers considerable potential for sustainable agricultural applications.

Abstract This experiment was conducted in the Al-Abayji district, Baghdad Governorate, Iraq, during the winter season of 2022-2023, to study the effect of bacterial inoculum, zeolite and organic fertilizer on secondary metabolites in fenugreek seeds, which consists of (24) treatments resulting from the interaction of bacterial inoculum (Sinorhizobium meliloti) without inoculation 0, 200 ml and 400 ml/25 kg seeds) with combinations of zeolite and organic fertilizer. These combinations are as follows: control treatment and 25 kg. dunum-1 zeolite and 4 tons. dun-1 organic fertilizer and 6 tons. dun-1 organic fertilizer and 2 tons. dun-1 organic fertilizer and 25 kg. dun-1 zeolite + 6 tons. dun-1 organic fertilizer and 25 kg. dun-1 zeolite + 4 tons. dun-1 of organic fertilizer and 25 kg. 1 dunum of zeolite + 2 tons. 1 dunum of organic fertilizer. (The results revealed that among the different treatments, the bacterial inoculation treatment (S. meliloti) at a rate of 400 ml was significantly superior in secondary metabolites represented by trichonelline (47.484 ppm), diosgenin (54.952 ppm), glutathione (38.946 ppm) and arginine (32.217 ppm) compared to the control treatment. The use of a combination of zeolite 25 kg. 1 dunum of + organic fertilizer 6 tons. 1 dunum of organic fertilizer resulted in a significant superiority in secondary metabolites represented by trichonelline (48.701 ppm), Diosgenin (56.085 ppm), Glutathione (40.126 ppm) and Arginine (33.172 ppm) compared to other blends and control.

BackgroundNitrogen-fixing bacteria (NFBs) play a critical role in biological nitrogen fixation for supplying essential nitrogen nutrients to plants in agriculture and natural ecosystems. Especially, these bacteria and Leguminosae plants form symbiosis to improve plant growth and soil fertility. Theoretically, the inoculation of NFBs into soils increases biological nitrogen fixation, but the efficiency of NFBs is frequently compromised by the low capacity of NFB root colonization. In this study, we introduced the synthetic bacterium EcCMC, which was genetically engineered to express the surface-displayed artificial polysaccharide (PS)-recognizing protein Cmc, to test if it can improve NFBs root colonization in representative Leguminosae plants, including Astragalus sinicus and Medicago sativa. Rhizosphere microbiomes, biochemical indicators, and plant yields were evaluated after 28 days in the three treatments, i.e., the control group without addition of any exogenous bacterium, the NFBs plus EcM (bacteria only expressing mCherry rather than Cmc) group, and the NFBs plus EcCMC group (n = 3).ResultsOwing to its polysaccharide-binding capacity, EcCMC strongly bound to the surface of A. sinicus roots. This binding was followed by the increased recruitment of the exogenous NFBs, Sinorhizobium meliloti and Sphingomonas endophytica, on the roots. As revealed by amplicon sequencing of the 16S rRNA gene, a combined inoculation of EcCMC and the NFBs increased the relative abundance of both Rhizobiales and Sphingomonadales, two important bacterial groups involved in nitrogen fixation. Consistently, metabolomic analysis showed that the metabolites involved in nitrogen fixation remarkably accumulated in the rhizosphere soils inoculated with NFBs plus EcCMC. Moreover, inoculation of NFBs plus EcCMC increased the activity of nitrogenase from 10.8 ~ 11.3 to 16.2 nmol/min/g (significant difference, p < 0.05, t-test), together with the total soil nitrogen levels from 217 ~ 258 to 414 mg/kg (significant difference, p < 0.05), and the soil organic matter levels from 19.5 ~ 20.8 to 23.6 mg/kg (significant difference, p < 0.05). Consequently, the yield of A. sinicus was remarkably improved by the inoculation of NFBs plus EcCMC. Similar results were observed in the experiments using Medicago sativa.ConclusionsThis study sheds a novel light on a synthetic biology-assisted regulation of rhizosphere microbiomes for enhanced nitrogen fixation and soil fertility in Leguminous plants. The designed polysaccharide-binding protein may be used as a universal tool to promote plant growth and enhance crop resilience in the future.Video Graphical Supplementary InformationThe online version contains supplementary material available at 10.1186/s40168-025-02189-5.

