Precise deletion of tagD and controlled depletion of its product, glycerol 3-phosphate cytidylyltransferase, leads to irregular morphology and lysis of Bacillus subtilis grown at physiological temperature

Abstract

Using a previously reported conditional expression system for use in Bacillus subtilis (A. P. Bhavsar, X. Zhao, and E. D. Brown, Appl. Environ. Microbiol. 67:403-410, 2001), we report the first precise deletion of a teichoic acid biosynthesis (tag) gene, tagD, in B. subtilis. This teichoic acid mutant showed a lethal phenotype when characterized at a physiological temperature and in a defined genetic background. This tagD mutant was subject to full phenotypic rescue upon expression of the complementing copy of tagD. Depletion of the tagD gene product (glycerol 3-phosphate cytidylyltransferase) via modulated expression of tagD from the amyE locus revealed structural defects centered on shape, septation, and division. Thickening of the wall and ultimately lysis followed these events.

FIG. 1

Xylose dependence of tagD deletion strain EB240. Strains EB124 and EB240 were plated on LB-CHL medium in the presence or absence of 2% xylose. Strains were grown overnight at 30°C.

FIG. 2

Characterization of a tagD deletion strain by microscopy. EB240 (TagD depleted) and EB124 (TagD wild type) were visualized by differential interference contrast microscopy (A) and transmission electron microscopy (B). EB240 and EB124 were also examined by scanning electron microscopy (C). Size bars are 500 nm.

FIG. 3

Growth curve and morphology of TagD depletion. (Top) EB240 was inoculated into LB-CHL-SPC medium with 2% xylose (B), 0.2% xylose (C), 0.06% xylose (D), 0.02% xylose (E), and no xylose (F). EB124 was also inoculated into LB-CHL medium with 2% xylose (A). Culture growth was monitored for 19 h, and samples were immediately prepared for transmission electron microscopy. The size bar (bottom right) is 500 nm.