Exopolysaccharides from Sinorhizobium meliloti can protect against H2O2-dependent damage.
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ABSTRACT:
SUBMITTER: Lehman AP
PROVIDER: S-EPMC3837946 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Sinorhizobium meliloti is 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 in rpoA corresponding to the domain of the RNA polymerase alpha subunit known to interact with transcription regulators.IMPORTANCE The alphaproteobacterium Sinorhizobium meliloti converts dinitrogen to ammonium while inhabiting specialized plant organs termed root nodules. The transformation of S. meliloti from 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, the S. meliloti cell surface plays a critical role in adaptation to varied soil environments and in interaction with plant hosts. We isolated and characterized S. meliloti mutants 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.
| S-EPMC5763050 | biostudies-literature
Project description:Sinorhizobium meliloti 1021 carries two megaplasmids, pSymA of 1,354 kb and pSymB of 1,683 kb, which are essential in establishing symbiosis with its legume hosts and important for bacterial fitness in the rhizosphere. We have previously shown that pSymA is self-transmissible and that its conjugal functions are regulated by the transcriptional repressor RctA. Here, we show conjugal transfer of pSymB as an in trans mobilization event that requires the type IV secretion system encoded by pSymA. pSymB carries a functional oriT and an adjacent relaxase gene, traA2, that is also transcriptionally repressed by rctA. Both symbiotic megaplasmids would require the relaxase genes in cis with their respective oriTs to achieve the highest transfer efficiencies.
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Project description: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.
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Project description:The number of solute-binding protein-dependent transporters in rhizobia is dramatically increased compared with the majority of other bacteria so far sequenced. This increase may be due to the high affinity of solute-binding proteins for solutes, permitting the acquisition of a broad range of growth-limiting nutrients from soil and the rhizosphere. The transcriptional induction of these transporters was studied by creating a suite of plasmid and integrated fusions to nearly all ATP-binding cassette (ABC) and tripartite ATP-independent periplasmic (TRAP) transporters of Sinorhizobium meliloti. In total, specific inducers were identified for 76 transport systems, amounting to approximately 47% of the ABC uptake systems and 53% of the TRAP transporters in S. meliloti. Of these transport systems, 64 are previously uncharacterized in Rhizobia and 24 were induced by solutes not known to be transported by ABC- or TRAP-uptake systems in any organism. This study provides a global expression map of one of the largest transporter families (transportome) and an invaluable tool to both understand their solute specificity and the relationships between members of large paralogous families.
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Project description:Reactive oxygen species such as peroxides play an important role in plant development, cell wall maturation, and defense responses. During nodulation with the host plant Medicago sativa, Sinorhizobium meliloti cells are exposed to H2O2 in infection threads and developing nodules (R. Santos, D. Hérouart, S. Sigaud, D. Touati, and A. Puppo, Mol Plant Microbe Interact 14:86-89, 2001, https://doi.org/10.1094/MPMI.2001.14.1.86). S. meliloti cells likely also experience oxidative stress, from both internal and external sources, during life in the soil. Here, we present microarray transcription data for S. meliloti wild-type cells compared to a mutant deficient in the key oxidative regulatory protein OxyR, each in response to H2O2 treatment. Several alternative sigma factor genes are upregulated in the response to H2O2; the stress sigma gene rpoE2 shows OxyR-dependent induction by H2O2, while rpoH1 expression is induced by H2O2 irrespective of the oxyR genotype. The activity of the RpoE2 sigma factor in turn causes increased expression of two more sigma factor genes, rpoE5 and rpoH2 Strains with deletions of rpoH1 showed improved survival in H2O2 as well as increased levels of oxyR and total catalase expression. These results imply that ΔrpoH1 strains are primed to deal with oxidative stress. This work presents a global view of S. meliloti gene expression changes, and of regulation of those changes, in response to H2O2IMPORTANCE Like all aerobic organisms, the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti experiences oxidative stress throughout its complex life cycle. This report describes the global transcriptional changes that S. meliloti makes in response to H2O2 and the roles of the OxyR transcriptional regulator and the RpoH1 sigma factor in regulating those changes. By understanding the complex regulatory response of S. meliloti to oxidative stress, we may further understand the role that reactive oxygen species play as both stressors and potential signals during symbiosis.
