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Role of calcium and exopolysaccharide production in acid tolerance by Sinorhizobium meliloti

Vivas-Marfisi, Ana Isabel (2001) Role of calcium and exopolysaccharide production in acid tolerance by Sinorhizobium meliloti. PhD thesis, Murdoch University.

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This thesis reports on the role(s) of exopolysaccharides (EPS) in acid tolerance in Sinorhizobium meliloti, whether the presence of calcium in the growth medium influences the type and amount of EPS produced at low pH, and the significance of the exoH gene in acid tolerance.

The strains used were the wild type WSM419 and a series of transposon Tn5-induced mutants which varied in sensitivity to low pH, and in their growth response to calcium at low pH. The EPS produced by the wild type and the mutants grown at pH 7.0 and pH 6.0, in the presence of 1 or 3 mM CaCl2 was analyzed by 1H NMR spectroscopy. The relative amounts of the acetyl and pyruvyl modifications to EPS were significantly lower at pH 6.0 than at pH 7.0 for the wild type, WRR2, TG2-6 and TG5-45. There was no effect of pH on the relative amount of succinylation nor of calcium concentration on the relative amount of the modifications.

The rates of EPS synthesis by cultures of WSM419 and its mutants were also measured. At neutral pH, the rates of EPS synthesis for all the mutants were unaffected by calcium concentration. At pH 6.0 and 1 mM CaCI2, the rate of synthesis of EPS by most mutants was up to three-fold that of the wild type WSM419.

For WSM419, the rate of EPS production at pH 6.0 was stimulated approximately 40% when calcium concentration was increased from 1 mM to 3 mM. By contrast, the rate of EPS synthesis shown by most of the mutants decreased when the calcium concentration went from 1 mM to 3 mM. For WRR1, the rate of synthesis of EPS was not affected by the calcium concentration at either pH 7.0 or 6.0.

Gel filtration assays were used to evaluate the molecular weight distribution of EPS produced by S. meliloti WSM419 and its exoH mutant WRR1 at pH 7.0 and 6.0, and with calcium concentrations of 0.3 mM and 3 mM. EPS produced by WRR1 was predominantly high-molecular weight. Integration of the areas of the gel filtration peaks indicated that the relative amounts of high and low molecular weight succinoglycan produced by the wild type WSM419 and WRR1 are unaffected by pH.

As well as being acid-sensitive and failing to grow below pH 6.0, the mutant WRR1 also shows a zinc-sensitive phenotype. This mutant has been shown to have a single Tn5-insertion disrupting the exoH gene. A large plasmid, (pEX154), which carries 18 kb of DNA from S. meliloti Rm1021 (including many of the EPS biosynthetic exo genes), complements both the acid- and zinc-sensitivity of WRR1.

Sub-cloning of fragments of pEX154 was used to localise the complementing gene(s). The plasmid pAV-1C carried an 11.5 kb DNA fragment (including exoTIHKLA) and also complemented the mutation in WRR1. Smaller fragments cloned into the vector pSW213 did not complement WRR1. These results raised the possibility that the lack of complementation observed could be the result of a weak transcription of the exo genes cloned into the vector pSW213.

The involvement of the gene in EPS I biosynthesis and acid and zinc sensitivity was explored further using another vector. The plasmids pEX41 and pEX42 carrying exoH and exoK genes as well as part of the exoL locus, were successful in complementing both the pH and zinc-sensitivity defects of WRR1. The complementation responses obtained with smaller clones derived from those plasmids has suggested that exoH alone is able to restore the phenotypic defects in WRR1.

WRR1 did not grow when EPS produced by the wild type WSM419 was added, nor when it was inoculated into minimal medium at pH 6.0 with the wild type WSM419. When the exoY gene in WSM419 was inactivated, the mutant failed to produce EPS I, but it was able to grow both at low pH and on zinc supplemented media. These results suggest that EPS is not involved in restoring the acid- and zinc-sensitivity defects in WRR1 and that the exoH gene may code for a protein with cellular functions other than that of succinylating EPS.

Item Type: Thesis (PhD)
Murdoch Affiliation: Division of Science and Engineering
Supervisor(s): Dilworth, Michael and Glenn, Andrew
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