Effects of Engineered Sinorhizobium meliloti on Cytokinin Synthesis and Tolerance of Alfalfa to Extreme Drought Stress

ABSTRACTCytokinin is required for the initiation of leguminous nitrogen fixation nodules elicited by rhizobia and the delay of the leaf senescence induced by drought stress. A few free-living rhizobia have been found to produce cytokinin. However, the effects of engineered rhizobia capable of synthesizing cytokinin on host tolerance to abiotic stresses have not yet been described. In this study, two engineeredSinorhizobiumstrains overproducing cytokinin were constructed. The tolerance of inoculated alfalfa plants to severe drought stress was assessed. The engineered strains, which expressed theAgrobacterium iptgene under the control of different promoters, synthesized more zeatins than the control strain under free-living conditions, but their own growth was not affected. After a 4-week inoculation period, the effects of engineered strains on alfalfa growth and nitrogen fixation were similar to those of the control strain under nondrought conditions. After being subjected to severe drought stress, most of the alfalfa plants inoculated with engineered strains survived, and the nitrogenase activity in their root nodules showed no apparent change. A small elevation in zeatin concentration was observed in the leaves of these plants. The expression of antioxidant enzymes increased, and the level of reactive oxygen species decreased correspondingly. Although theiptgene was transcribed in the bacteroids of engineered strains, the level of cytokinin in alfalfa nodules was identical to that of the control. These findings suggest that engineeredSinorhizobiumstrains synthesizing more cytokinin could improve the tolerance of alfalfa to severe drought stress without affecting alfalfa nodulation or nitrogen fixation.

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Mutation in the ntrR Gene, a Member of the vap Gene Family, Increases the Symbiotic Efficiency of Sinorhizobium meliloti

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.7.887 ◽

2001 ◽

Vol 14 (7) ◽

pp. 887-894 ◽

Cited By ~ 25

Author(s):

Boglárka Oláh ◽

Erno Kiss ◽

Zoltán Györgypál ◽

Judit Borzi ◽

Gyöngyi Cinege ◽

...

Keyword(s):

Nitrogen Fixation ◽

Pathogenic Bacteria ◽

Sinorhizobium Meliloti ◽

Root Nodules ◽

Nifh Gene ◽

Dry Weight ◽

Nodule Occupancy ◽

Gene Encoding ◽

Symbiotic Efficiency ◽

Host Interactions

In specific plant organs, namely the root nodules of alfalfa, fixed nitrogen (ammonia) produced by the symbiotic partner Sinorhizobium meliloti supports the growth of the host plant in nitrogen-depleted environment. Here, we report that a derivative of S. meliloti carrying a mutation in the chromosomal ntrR gene induced nodules with enhanced nitrogen fixation capacity, resulting in an increased dry weight and nitrogen content of alfalfa. The efficient nitrogen fixation is a result of the higher expression level of the nifH gene, encoding one of the subunits of the nitrogenase enzyme, and nifA, the transcriptional regulator of the nif operon. The ntrR gene, controlled negatively by its own product and positively by the symbiotic regulator syrM, is expressed in the same zone of nodules as the nif genes. As a result of the nitrogen-tolerant phenotype of the strain, the beneficial effect of the mutation on efficiency is not abolished in the presence of the exogenous nitrogen source. The ntrR mutant is highly competitive in nodule occupancy compared with the wild-type strain. Sequence analysis of the mutant region revealed a new cluster of genes, termed the “ntrPR operon,” which is highly homologous to a group of vap-related genes of various pathogenic bacteria that are presumably implicated in bacterium-host interactions. On the basis of its favorable properties, the strain is a good candidate for future agricultural utilization.

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Paraburkholderia phymatum Homocitrate Synthase NifV Plays a Key Role for Nitrogenase Activity during Symbiosis with Papilionoids and in Free-Living Growth Conditions

Cells ◽

10.3390/cells10040952 ◽

2021 ◽

Vol 10 (4) ◽

pp. 952

Author(s):

Paula Bellés-Sancho ◽

Martina Lardi ◽

Yilei Liu ◽

Sebastian Hug ◽

Marta Adriana Pinto-Carbó ◽

...