In this study, succinoglycan (SG), an anionic exopolysaccharide derived from Sinorhizobium meliloti Rm1021, was chemically modified to introduce boronic acid groups, creating a boronic-acid-functionalized polysaccharide (SG-APBA). The degree of substitution varied from 4.24% to 24.3%, depending on APBA concentration, with SG-APBA 2 identified as the optimal formulation. The properties of SG-APBA were characterized using 1H NMR, FTIR, TGA, and XRD, along with rheological analysis to assess changes in the polymer’s behavior. The hydrogel, referred to as SAT, was formed through dynamic boronate-ester bonds and hydrogen bonds between SG-APBA and tannic acid (TA). This hydrogel demonstrated excellent injectability, self-healing capacity, and biocompatibility. Incorporation of boronic acid groups allowed the hydrogel to respond to variations in glucose levels and pH, enabling controlled TA release and enhancing its stimulus-responsive antioxidant and antibacterial activities. Antioxidant performance was confirmed through DPPH and ABTS radical scavenging assays, achieving respective activities of 89.8% and 96.4%. Antibacterial effectiveness was validated via inhibition zone tests. Additionally, the SAT hydrogel exhibited dual responsiveness to pH and glucose, with TA release percentages of 55.4% at pH 9.0, 62.7% at pH 7.4, and 69.9% at pH 5.0; and 62.7% at 0 mM glucose, 68.9% at 5 mM, and 72.5% at 25 mM glucose after 120 h. Moreover, combined alterations in pH and glucose triggered a synergistic double-shock effect, markedly accelerating TA release relative to individual stimuli. Overall, these results indicate that the SG-APBA/TA hydrogel has strong potential as a stimuli-responsive platform for drug delivery and biomedical applications.

The soil bacterium Sinorhizobium meliloti can proliferate by leveraging its nitrogen-fixing symbiosis with legumes that form indeterminate root nodules, such as Medicago sativa (alfalfa) and M. truncatula. In contrast to determinate-nodulating legumes, such as Glycine max (soybean) and Lotus japonicus, indeterminate-nodulating legumes impose terminal differentiation on nitrogen-fixing (N2-fixing) rhizobia. Thus, the bacterial population is split between those that benefit the plant by N2 fixation, but are a reproductive dead end, and those that are undifferentiated, capable of resuming free-living growth but not fixing nitrogen. We show that in mixed nodules colonized by nearly isogenic strains, with one N2-fixing and one unable to fix N2 (Fix-), alfalfa do not preferentially penalize the Fix- strain, allowing "cheating" at the expense of the plant and the N2 fixer. Thus, a Fix- strain that successfully conodulates with an N2-fixing strain can benefit from resources the host provides to the nodule in response to N2 fixed by the conodulating strain. Coinvasion of alfalfa nodules with an N2-fixing strain may be a successful strategy for a Fix- strain to cheat both the plant that provides fixed carbon and the N2-fixing strain. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Sinorhizobium meliloti forms a robust N2-fixing root-nodule symbiosis with Medicago sativa. We are interested in identifying the minimal symbiotic genome of the model strain S. meliloti Rm1021. This gene set refers to the minimal genetic determinants required to form a robust N2-fixing symbiosis. Many symbiotic genes are located on the 1,354-kb pSymA megaplasmid of S. meliloti Rm1021. We recently constructed a minimalized pSymA, minSymA2.1, that lacked over 90% of the pSymA genes. Relative to the wild type, minSymA2.1 showed a reduction in M. sativa shoot biomass production and nodule size with an increase in total nodule number. Here, we show that the addition of either the stachydrine (stc) or trigonelline (trc) catabolism genes from pSymA to minSymA2.1 restores nodule size and total nodule number to levels indistinguishable from the wild type but does not restore reduced shoot biomass production. In the context of the complete Rm1021 genome, removing the stc genes reduced the nodule size and increased the total nodule number, whereas removal of the trc genes alone had no apparent effect. Together, these observations implicate stachydrine catabolism as an important determinant of root nodule symbiosis between S. meliloti and M. sativa, whereas trigonelline catabolism seems to contribute in a more conditional manner, in the context of the minimized genome. These findings highlight the minimal symbiotic genome as a tool for investigating the impact of individual genetic determinants in conferring an optimal symbiosis, factors whose impact, in the context of a complete genome, may be hidden or dampened due to redundancies. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Three symbiotic bacteria (K101T, C101 and M103) were obtained from nodule-trapping experiments using Phaseolus vulgaris, which was inoculated with soil samples from three distinct field sites in Manitoba, Canada. Here, we provide a phenotypic characterization and genomic analysis of these bacteria. Based on a core phylogeny (424 core genes), digital DNA–DNA hybridization and average nucleotide alignment, these isolates group within the Sinorhizobium clade and are closely related to Sinorhizobium meliloti. Each strain contains four replicons that include a chromosome (3.5 Mb), a putative chromid (1.7 Mb) and two plasmids (plasmid A, 0.56 Mb; plasmid B, 0.77 Mb). The chromosome, chromid and plasmid B are closely related to the replicons found in S. meliloti, as shown by phylogenies constructed from the concatenation of either the parAB genes for the chromosome or the repABC genes for the chromid and plasmid B. The remaining plasmid was found to group with a plasmid from Sinorhizobium americanum. Consistent with this, the nodulation genes on this plasmid were also more similar to those in S. americanum, as seen in a phylogeny generated from the concatenation of the nodABC genes. Examination of the nodC phylogeny suggests a close association with the mediterranensis symbiovar. All three isolates were capable of symbiotic nitrogen fixation with P. vulgaris. Based on genomic and phenotypic data, we propose these isolates as a novel species within the Sinorhizobium clade, named Sinorhizobium prairiense sp. nov., for which the type strain is K101T (=LMG 33767T=DSM 118657T).

