Deeraksa Arpaporn, Moonmangmee Somporn, Toyama Hirohide, Yamada Mamoru, Adachi Osao, Matsushita Kazunobu
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan.
Department of Biotechnology, Thailand Institute of Scientific and Technological Research, Khlong Luang, Pathumthani 12120, Thailand.
Microbiology (Reading). 2005 Dec;151(Pt 12):4111-4120. doi: 10.1099/mic.0.28350-0.
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.
热带醋酸杆菌SKU1100产生一种附着于细胞表面的、由半乳糖、葡萄糖和鼠李糖组成的菌膜多糖。该菌株在琼脂平板上形成两种类型的菌落：表面粗糙的菌落（R菌株）和黏液状表面光滑的菌落（S菌株）。R菌株形成菌膜，使其在静置培养时能漂浮在培养基表面，而S菌株则不能。菌膜是由与细胞表面的荚膜多糖（CPS）紧密相连的细胞组成的聚集体。在本研究中，通过使用Tn10进行转座子诱变，研究了R菌株形成菌膜所需的基因。产生的突变体命名为Pel-，其菌落表面光滑，与S菌株一样，在菌膜形成方面存在缺陷。该突变体产生的多糖反而作为胞外多糖（EPS）分泌到培养基中。在Tn10插入位点鉴定出一个开放阅读框，命名为polE，其上游还发现了polABCD基因。polABCD推导的氨基酸序列与参与dTDP-鼠李糖合成的rfbBACD的氨基酸序列具有高度同源性，而polE与糖基转移酶的同源性相对较低。在本研究中，还制备了一个polB（rfbA）缺失突变体，该突变体既不产生CPS也不产生EPS。携带polE或polB基因的质粒可以分别在Pel（-）突变体和S菌株以及DeltapolB突变体中恢复菌膜形成。因此，polE和polB显然都参与了菌膜形成，最有可能分别是通过将多糖锚定到细胞表面和通过产生dTDP-鼠李糖来实现的。Pel-和DeltapolB突变体在静置培养中无法生长，并且由于菌膜形成的丧失而对乙酸变得更加敏感。此外，本研究确定了从原始培养物中自发分离出的几个S菌株的突变位点，发现它们集中在polE编码序列中7个C残基的序列中，单个C核苷酸发生缺失或添加。
