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Induction of L-form-like cell shape change of Bacillus subtilis under microculture conditions

¹ Department of Oral Microbiology, Okayama University Graduate School of Medicine and Dentistry, Shikata-cho, Okayama 700-8525, Japan
² Department of Bioresource Science, Ibaraki University, School of Agriculture, Ami, Ibaraki 300-0393, Japan
³ Department of Intercellular Communication, Graduate School of Biological Science, Nara Institute of Science and Technology, Ikoma 630-0101, Japan
⁴ Department of Bioinformatics and Genomics, Graduate School of Information Science, Nara Institute of Science and Technology, Ikoma 630-0101, Japan
⁵ Department of Operative Dentistry, Okayama University Graduate School of Medicine and Dentistry, Shikata-cho, Okayama 700-8525, Japan

Correspondence
Ryuji Shingaki
shingaki{at}md.okayama-u.ac.jp

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 -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.

A table of proteins detected by LC/MS/MS in cells grown in microculture on CI and CI+KNO₃ is available as supplementary data with the online version of this paper at http://mic.sgmjournals.org.

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