Naturally occurring root-nodule bacteria (rhizobia) vary substantially in their effectiveness at promoting growth of different plant hosts via symbiotic nitrogen fixation. These variations in rhizobial partner quality have important implications for the productivity of nitrogen-fixing symbioses in natural and agricultural ecosystems, yet we have a limited understanding of the genetic basis for this variation. In a case of host-specific reduction in symbiotic effectiveness (N2-fixation) with Medicago sativa, we identified the causative genetic elements from the pSymA replicon of Sinorhizobum meliloti HM006 and show them to be involved in polyhydroxyalkanoate (PHA) production in nitrogen-fixing bacteroids. Transfer of this gene region to a strain that forms an effective symbiosis with Medicago sativa resulted in a complete loss of symbiotic N2-fixation. We showed the mechanism for symbiotic collapse is the diversion of succinate semialdehyde pools in the bacteroid to gamma-hydroxybutyrate (GHB) by an iron-containing dehydrogenase, GhbD. These findings reveal unexpected impacts of carbon metabolism changes in nodules on symbiont performance and provide a rare example of mechanism for variation in rhizobium partner quality, suggesting that host-specific metabolic incompatibility may be a key player in the variations in partner quality observed in nature.

Nodule-forming bacteria play crucial roles in plant health and nutrition by providing fixed nitrogen to leguminous plants. Despite the importance of this relationship, how nodule-forming bacteria are affected by plant exudates and soil minerals is not fully characterized. Here, the effects of plant-derived methanol and lanthanide metals on the growth of nitrogen-fixing Rhizobiales are examined. Prior work has demonstrated that select Bradyrhizobium strains can assimilate methanol only in the presence of lanthanide metals; however, the pathway enabling assimilation remains unknown. In this study, we characterize Bradyrhizobium diazoefficiens USDA 110, Bradyrhizobium sp. USDA 3456, and Sinorhizobium meliloti 2011 to determine the pathways involved in methanol metabolism in previously characterized strains, other clades of Bradyrhizobium, and the more distantly related Sinorhizobium. Based on genomic analyses, we hypothesized that methanol assimilation in these organisms occurs via the lanthanide-dependent methanol dehydrogenase XoxF, followed by oxidation of formaldehyde via the glutathione-linked oxidation pathway, subsequent oxidation of formate via formate dehydrogenases, and finally assimilation of CO2 via the Calvin-Benson-Bassham (CBB) cycle. Transcriptomics revealed upregulation of the aforementioned pathways in Bradyrhizobium sp. USDA 3456 during growth with methanol. Enzymatic assays demonstrated increased activity of the glutathione-linked oxidation pathway and formate dehydrogenases in all strains during growth with methanol compared to succinate. 13C-labeling studies confirmed the presence of CBB intermediates and label incorporation during growth with methanol. Our findings provide multiple lines of evidence supporting the proposed XoxF-CBB pathway and, combined with genomic analyses, suggest that this metabolism is widespread among Bradyrhizobium and Sinorhizobium species.IMPORTANCENitrogen-fixing soil bacteria such as Bradyrhizobium and Sinorhizobium promote plant growth while reducing dependence on artificial, energy-intensive fertilizers. Numerous studies have attempted to increase bacterial nitrogen fixation and colonization of plant tissues by identifying the micronutrients and plant exudates that promote successful symbiotic relationships. Among the compounds encountered by rhizobacteria, lanthanides have received little attention, despite reports that plant growth is affected by the presence of lanthanides. We characterized three agriculturally relevant Bradyrhizobium and Sinorhizobium strains, demonstrated that they gain the capacity to utilize methanol when lanthanides are present, and experimentally determined the pathway by which this metabolism occurs. This study provides a foundation for understanding the lanthanide-dependent metabolism of Bradyrhizobium and Sinorhizobium, which may influence their physiology and abundance in the environment.

