Complete Genome Sequences of Rhizobium gallicum M101 and Two Potential New Rhizobium Species Isolated from Soils in Central Canada.

Here, we report the genome sequences of Rhizobium gallicum M101, Rhizobium sp. strain C104, and Rhizobium sp. strain K102. These bacteria were isolated from three locations in Manitoba, Canada. The M101 genome meets the criteria for R. gallicum based on average nucleotide identity and DNA-DNA hybridization; the genomes of C104 and K102 are below the thresholds to be matched to known type strains.

The Rhizobium-Legume Symbiosis: Co-opting Successful Stress Management.

The interaction of bacteria with plants can result in either a positive, negative, or neutral association. The rhizobium-legume interaction is a well-studied model system of a process that is considered a positive interaction. This process has evolved to require a complex signal exchange between the host and the symbiont. During this process, rhizobia are subject to several stresses, including low pH, oxidative stress, osmotic stress, as well as growth inhibiting plant peptides. A great deal of work has been carried out to characterize the bacterial response to these stresses. Many of the responses to stress are also observed to have key roles in symbiotic signaling. We propose that stress tolerance responses have been co-opted by the plant and bacterial partners to play a role in the complex signal exchange that occurs between rhizobia and legumes to establish functional symbiosis. This review will cover how rhizobia tolerate stresses, and how aspects of these tolerance mechanisms play a role in signal exchange between rhizobia and legumes.

Characterization of Mutations That Affect the Nonoxidative Pentose Phosphate Pathway in Sinorhizobium meliloti.

Sinorhizobium meliloti is a Gram-negative alphaproteobacterium that can enter into a symbiotic relationship with Medicago sativa and Medicago truncatula Previous work determined that a mutation in the tkt2 gene, which encodes a putative transketolase, could prevent medium acidification associated with a mutant strain unable to metabolize galactose. Since the pentose phosphate pathway in S. meliloti is not well studied, strains carrying mutations in either tkt2 and tal, which encodes a putative transaldolase, were characterized. Carbon metabolism phenotypes revealed that both mutants were impaired in growth on erythritol and ribose. This phenotype was more pronounced for the tkt2 mutant strain, which also displayed auxotrophy for aromatic amino acids. Changes in pentose phosphate pathway metabolite concentrations were also consistent with a mutation in either tkt2 or tal The concentrations of metabolites in central carbon metabolism were also found to shift dramatically in strains carrying a tkt2 mutation. While the concentrations of proteins involved in central carbon metabolism did not change significantly under any conditions, the levels of those associated with iron acquisition increased in the wild-type strain with erythritol induction. These proteins were not detected in either mutant, resulting in less observable rhizobactin production in the tkt2 mutant. While both mutants were impaired in succinoglycan synthesis, only the tkt2 mutant strain was unable to establish symbiosis with alfalfa. These results suggest that tkt2 and tal play central roles in regulating the carbon flow necessary for carbon metabolism and the establishment of symbiosis.IMPORTANCESinorhizobium meliloti is a model organism for the study of plant-microbe interactions and metabolism, especially because it effects nitrogen fixation. The ability to derive the energy necessary for nitrogen fixation is dependent on an organism's ability to metabolize carbon efficiently. The pentose phosphate pathway is central in the interconversion of hexoses and pentoses. This study characterizes the key enzymes of the nonoxidative branch of the pentose phosphate pathway by using defined genetic mutations and shows the effects the mutations have on the metabolite profile and on physiological processes such as the biosynthesis of exopolysaccharide, as well as the ability to regulate iron acquisition.

Succinoglycan Production Contributes to Acidic pH Tolerance in Sinorhizobium meliloti Rm1021.