Keyword(s):

Nitrogenase Activity ◽

Root Nodules ◽

Growth Conditions ◽

Lysine Biosynthesis ◽

Metabolome Analysis ◽

Plant Host ◽

Free Living ◽

Bacterial Enzyme ◽

Homocitrate Synthase ◽

Iron Molybdenum Cofactor

Homocitrate is an essential component of the iron-molybdenum cofactor of nitrogenase, the bacterial enzyme that catalyzes the reduction of dinitrogen (N2) to ammonia. In nitrogen-fixing and nodulating alpha-rhizobia, homocitrate is usually provided to bacteroids in root nodules by their plant host. In contrast, non-nodulating free-living diazotrophs encode the homocitrate synthase (NifV) and reduce N2 in nitrogen-limiting free-living conditions. Paraburkholderia phymatum STM815 is a beta-rhizobial strain, which can enter symbiosis with a broad range of legumes, including papilionoids and mimosoids. In contrast to most alpha-rhizobia, which lack nifV, P. phymatum harbors a copy of nifV on its symbiotic plasmid. We show here that P. phymatum nifV is essential for nitrogenase activity both in root nodules of papilionoid plants and in free-living growth conditions. Notably, nifV was dispensable in nodules of Mimosa pudica despite the fact that the gene was highly expressed during symbiosis with all tested papilionoid and mimosoid plants. A metabolome analysis of papilionoid and mimosoid root nodules infected with the P. phymatum wild-type strain revealed that among the approximately 400 measured metabolites, homocitrate and other metabolites involved in lysine biosynthesis and degradation have accumulated in all plant nodules compared to uninfected roots, suggesting an important role of these metabolites during symbiosis.

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Succinate Transport Is Not Essential for Symbiotic Nitrogen Fixation by Sinorhizobium meliloti or Rhizobium leguminosarum

Applied and Environmental Microbiology ◽

10.1128/aem.01561-17 ◽

2017 ◽

Vol 84 (1) ◽

Cited By ~ 10

Author(s):

Michael J. Mitsch ◽

George C. diCenzo ◽

Alison Cowie ◽

Turlough M. Finan

Keyword(s):

Nitrogen Fixation ◽

Carbon Source ◽

Sinorhizobium Meliloti ◽

Rhizobium Leguminosarum ◽

Symbiotic Nitrogen Fixation ◽

Sequencing Analysis ◽

Wild Type ◽

Content Type ◽

Transcriptional Changes ◽

Symbiotic Properties

ABSTRACTSymbiotic nitrogen fixation (SNF) is an energetically expensive process performed by bacteria during endosymbiotic relationships with plants. The bacteria require the plant to provide a carbon source for the generation of reductant to power SNF. While C4-dicarboxylates (succinate, fumarate, and malate) appear to be the primary, if not sole, carbon source provided to the bacteria, the contribution of each C4-dicarboxylate is not known. We address this issue using genetic and systems-level analyses. Expression of a malate-specific transporter (MaeP) inSinorhizobium melilotiRm1021dctmutants unable to transport C4-dicarboxylates resulted in malate import rates of up to 30% that of the wild type. This was sufficient to support SNF withMedicago sativa, with acetylene reduction rates of up to 50% those of plants inoculated with wild-typeS. meliloti.Rhizobium leguminosarumbv. viciae 3841dctmutants unable to transport C4-dicarboxylates but expressing themaePtransporter had strong symbiotic properties, withPisum sativumplants inoculated with these strains appearing similar to plants inoculated with wild-typeR. leguminosarum. This was despite malate transport rates by the mutant bacteroids being 10% those of the wild type. An RNA-sequencing analysis of the combinedP. sativum-R. leguminosarumnodule transcriptome was performed to identify systems-level adaptations in response to the inability of the bacteria to import succinate or fumarate. Few transcriptional changes, with no obvious pattern, were detected. Overall, these data illustrated that succinate and fumarate are not essential for SNF and that, at least in specific symbioses,l-malate is likely the primary C4-dicarboxylate provided to the bacterium.IMPORTANCESymbiotic nitrogen fixation (SNF) is an economically and ecologically important biological process that allows plants to grow in nitrogen-poor soils without the need to apply nitrogen-based fertilizers. Much research has been dedicated to this topic to understand this process and to eventually manipulate it for agricultural gains. The work presented in this article provides new insights into the metabolic integration of the plant and bacterial partners. It is shown that malate is the only carbon source that needs to be available to the bacterium to support SNF and that, at least in some symbioses, malate, and not other C4-dicarboxylates, is likely the primary carbon provided to the bacterium. This work extends our knowledge of the minimal metabolic capabilities the bacterium requires to successfully perform SNF and may be useful in further studies aiming to optimize this process through synthetic biology approaches. The work describes an engineering approach to investigate a metabolic process that occurs between a eukaryotic host and its prokaryotic endosymbiont.

