Characterization and spontaneous mutation of a novel gene, polE, involved in pellicle formation in Acetobacter tropicalis SKU1100

Abstract

Acetobacter tropicalis 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 Tn10. 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 Tn10 insertion site, designated polE, upstream of which polABCD genes were also found. The deduced amino acid sequences of polABCD showed a high level of homology to those of rfbBACD which are involved in dTDP-rhamnose synthesis, whereas polE had a relatively low level of homology to glycosyltransferase. In this study a polB (rfbA) disruptant was also prepared, which lacked both CPS and EPS production. A plasmid harbouring the polE or polB genes could restore pellicle formation in the Pel(-) mutant and S strains, and in the DeltapolB mutant, respectively. Thus both polE and polB 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 DeltapolB 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 polE, with the deletion or addition of a single C nucleotide.

Adaptation and tolerance of bacteria against acetic acid

“…Strains of A. pasteurianus able to cover the cells with capsular polysaccharides were found to tolerate higher concentrations of acetic acid in comparison with corresponding mutants that were not able to produce this pellicle (Deeraksa et al 2005). This protection against acetic acid appeared at the late exponential and stationary growth phase but was not present during the acetate consumption, indicating that the polysaccharides covering the cells reduce diffusion of acetic acid into the cell during ethanol oxidation phase (Kanchanarach et al 2010).…”

Structural characterization of pellicle polysaccharides of Acetobacter tropicalis SKU1100 wild type and mutant strains

“…tropicalis SKU1100 (R) strain and its mutants polE and galE (Deeraksa et al, 2005(Deeraksa et al, , 2006 were used in this study. In polE mutant, the polE gene was disrupted by insertion of Tn10 transposon, while, in galE, the galE gene (UDP-galactose 4-epimerase) was disrupted by insertion of non-polar kanamycin cassette.…”

“…In polE mutant, the polE gene was disrupted by insertion of Tn10 transposon, while, in galE, the galE gene (UDP-galactose 4-epimerase) was disrupted by insertion of non-polar kanamycin cassette. All strains were grown at 30 • C in potato medium (Deeraksa et al, 2005). Antibiotics, kanamycin or tetracycline, were added at the concentration of 50 g/ml, or 12.5 g/ml, respectively.…”

“…The genetic study of polysaccharides in acetic acid bacteria has shown that the acs (Saxena, Kudlicka, Okuda, & Brown, 1994) and the bcs (Wong et al, 1990) operons, in addition to ORF2 gene (Nakai, Nishiyama, Kuga, Sugano, & Shoda, 2002), are involved in cellulose biosynthesis, and the aceRQP operon in acetan biosynthesis in Ga. xylinus (Ishida, Sugano, & Shoda, 2002). Moreover, a gene cluster, polABCDE, required for pellicle formation in the R strain of A. tropicalis SKU1100, has been identified (Deeraksa et al, 2005). In this operon, polABCD showed high similarity to rfbBACD genes which are involved in dTDP-l-rhamnose biosynthesis, while polE, a novel gene downstream of polABCD, had a relatively low similarity to glycosyltransferases.…”

Determinative Factors of Competitive Advantage between Aerobic Bacteria for Niches at the Air-Liquid Interface

“…Therefore, pellicles are considered advantageous to aerobic bacteria for acquiring niches at the air-liquid interface. Pellicles of acetic acid bacteria (2), Bacillus sp. (1,10), and Pseudomonas sp.…”