Shingaki Ryuji, Kasahara Yasuhiro, Iwano Megumi, Kuwano Masayoshi, Takatsuka Tomomasa, Inoue Tetsuyoshi, Kokeguchi Susumu, Fukui Kazuhiro
Department of Oral Microbiology, Okayama University Graduate School of Medicine and Dentistry, Shikata-cho, Okayama 700-8525, Japan.
Department of Bioresource Science, Ibaraki University, School of Agriculture, Ami, Ibaraki 300-0393, Japan.
Microbiology (Reading). 2003 Sep;149(Pt 9):2501-2511. doi: 10.1099/mic.0.26259-0.
A remarkable cell shape change was observed in Bacillus subtilis strain 168 under microculture conditions on CI agar medium (Spizizen's minimal medium supplemented with a trace amount of yeast extract and Casamino acids). Cells cultured under a cover glass changed in form from rod-shaped to spherical, large and irregular shapes that closely resembled L-form cells. The cell shape change was observed only with CI medium, not with Spizizen's minimum medium alone or other rich media. The whole-cell protein profile of cells grown under cover glass and cells grown on CI agar plates differed in several respects. Tandem mass analysis of nine gel bands which differed in protein expression between the two conditions showed that proteins related to nitrate respiration and fermentation were expressed in the shape-changed cells grown under cover glass. The cell shape change of CI cultures was repressed when excess KNO3 was added to the medium. Whole-cell protein analysis of the normal rod-shaped cells grown with 0.1% KNO3 and the shape-changed cells grown without KNO3 revealed that the expression of the branched-chain alpha-keto acid dehydrogenase complex (coded by the bfmB gene locus) was elevated in the shape-changed cells. Inactivation of the bfmB locus resulted in the repression of cell shape change, and cells in which bfmB expression was induced by IPTG did show changes in shape. Transmission electron microscopy of ultrathin sections demonstrated that the shape-changed cells had thin walls, and plasmolysis of cells fixed with a solution including 0.1 M sucrose was observed. Clarifying the mechanism of thinning of the cell wall may lead to the development of a new type of cell wall biosynthetic inhibitor.
在CI琼脂培养基（添加微量酵母提取物和酪蛋白氨基酸的Spizizen基本培养基）上进行微培养时，枯草芽孢杆菌168菌株中观察到显著的细胞形态变化。在盖玻片下培养的细胞形态从杆状变为球形、大的不规则形状，与L型细胞非常相似。仅在CI培养基中观察到细胞形态变化，单独的Spizizen基本培养基或其他丰富培养基中未观察到。在盖玻片下生长的细胞和在CI琼脂平板上生长的细胞的全细胞蛋白质谱在几个方面存在差异。对两种条件下蛋白质表达不同的九条凝胶带进行串联质谱分析表明，与硝酸盐呼吸和发酵相关的蛋白质在盖玻片下生长的形态改变细胞中表达。当向培养基中添加过量的KNO3时，CI培养物的细胞形态变化受到抑制。对在0.1% KNO3条件下生长的正常杆状细胞和在无KNO3条件下生长的形态改变细胞进行全细胞蛋白质分析发现，支链α-酮酸脱氢酶复合物（由bfmB基因座编码）在形态改变细胞中的表达升高。bfmB基因座的失活导致细胞形态变化受到抑制，而通过IPTG诱导bfmB表达的细胞确实出现了形态变化。超薄切片的透射电子显微镜观察表明，形态改变的细胞具有薄壁，并且观察到用含0.1 M蔗糖的溶液固定的细胞发生了质壁分离。阐明细胞壁变薄的机制可能会导致新型细胞壁生物合成抑制剂的开发。