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Sinorhizobium meliloti Controls Nitric Oxide–Mediated Post-Translational Modification of a Medicago truncatula Nodule Protein

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-05-15-0118-r ◽

2015 ◽

Vol 28 (12) ◽

pp. 1353-1363 ◽

Cited By ~ 21

Author(s):

Pauline Blanquet ◽

Liliana Silva ◽

Olivier Catrice ◽

Claude Bruand ◽

Helena Carvalho ◽

...

Keyword(s):

Nitric Oxide ◽

Nitrogen Fixation ◽

Medicago Truncatula ◽

Sinorhizobium Meliloti ◽

Molecular Mechanisms ◽

Root Nodules ◽

Plant Protein ◽

Post Translational Modification ◽

Model Legume ◽

Bacterial Mutants

Nitric oxide (NO) is involved in various plant-microbe interactions. In the symbiosis between soil bacterium Sinorhizobium meliloti and model legume Medicago truncatula, NO is required for an optimal establishment of the interaction but is also a signal for nodule senescence. Little is known about the molecular mechanisms responsible for NO effects in the legume-rhizobium interaction. Here, we investigate the contribution of the bacterial NO response to the modulation of a plant protein post-translational modification in nitrogen-fixing nodules. We made use of different bacterial mutants to finely modulate NO levels inside M. truncatula root nodules and to examine the consequence on tyrosine nitration of the plant glutamine synthetase, a protein responsible for assimilation of the ammonia released by nitrogen fixation. Our results reveal that S. meliloti possesses several proteins that limit inactivation of plant enzyme activity via NO-mediated post-translational modifications. This is the first demonstration that rhizobia can impact the course of nitrogen fixation by modulating the activity of a plant protein.

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HPrK Regulates Succinate-Mediated Catabolite Repression in the Gram-Negative Symbiont Sinorhizobium meliloti

Journal of Bacteriology ◽

10.1128/jb.01115-08 ◽

2008 ◽

Vol 191 (1) ◽

pp. 298-309 ◽

Cited By ~ 24

Author(s):

Catalina Arango Pinedo ◽

Daniel J. Gage

Keyword(s):

Catabolite Repression ◽

Sinorhizobium Meliloti ◽

Root Nodules ◽

Phosphotransferase System ◽

Free Living ◽

Gene Encoding ◽

Common Component ◽

Gram Negative ◽

Gram Positive Bacteria ◽

Living Organisms

ABSTRACT The HPrK kinase/phosphatase is a common component of the phosphotransferase system (PTS) of gram-positive bacteria and regulates catabolite repression through phosphorylation/dephosphorylation of its substrate, the PTS protein HPr, at a conserved serine residue. Phosphorylation of HPr by HPrK also affects additional phosphorylation of HPr by the PTS enzyme EI at a conserved histidine residue. Sinorhizobium meliloti can live as symbionts inside legume root nodules or as free-living organisms and is one of the relatively rare gram-negative bacteria known to have a gene encoding HPrK. We have constructed S. meliloti mutants that lack HPrK or that lack key amino acids in HPr that are likely phosphorylated by HPrK and EI. Deletion of hprK in S. meliloti enhanced catabolite repression caused by succinate, as did an S53A substitution in HPr. Introduction of an H22A substitution into HPr alleviated the strong catabolite repression phenotypes of strains carrying ΔhprK or hpr(S53A) mutations, demonstrating that HPr-His22-P is needed for strong catabolite repression. Furthermore, strains with a hpr(H22A) allele exhibited relaxed catabolite repression. These results suggest that HPrK phosphorylates HPr at the serine-53 residue, that HPr-Ser53-P inhibits phosphorylation at the histidine-22 residue, and that HPr-His22-P enhances catabolite repression in the presence of succinate. Additional experiments show that ΔhprK mutants overproduce exopolysaccharides and form nodules that do not fix nitrogen.

