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.
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
Acetobacter strains able to produce a thick pellicle at 379 C were screened among many thermotolerant strains isolated from fruits in Thailand. As a result, Acetobacter sp. SKU 1100 was selected as the producer of a relatively thick pellicle even when cultured at higher temperatures such as 379 C or 409 C. This strain could produce a pellicle polysaccharide in a shaking submerged culture as well as under static culture conditions. The polysaccharide was found to be attached to the bacterial cells. Although the polysaccharide production was higher at 309 C than at 379 C in shaking submerged culture, the productivity in static culture was not decreased even at higher temperatures. The membrane-attached polysaccharide was puriˆed from the SKU 1100 strain by cell disruptions using either ultrasonic treatment or lysozyme treatment, followed by ultracentrifugation, enzyme treatments, dialysis against SDS, DEAE-cellulose column chromatography, alcohol precipitation, and gel ltration chromatography. The polysaccharide puriˆed by the sonic treatment and also by the mild conditions using lysozyme treatment had the same average molecular mass of 120 kDa. The puriˆed polysaccharide was composed of three diŠerent monosaccharides; glucose, galactose, and rhamnose, in an approximately equimolar ratio of 1:1:1.
A Gram-positive and catalase-negative coccus that formed chains, strain FP15-1 T , isolated from fermented tea leaves ('miang'), was studied systematically. The strain was facultatively anaerobic and produced L-lactic acid from glucose. Demethylmenaquinone (DMK-7) was the major menaquinone. Straight-chain unsaturated fatty acids C 16 : 1 and C 18 : 1 were the dominant components. The DNA G+C content was 37.8 mol%. On the basis of 16S rRNA and RNA polymerase a subunit (rpoA) gene sequence analysis, strain FP15-1 T was closely related toEnterococcus italicus KCTC 5373 T , with 99.2 and 93.8 % similarity, respectively. The strain could be clearly distinguished from E. italicus ATCC 5373T by low DNA-DNA relatedness (¡33.8 %)and phenotypic characteristics. Therefore, this strain represent a novel species of the genus Enterococcus, for which the name Enterococcus camelliae sp. nov. is proposed. The enterococci comprise an important group of lactic acid bacteria found ubiquitously in the environment, the gastrointestinal tract, traditional fermented foods and dairy products. The classification of the genus Enterococcus has undergone considerable changes as a consequence of the increasing number of species and also improvements in methods to discriminate between species (Baele et al., 2000;Merquior et al., 1994;Naser et al., 2005 (Fortina et al., 2004), by comparison of partial sequences for three housekeeping genes, phenylalanyl-tRNA synthase a subunit (pheS), RNA polymerase a subunit (rpoA) and the a subunit of ATP synthase (atpA), and as confirmed by DNA-DNA hybridization (Naser et al., 2006). In the present paper, we describe a novel Enterococcus strain from fermented tea leaves ('miang') based on phenotypic and chemotaxonomic characteristics, DNA-DNA relatedness and 16S rRNA and rpoA gene sequence analysis.Samples of fermented tea leaves were collected from Chiangmai province, in the northern part of Thailand. Cocci in chains were isolated from the samples using GYPCaCO 3 agar (Tanasupawat et al., 1992). GYP-sodium acetate-mineral salt broth (Tanasupawat et al., 1992) (pH 7.2) was used for working cultures. All tests were performed by incubating the cultures at 30 u C. Cell shape, size and arrangement and colony appearance were examined using cells grown on GYP agar for 3 days. Gram staining was done as described by Hucker & Conn (1923). Spore formation was examined in the Gram-stained specimen.Results of the oxidation/fermentation test and motility were examined in soft agar (Whittenbury, 1963). Catalase activity,The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and rpoA gene sequences of strain FP15-1 T are respectively EF154454 and EF197993.16S rRNA gene sequence-based maximum-likelihood and maximumparsimony trees are available as supplementary material with the online version of this paper. hydrolysis of gelatin, aesculin, arginine and starch, nitrate reduction, production of gas from glucose, gluconate and citrate and acid formation from carbohydrates were tested as reported by Tanasupawat et al. (1992)...
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