1887

Abstract

SKU1100 produces a pellicle polysaccharide, consisting of galactose, glucose and rhamnose, which attaches to the cell surface. This strain forms two types of colony on agar plates: a rough-surfaced colony (R strain) and a mucoid smooth-surfaced colony (S strain). The R strain forms a pellicle, allowing it to float on the medium surface in static culture, while the S strain does not. The pellicle is an assemblage of cells which are tightly associated with capsular polysaccharides (CPS) on the cell surface. In this study, a gene required for pellicle formation by the R strain was investigated by transposon mutagenesis using Tn. The resulting mutant, designated Pel, has a smooth-surfaced colony and a defect in pellicle formation, as for the S strain. The mutant produced polysaccharide which was instead secreted into the culture medium as extracellular polysaccharide (EPS). An ORF was identified at the Tn insertion site, designated , upstream of which genes were also found. The deduced amino acid sequences of showed a high level of homology to those of which are involved in dTDP-rhamnose synthesis, whereas had a relatively low level of homology to glycosyltransferase. In this study a () disruptant was also prepared, which lacked both CPS and EPS production. A plasmid harbouring the or genes could restore pellicle formation in the Pel mutant and S strains, and in the Δ mutant, respectively. Thus both and are evidently involved in pellicle formation, most likely by anchoring polysaccharide to the cell surface and through the production of dTDP-rhamnose, respectively. The Pel and Δ mutants were unable to grow in static culture and became more sensitive to acetic acid due to the loss of pellicle formation. Additionally, this study identified the mutation sites of several S strains which were spontaneously isolated from the original culture and found them to be concentrated in a sequence of 7 C residues in the coding sequence of , with the deletion or addition of a single C nucleotide.

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2005-12-01
2019-10-14
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vol. 151, part 12, pp. 4111-4120

Tables showing the bacterial strains, plasmids and PCR primers used in this study are available as an Acrobat PDF file.



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