Chemotaxis is crucial for the establishment of beneficial plant-microbe associations, yet mechanistic studies of chemotaxis have been limited to a handful of soil bacterial models, namely Azospirillum brasilense , Sinorhizobium meliloti , and Rhizobium leguminosarum . These three models represent only a fraction of the diversity found among plant- beneficial bacteria and agricultural inoculants. The soybean symbiont Bradyrhizobium diazoefficiens USDA110 is a commonly used soybean inoculant with exceptional nitrogen fixation efficiency, but the genetic control of chemotaxis in B. diazoefficiens has not been examined. Establishing B. diazoefficiens as a model of chemotaxis provides an opportunity to understand how multiple chemotaxis systems coordinate root colonization in this major agricultural symbiont and can enable comparative analyses of plant-microbe recognition strategies across agricultural bacteria.

ABSTRACTClimate change is altering both soil microbial communities and the ecological context of plant–microbe interactions. Heat, drought, and their legacies can alter soil microbiomes and potential plant symbionts, but the direct consequences of these microbial changes on plant performance and plant investment in symbiosis remain underexplored. Predicting how soil microbes modulate plant resilience to heat and drought is critical to mitigating the negative effects of climate change on ecosystems and agriculture. In this proof of concept study, we conducted growth chamber experiments to isolate the microbially mediated indirect effects of heat and drought on plant performance and symbiosis. In the first experiment, focused on drought, we found that drought and drought‐treated microbes, along with their interaction, significantly decreased the biomass of Medicago lupulina plants compared to well‐watered microbiomes and conditions. In a second experiment, we then tested how the addition of a well‐known microbial mutualist, Sinorhizobium meliloti, affected heat‐ and drought‐treated microbiomes' impact on M. lupulina. We found that drought‐adapted microbiomes negatively impacted legume performance by increasing mortality and reducing branch number, but that adding rhizobia erased differences in plant responses to climate‐treated soils. In contrast, heat‐adapted microbiomes did not differ significantly from control microbiomes in their effects on a legume. Our results suggest microbial legacy effects, mutualist partners, and their interactions are important in mediating plant responses to drought, with some mutualists equalizing plant responses across microbial legacies.

Poly(3-hydroxybutyrate) (PHB) is a carbon and energy storage polymer, whose accumulation under nutrient imbalances with excess carbon is common in bacteria. PhaR is a conserved transcriptional regulator that associates with PHB granules in several species. Although its role in modulating PHB storage and metabolism has been extensively studied across the bacterial phylogeny, a systems-level view of PhaR's dual function as a metabolic sensor and regulator is lacking. Here, we integrated co-expression network analysis with proteome profiling across multiple mutant backgrounds (lack of PhaR [AniA] and/or PHB synthesis) in the free-living state of the PHB-accumulating α-proteobacterial root nodule symbiont Sinorhizobium meliloti. This analysis was enriched by identifying direct regulatory targets of PhaR through a regulon-centric computational multistep search for DNA-binding site motifs combined with PhaR-DNA-binding and promoter-reporter assays. We confirmed that the model of accumulated PHB sequestering PhaR, and thereby relieving phasin and PHB depolymerase gene repression to control cellular PHB levels, also applies to S. meliloti and showed that PhaR-mediated regulation also occurs in the symbiotic state. Our integrated analyses of the impact of PHB-mediated PhaR titration on cellular functions revealed exopolysaccharide production as well as central carbon metabolism (pdh and bkd), gluconeogenesis (ppdK and pyc), entry into the TCA cycle (gltA), and the initial steps of the Entner-Doudoroff (ED) pathway (zwf, pgl, and edd) as major regulatory targets, along with target genes of yet unknown function. Our findings highlight a pivotal role for PhaR in orchestrating carbon metabolism.IMPORTANCEPoly(3-hydroxybutyrate) (PHB) is a carbon and energy storage polymer typically associated with bacterial survival under nutrient-limited conditions. Its accumulation reflects the cellular metabolic balance, and the transcriptional regulator PhaR has been shown to bind PHB and control the expression of genes involved in its metabolism. At the same time, PhaR has been implicated in broader regulatory roles affecting global gene expression, although the connection between this function and its ability to sense PHB has remained unresolved. In this study, we used the model legume symbiont Sinorhizobium meliloti to bridge this gap. We demonstrated that PhaR modulates global gene expression in response to the metabolic state signaled by PHB accumulation. Our findings highlight PHB not only as a storage compound, but also as a key integrator of metabolic status that links nutrient availability to coordinated transcriptional responses.