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Project description:Sinorhizobium meliloti Rm41 nodulates alfalfa plants, forming indeterminate type nodules. It is characterized by a strain-specific K-antigen able to replace exopolysaccharides in promotion of nodule invasion. We present the Rm41 genome, composed of one chromosome, the chromid pSymB, the megaplasmid pSymA, and the nonsymbiotic plasmid pRme41a.
| S-EPMC3556828 | biostudies-literature
Project description:Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in piglets; ETEC cells colonize the intestinal mucosa with adhesins and deliver toxins that cause fluid loss. This study determined the antiadhesive properties of bacterial exopolysaccharides (reuteran and levan) and related glycans (dextran and inulin) in a small intestinal segment perfusion (SISP) model. The SISP model used 10 jejunal segments from 5-week-old piglets. Five segments were infected with ETEC expressing K88 fimbriae (ETEC K88), while five segments were treated with saline. Every two segments (ETEC and non-ETEC infected) were infused with 65 ml of 10 g liter(-1) of glycans or saline (control) for 8 h. High-resolution melting-curve (HRM) quantitative PCR (qPCR) indicated that E. coli is the dominant bacterium in infected segments, while other bacteria were predominant in noninfected segments. Infection by ETEC K88 was also verified by qPCR; gene copy numbers of K88 fimbriae and the heat-labile toxin (LT) in mucosal scrapings and outflow fluid of infected segments were significantly higher than those in noninfected segments. Genes coding for K88 fimbriae and LT were also detected in noninfected segments. LT amplicons from infected and noninfected segments were 99% identical over 481 bp, demonstrating the presence of autochthonous ETEC K88. All glycans reduced fluid loss caused by ETEC K88 infection. Reuteran tended (P = 0.06) to decrease ETEC K88 levels in mucosal scraping sample, as judged by qPCR. Fluorescent in situ hybridization analysis demonstrated that reuteran significantly (P = 0.012) decreased levels of adherent ETEC K88. Overall, reuteran may prevent piglet diarrhea by reducing adhesion of ETEC K88.
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Project description:Vitamin B(12) (cobalamin) is a critical cofactor for animals and protists, yet its biosynthesis is limited to prokaryotes. We previously showed that the symbiotic nitrogen-fixing alphaproteobacterium Sinorhizobium meliloti requires cobalamin to establish a symbiotic relationship with its plant host, Medicago sativa (alfalfa). Here, the specific requirement for cobalamin in the S. meliloti-alfalfa symbiosis was investigated. Of the three known cobalamin-dependent enzymes in S. meliloti, the methylmalonyl CoA mutase (BhbA) does not affect symbiosis, whereas disruption of the metH gene encoding the cobalamin-dependent methionine synthase causes a significant defect in symbiosis. Expression of the cobalamin-independent methionine synthase MetE alleviates this symbiotic defect, indicating that the requirement for methionine synthesis does not reflect a need for the cobalamin-dependent enzyme. To investigate the function of the cobalamin-dependent ribonucleotide reductase (RNR) encoded by nrdJ, S. meliloti was engineered to express an Escherichia coli cobalamin-independent (class Ia) RNR instead of nrdJ. This strain is severely defective in symbiosis. Electron micrographs show that these cells can penetrate alfalfa nodules but are unable to differentiate into nitrogen-fixing bacteroids and, instead, are lysed in the plant cytoplasm. Flow cytometry analysis indicates that these bacteria are largely unable to undergo endoreduplication. These phenotypes may be due either to the inactivation of the class Ia RNR by reactive oxygen species, inadequate oxygen availability in the nodule, or both. These results show that the critical role of the cobalamin-dependent RNR for survival of S. meliloti in its plant host can account for the considerable resources that S. meliloti dedicates to cobalamin biosynthesis.
| S-EPMC2979309 | biostudies-literature
Project description:Fur is a transcriptional regulator involved in iron-dependent control of gene expression in many bacteria. In this work we analyzed the phenotype of a fur mutant in Sinorhizobium meliloti, an alpha-proteobacterium that fixes N(2) in association with host plants. We demonstrated that some functions involved in high-affinity iron transport, siderophore production, and iron-regulated outer membrane protein expression respond to iron in a Fur-independent manner. However, manganese-dependent expression of the MntABCD manganese transport system was lost in a fur strain as discerned by constitutive expression of a mntA::gfp fusion reporter gene in the mutant. Thus, Fur directly or indirectly regulates a manganese-dependent function. The data indicate a novel function for a bacterial Fur protein in mediating manganese-dependent regulation of gene expression.
| S-EPMC444773 | biostudies-literature
Project description:The availability of bacterial genome sequences has created a need for improved methods for sequence-based functional analysis to facilitate moving from annotated DNA sequence to genetic materials for analyzing the roles that postulated genes play in bacterial phenotypes. A powerful cloning method that uses lambda integrase recombination to clone and manipulate DNA sequences has been adapted for use with the gram-negative alpha-proteobacterium Sinorhizobium meliloti in two ways that increase the utility of the system. Adding plasmid oriT sequences to a set of vehicles allows the plasmids to be transferred to S. meliloti by conjugation and also allows cloned genes to be recombined from one plasmid to another in vivo by a pentaparental mating protocol, saving considerable time and expense. In addition, vehicles that contain yeast Flp recombinase target recombination sequences allow the construction of deletion mutations where the end points of the deletions are located at the ends of the cloned genes. Several deletions were constructed in a cluster of 60 genes on the symbiotic plasmid (pSymA) of S. meliloti, predicted to code for a denitrification pathway. The mutations do not affect the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa (alfalfa) roots.
| S-EPMC404432 | biostudies-literature