In this work, the hypothesis that exopolysaccharide plays a role in the survival of Sinorhizobium meliloti at low pH levels is addressed. When S. meliloti was grown at pH 5.75, synthesis of succinoglycan increased, whereas synthesis of galactoglucan decreased. Succinoglycan that was isolated from cultures grown at low pH had a lower degree of polymerization relative to that which was isolated from cultures grown at neutral pH, suggesting that low-molecular weight (LMW) succinoglycan might play a role in adaptation to low pH. Mutants unable to produce succinoglycan or only able to produce high-molecular weight polysaccharide were found to be sensitive to low pH. However, strains unable to produce LMW polysaccharide were 10-fold more sensitive. In response to low pH, transcription of genes encoding proteins for succinoglycan, glycogen, and cyclic ß(1-2) glucans biosynthesis increased, while those encoding proteins necessary for the biosynthesis of galactoglucan decreased. While changes in pH did not affect the production of glycogen or cyclic ß(1-2) glucan, it was found that the inability to produce cyclic ß(1-2) glucan did contribute to pH tolerance in the absence of succinoglycan. Finally, in addition to being sensitive to low pH, a strain carrying mutations in exoK and exsH, which encode the glycanases responsible for the cleavage of succinoglycan to LMW succinoglycan, exhibited a delay in nodulation and was uncompetitive for nodule occupancy. Taken together, the data suggest that the role for LMW succinoglycan in nodule development may be to enhance survival in the colonized curled root hair.

Common dyes used to determine bacterial polysaccharides on agar are affected by medium acidification.

In this work, we highlight effects of pH on bacterial phenotypes when using the bacteriological dyes Aniline blue, Congo red, and Calcofluor white to analyze polysaccharide production. A study of galactose catabolism in Sinorhizobium meliloti led to the isolation of a mutation in dgoK1, which was observed to overproduce exopolysaccharides when grown in the presence of galactose. When this mutant strain was spotted onto plates containing Aniline blue, Congo red, or Calcofluor white, the intensity of the associated staining was strikingly different from that of the wild type. Additionally, a Calcofluor dull phenotype was observed, suggesting production of a polysaccharide other than succinoglycan. Further investigation of this phenotype revealed that these results were dependent on medium acidification, as buffering at pH 6 had no effect on these phenotypes, while medium buffered at pH 6.5 resulted in a reversal of the phenotypes. Screening for mutants of the dgoK1 strain that were negative for the Aniline blue phenotype yielded a strain carrying a mutation in tkt2, which is annotated as a putative transketolase. Consistent with the plate phenotypes, when this mutant was grown in broth cultures, it did not acidify its growth medium. Overall, this work shows that caution should be exercised in evaluating polysaccharide phenotypes based strictly on the use of dyes.

Characterisation of a gene encoding a membrane protein that affects exopolysaccharide production and intracellular Mg2+ concentrations in Ensifer meliloti.

Exopolysaccharides play an important role in the physiology of a bacterial cell. Ensifer meliloti is capable of producing at least two types of exopolysaccharides (EPS): succinoglycan and galactoglucan. In E. meliloti, EPS are best known for their role in mediating interaction with its symbiotic hosts. It was previously shown that high concentrations of Mg2+ or K+ were capable of suppressing the mucoid phenotype associated with galactoglucan production in an expR+ derivative of Rm1021. In an attempt to determine how Mg2+ regulates galactoglucan production, SRmD363 was mutagenised and screened for mutants which were visibly mucoid at high concentrations of magnesium. Tn5 mutations in genes exoX, emmB, phoC and SMc00722 were isolated. SMc00722 is annotated as a hypothetical transmembrane protein that is conserved in the α-proteobacteria. Characterisation of SMc00722 in Rm1021 showed that the increased mucoidy was due to succinoglycan. Strains carrying mutations in SMc00722 showed increased biofilm production, and were more sensitive to high Mg2+ concentrations and deoxycholate. In addition, we show that strains carrying a mutation in SMc00722 have elevated intracellular Mg2+ concentrations. Taken together, the data are consistent with the hypothesis that SMc0722 may play a role in maintaining intracellular magnesium concentration, and we suggest that this gene be tentatively annotated as mhrA (magnesium homeostasis related).