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.

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Acetobacter aceti IFO 3284 has been shown to have two types of strains: one forms a smooth-surfaced colony (S strain) and the other forms a rough-surfaced colony (R strain) (Matsushita et al., 1992). In this study, both S and R strains were isolated and characterized. The S strain grew well in submerged culture but very poorly in static culture. In contrast, the R strain grew well in static culture by floating on the surface of the culture medium, as well as in shaking submerged culture. Scanning electron microscopy revealed that the R strain was covered by some amorphous materials that were not seen in the S strain. The R strain produced 5-fold higher levels of sugars related to polysaccharides responsible for pellicle formation than the S strain did. Unlike cellulose of Acetobacter xylinum, the polysaccharides of the R strain were cellulase-resistant and alkaline-sensitive. The polysaccharides were not secreted into the culture medium, and more than 90% of them were retained in the membrane fraction when the cells were disrupted under mild conditions by lysozyme treatment. Furthermore, the polysaccharides were shown to be mainly attached to the outer membrane when separated. After solubilization with beta-octylglucoside, the membrane-attached polysaccharides were purified by several steps including enzyme treatment, column chromatography and alcohol precipitation. The purified polysaccharide was estimated to have an apparent molecular mass of 700-kDa based on Sephacryl S-500 column chromatography, and to be composed of two monosaccharides, glucose and rhamnose, at an approximately equimolar ratio. Thus, in this study, we clarified that the A. aceti R strain produced a polysaccharide associated with the flotation of the cells on the medium surface, like A. xylinum, and that the polysaccharide was a novel one consisting of glucose and rhamnose.

Two strains of strictly aerobic, moderately halophilic Gram-positive rods were isolated from fermented shrimp paste ('ka-pi') produced in Thailand. They produced a red pigment and grew optimally in the presence of 5-30 % NaCl. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The predominant menaquinone was MK-7. The major cellular fatty acid was anteiso-C 15 : 0 . Phosphatidylglycerol, diphosphatidylglycerol and two unidentified glycolipids were found to be the major polar lipid components. The DNA G+C content was 41.2-41.6 mol%. Comparative 16S rRNA gene sequence analyses showed that strain PN7-6 T was most closely related to Lentibacillus salarius KCTC 3911 T with 96.5 % sequence similarity. On the basis of phenotypic and molecular properties, the two isolates represent a novel species of the genus Lentibacillus, for which the name Lentibacillus kapialis sp. nov. is proposed. The type strain is PN7-6 T (=JCM 12580Moderately halophilic, alkaliphilic, and related aerobic endospore-forming, Gram-positive, rod-shaped bacteria, including cocci, have been isolated from various salty environments. These isolates have included members of the genera Marinococcus (Hao et al