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Ineffectiveness of viomycin-resistant mutants of Rhizobium meliloti

Canadian Journal of Microbiology ◽

10.1139/m69-119 ◽

1969 ◽

Vol 15 (7) ◽

pp. 671-675 ◽

Cited By ~ 12

Author(s):

G. S. Hendry ◽

D. C. Jordan

Keyword(s):

Cell Wall ◽

Nitrogen Fixation ◽

Parent Strain ◽

Rhizobium Meliloti ◽

Nitrogenase Activity ◽

Root Nodules ◽

Effective Strain ◽

One Step ◽

Increased Sensitivity ◽

Resistant Mutants

Under clearly defined conditions one-step acquisition of viomycin resistance by a normally effective strain of Rhizobium meliloti resulted in one-step acquisition of ineffectiveness in nitrogen fixation, which probably occurred with a one-gene change in the R. meliloti genome. Two-step mutants retained their ability to produce root nodules but such nodules also were ineffective. Increased sensitivity of the viomycin-resistant mutants to glycine and D-alanine was not noted. Bacteroids were not seen in nodules formed by the viomycin-resistant mutants on their homologous host plant. Nitrogenase activity was not detected, by acetylene reduction, in detached ineffective nodules, whereas effective nodules formed 10.6 μmoles of ethylene per hour per gram of nodules. Growth of the effective parent strain in a low concentration of viomycin resulted in elongation and swelling of the cells so that they appeared as artificially produced bacteroids. Viomycin-resistant mutants did not undergo this transformation. Antigens could be readily extracted by hot- and cold-saline extraction of wet packed cells of both resistant and sensitive cultures but antigenic differences, which may have indicated cell wall differences, were not noted.

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The reaction of self-fertile alfalfa lines to inoculation with nodule bacteria

V N Karazin National University Series “Biology” ◽

10.26565/2075-5457-2020-34-17 ◽

2020 ◽

Keyword(s):

Nitrogen Fixation ◽

Sinorhizobium Meliloti ◽

Root Nodules ◽

Growing Season ◽

Dry Mass ◽

Nodule Bacteria ◽

Forage Crop ◽

Nitrogen Fixation Activity ◽

Fixation Activity ◽

Different Strains

Under the conditions of a model pot experiments, the reaction of the self-fertile lines of alfalfa Kishvardy 46, Kishvardy 27, Vertus and Ziguen to inoculation with nodule bacteria Sinorhizobium meliloti AC48 and AC88 was studied. As a result of studies, it was found that the intensity of assimilation of N2 by symbiotic systems created with the participation of various genotypes of alfalfa and active strains of S. meliloti is one of the main factors that affects the vegetative mass yield of this important forage crop. Self-fertile lines of Medicago sativa L. plants, inoculated with different strains of rhizobia were characterized by higher rates of the mass formed on the root nodules, compared to the control plants of the alfalfa variety Yaroslavna. The traditional dynamics of nitrogen-fixation activity of root nodules was maintained in all the symbiotic systems studied by us, with low values in the stems formation stage and intensive growth in the budding and flowering stages. The highest level of nitrogen fixation and vegetative growth of plants (values of plants green and dry mass, roots and root nodules mass) was established by inoculation of alfalfa line Kishvardy 46 with strain S. meliloti AC48. During the growing season the indices of the mass of nodules formed on the roots of these plants were higher by 1.8–2.3 times, the green mass by 1.2–1.6 times and the height of the plants 1.2–1.4 times as compared to the control. In the flowering stages the nitrogen-fixation activity of the symbiotic complex of plants of the Kishvardy line 27 and nodule bacteria S. meliloti AC48 exceeded the values in the symbiotic systems formed with the participation of the same strain and plants of the Ziguen and Vertus lines by 13.0 and 39.4 %. The lowest values of nitrogen fixation activity were observed by inoculation of plants of the Vertus and Ziguen lines with active strains S. meliloti AC48 and AC88 compared to the symbioses formed by the plants of the Kishvardy lines 27 and 46, as well as of the control-variety Yaroslavna with the noted strains. A stimulating effect of inoculation of alfalfa seeds of different genotypes on the growth and development of plants was noted, as evidenced by the positive dynamics of the increase in above-ground mass, the accumulation of dry matter and higher than the control values (indicators) of plant height during the growing season.

