Dataset Information

The molecular weight distribution of succinoglycan produced by Sinorhizobium meliloti is influenced by specific tyrosine phosphorylation and ATPase activity of the cytoplasmic domain of the ExoP protein.

ABSTRACT: It is thought that in the gram-negative soil bacterium Sinorhizobium meliloti the protein ExoP is involved in biosynthesis of the acidic exopolysaccharide succinoglycan (EPS I). The amounts and compositions of EPS I produced by mutants expressing ExoP proteins characterized by specific amino acid substitutions in the C-terminal cytoplasmic domain were analyzed. The cytoplasmic domain of the ExoP protein was shown to have ATPase activity. Mutations in the highly conserved Walker A ATP-binding motif prevented ATPase activity of the ExoP protein. Phenotypically, these mutations resulted in much lower levels of succinoglycan which consisted only of monomers of the octasaccharide repeating unit. The ExoP protein has similarities to proteins with autophosphorylating protein tyrosine kinase activity. We found that ExoP was phosphorylated on tyrosine and that site-directed mutagenesis of specific tyrosine residues in the cytoplasmic domain of ExoP resulted in an altered ratio of low-molecular-weight succinoglycan to high-molecular-weight succinoglycan.

SUBMITTER: Niemeyer D

PROVIDER: S-EPMC95393 | biostudies-literature | 2001 Sep

REPOSITORIES: biostudies-literature

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The molecular weight distribution of succinoglycan produced by Sinorhizobium meliloti is influenced by specific tyrosine phosphorylation and ATPase activity of the cytoplasmic domain of the ExoP protein.

Niemeyer D D Becker A A

Journal of bacteriology 20010901 17

It is thought that in the gram-negative soil bacterium Sinorhizobium meliloti the protein ExoP is involved in biosynthesis of the acidic exopolysaccharide succinoglycan (EPS I). The amounts and compositions of EPS I produced by mutants expressing ExoP proteins characterized by specific amino acid substitutions in the C-terminal cytoplasmic domain were analyzed. The cytoplasmic domain of the ExoP protein was shown to have ATPase activity. Mutations in the highly conserved Walker A ATP-binding mot ...[more]

PMID: 11489870

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Project description:UnlabelledThe acidic polysaccharide succinoglycan produced by the rhizobial symbiont Sinorhizobium meliloti 1021 is required for this bacterium to invade the host plant Medicago truncatula and establish a nitrogen-fixing symbiosis. S. meliloti mutants that cannot make succinoglycan cannot initiate invasion structures called infection threads in plant root hairs. S. meliloti exoH mutants that cannot succinylate succinoglycan are also unable to form infection threads, despite the fact that they make large quantities of succinoglycan. Succinoglycan produced by exoH mutants is refractory to cleavage by the glycanases encoded by exoK and exsH, and thus succinoglycan produced by exoH mutants is made only in the high-molecular-weight (HMW) form. One interpretation of the symbiotic defect of exoH mutants is that the low-molecular-weight (LMW) form of succinoglycan is required for infection thread formation. However, our data demonstrate that production of the HMW form of succinoglycan by S. meliloti 1021 is sufficient for invasion of the host M. truncatula and that the LMW form is not required. Here, we show that S. meliloti strains deficient in the exoK- and exsH-encoded glycanases invade M. truncatula and form a productive symbiosis, although they do this with somewhat less efficiency than the wild type. We have also characterized the polysaccharides produced by these double glycanase mutants and determined that they consist of only HMW succinoglycan and no detectable LMW succinoglycan. This demonstrates that LMW succinoglycan is not required for host invasion. These results suggest succinoglycan function is not dependent upon the presence of a small, readily diffusible form.ImportanceSinorhizobium meliloti is a bacterium that forms a beneficial symbiosis with legume host plants. S. meliloti and other rhizobia convert atmospheric nitrogen to ammonia, a nutrient source for the host plant. To establish the symbiosis, rhizobia must invade plant roots, supplying the proper signals to prevent a plant immune response during invasion. A polysaccharide, succinoglycan, produced by S. meliloti is required for successful invasion. Here, we show that the critical feature of succinoglycan that allows infection to proceed is the attachment of a "succinyl" chemical group and that the chain length of succinoglycan is much less important for its function. We also show that none of the short-chain versions of succinoglycan is produced in the absence of two chain-cleaving enzymes.

Project description:Sinorhizobium meliloti enters into beneficial symbiotic interactions with Medicago species of legumes. Bacterial exopolysaccharides play critical signaling roles in infection thread initiation and growth during the early stages of root nodule formation. After endocytosis of S. meliloti by plant cells in the developing nodule, plant-derived nodule-specific cysteine-rich (NCR) peptides mediate terminal differentiation of the bacteria into nitrogen-fixing bacteroids. Previous transcriptional studies showed that the intensively studied cationic peptide NCR247 induces expression of the exo genes that encode the proteins required for succinoglycan biosynthesis. In addition, genetic studies have shown that some exo mutants exhibit increased sensitivity to the antimicrobial action of NCR247. Therefore, we investigated whether the symbiotically active S. meliloti exopolysaccharide succinoglycan can protect S. meliloti against the antimicrobial activity of NCR247. We discovered that high-molecular-weight forms of succinoglycan have the ability to protect S. meliloti from the antimicrobial action of the NCR247 peptide but low-molecular-weight forms of wild-type succinoglycan do not. The protective function of high-molecular-weight succinoglycan occurs via direct molecular interactions between anionic succinoglycan and the cationic NCR247 peptide, but this interaction is not chiral. Taken together, our observations suggest that S. meliloti exopolysaccharides not only may be critical during early stages of nodule invasion but also are upregulated at a late stage of symbiosis to protect bacteria against the bactericidal action of cationic NCR peptides. Our findings represent an important step forward in fully understanding the complete set of exopolysaccharide functions during legume symbiosis.IMPORTANCE Symbiotic interactions between rhizobia and legumes are economically important for global food production. The legume symbiosis also is a major part of the global nitrogen cycle and is an ideal model system to study host-microbe interactions. Signaling between legumes and rhizobia is essential to establish symbiosis, and understanding these signals is a major goal in the field. Exopolysaccharides are important in the symbiotic context because they are essential signaling molecules during early-stage symbiosis. In this study, we provide evidence suggesting that the Sinorhizobium meliloti exopolysaccharide succinoglycan also protects the bacteria against the antimicrobial action of essential late-stage symbiosis plant peptides.

Dataset Information

The molecular weight distribution of succinoglycan produced by Sinorhizobium meliloti is influenced by specific tyrosine phosphorylation and ATPase activity of the cytoplasmic domain of the ExoP protein.

Publications

The molecular weight distribution of succinoglycan produced by Sinorhizobium meliloti is influenced by specific tyrosine phosphorylation and ATPase activity of the cytoplasmic domain of the ExoP protein.

Similar Datasets

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