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Multiple groESL Operons Are Not Key Targets of RpoH1 and RpoH2 in Sinorhizobium meliloti

Journal of Bacteriology ◽

10.1128/jb.188.10.3507-3515.2006 ◽

2006 ◽

Vol 188 (10) ◽

pp. 3507-3515 ◽

Cited By ~ 20

Author(s):

Alycia N. Bittner ◽

Valerie Oke

Keyword(s):

Escherichia Coli ◽

Nitrogen Fixation ◽

High Temperature ◽

Sigma Factor ◽

Host Plants ◽

Sinorhizobium Meliloti ◽

Free Living ◽

Genes Encoding ◽

Multiple Copies ◽

Free Living Conditions

ABSTRACT Among the rhizobia that establish nitrogen-fixing nodules on the roots of host plants, many contain multiple copies of genes encoding the sigma factor RpoH and the chaperone GroEL/GroES. In Sinorhizobium meliloti there are two rpoH genes, four groESL operons, and one groEL gene. rpoH1 mutants are defective for growth at high temperature and form ineffective nodules, rpoH1 rpoH2 double mutants are unable to form nodules, and groESL1 mutants form ineffective nodules. To explore the roles of RpoH1 and RpoH2, we identified mutants that suppress both the growth and nodulation defects. These mutants do not suppress the nitrogen fixation defect. This implies that the functions of RpoH1 during growth and RpoH1/RpoH2 during the initiation of symbiosis are similar but that there is a different function of RpoH1 needed later during symbiosis. We showed that, unlike in Escherichia coli, overexpression of groESL is not sufficient to bypass any of the RpoH defects. Under free-living conditions, we determined that RpoH2 does not control expression of the groE genes, and RpoH1 only controls expression of groESL5. Finally, we completed the series of groE mutants by constructing groESL3 and groEL4 mutants and demonstrated that they do not display symbiotic defects. Therefore, the only groESL operon required by itself for symbiosis is groESL1. Taken together, these results suggest that GroEL/GroES production alone cannot explain the requirements for RpoH1 and RpoH2 in S. meliloti and that there must be other crucial targets.

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Proteome Profiling of the Rhodobacter capsulatus Molybdenum Response Reveals a Role of IscN in Nitrogen Fixation by Fe-Nitrogenase

Journal of Bacteriology ◽

10.1128/jb.00750-15 ◽

2015 ◽

Vol 198 (4) ◽

pp. 633-643 ◽

Cited By ~ 8

Author(s):

Marie-Christine Hoffmann ◽

Eva Wagner ◽

Sina Langklotz ◽

Yvonne Pfänder ◽

Sina Hött ◽

...

Keyword(s):

Nitrogen Fixation ◽

Biological Nitrogen Fixation ◽

Rhodobacter Capsulatus ◽

Nitrogenase Activity ◽

Central Process ◽

Content Type ◽

Proteome Profiling ◽

Biological Nitrogen ◽

Diazotrophic Growth

ABSTRACTRhodobacter capsulatusis capable of synthesizing two nitrogenases, a molybdenum-dependent nitrogenase and an alternative Mo-free iron-only nitrogenase, enabling this diazotroph to grow with molecular dinitrogen (N2) as the sole nitrogen source. Here, the Mo responses of the wild type and of a mutant lacking ModABC, the high-affinity molybdate transporter, were examined by proteome profiling, Western analysis, epitope tagging, andlacZreporter fusions. Many Mo-controlled proteins identified in this study have documented or presumed roles in nitrogen fixation, demonstrating the relevance of Mo control in this highly ATP-demanding process. The levels of Mo-nitrogenase, NifHDK, and the Mo storage protein, Mop, increased with increasing Mo concentrations. In contrast, Fe-nitrogenase, AnfHDGK, and ModABC, the Mo transporter, were expressed only under Mo-limiting conditions. IscN was identified as a novel Mo-repressed protein. Mo control of Mop, AnfHDGK, and ModABC corresponded to transcriptional regulation of their genes by the Mo-responsive regulators MopA and MopB. Mo control of NifHDK and IscN appeared to be more complex, involving different posttranscriptional mechanisms. In line with the simultaneous control of IscN and Fe-nitrogenase by Mo, IscN was found to be important for Fe-nitrogenase-dependent diazotrophic growth. The possible role of IscN as an A-type carrier providing Fe-nitrogenase with Fe-S clusters is discussed.IMPORTANCEBiological nitrogen fixation is a central process in the global nitrogen cycle by which the abundant but chemically inert dinitrogen (N2) is reduced to ammonia (NH3), a bioavailable form of nitrogen. Nitrogen reduction is catalyzed by nitrogenases found in diazotrophic bacteria and archaea but not in eukaryotes. All diazotrophs synthesize molybdenum-dependent nitrogenases. In addition, some diazotrophs, includingRhodobacter capsulatus, possess catalytically less efficient alternative Mo-free nitrogenases, whose expression is repressed by Mo. Despite the importance of Mo in biological nitrogen fixation, this is the first study analyzing the proteome-wide Mo response in a diazotroph. IscN was recognized as a novel member of the molybdoproteome inR. capsulatus. It was dispensable for Mo-nitrogenase activity but supported diazotrophic growth under Mo-limiting conditions.

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Novel Genes and Regulators That Influence Production of Cell Surface Exopolysaccharides inSinorhizobium meliloti

Journal of Bacteriology ◽

10.1128/jb.00501-17 ◽

2017 ◽

Vol 200 (3) ◽

Cited By ~ 5

Author(s):

Melanie J. Barnett ◽

Sharon R. Long

Keyword(s):

Cell Surface ◽

Biofilm Formation ◽

Sinorhizobium Meliloti ◽

Developmental Process ◽

Root Nodules ◽

Critical Role ◽

Nitrogen Fixing ◽

Symbiotic Interaction ◽

Content Type ◽

Mutant Phenotypes

ABSTRACTSinorhizobium melilotiis a soil-dwelling alphaproteobacterium that engages in a nitrogen-fixing root nodule symbiosis with leguminous plants. Cell surface polysaccharides are important both for adapting to stresses in the soil and for the development of an effective symbiotic interaction. Among the polysaccharides characterized to date, the acidic exopolysaccharides I (EPS-I; succinoglycan) and II (EPS-II; galactoglucan) are particularly important for protection from abiotic stresses, biofilm formation, root colonization, and infection of plant roots. Previous genetic screens discovered mutants with impaired EPS production, allowing the delineation of EPS biosynthetic pathways. Here we report on a genetic screen to isolate mutants with mucoid colonial morphologies that suggest EPS overproduction. Screening with Tn5-110, which allows the recovery of both null and upregulation mutants, yielded 47 mucoid mutants, most of which overproduce EPS-I; among the 30 unique genes and intergenic regions identified, 14 have not been associated with EPS production previously. We identified a new protein-coding gene,emmD, which may be involved in the regulation of EPS-I production as part of the EmmABC three-component regulatory circuit. We also identified a mutant defective in EPS-I production, motility, and symbiosis, where Tn5-110 was not responsible for the mutant phenotypes; these phenotypes result from a missense mutation inrpoAcorresponding to the domain of the RNA polymerase alpha subunit known to interact with transcription regulators.IMPORTANCEThe alphaproteobacteriumSinorhizobium meliloticonverts dinitrogen to ammonium while inhabiting specialized plant organs termed root nodules. The transformation ofS. melilotifrom a free-living soil bacterium to a nitrogen-fixing plant symbiont is a complex developmental process requiring close interaction between the two partners. As the interface between the bacterium and its environment, theS. meliloticell surface plays a critical role in adaptation to varied soil environments and in interaction with plant hosts. We isolated and characterizedS. melilotimutants with increased production of exopolysaccharides, key cell surface components. Our diverse set of mutants suggests roles for exopolysaccharide production in growth, metabolism, cell division, envelope homeostasis, biofilm formation, stress response, motility, and symbiosis.

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