The GTPase CpgA is implicated in the deposition of the peptidoglycan sacculus in Bacillus subtilis

Abstract

Depletion of the Bacillus subtilis GTPase CpgA produces abnormal cell shapes, nonuniform deposition of cell wall, and five- to sixfold accumulation of peptidoglycan precursors. Nevertheless, the inherent structure of the cell wall appeared mostly unchanged. The results are consistent with CpgA being involved in coordinating normal peptidoglycan deposition.

FIG. 1.

(A) Effect of depletion of CpgA on growth. Cultures were grown at 37°C for strain 168 trpC (•), its derivative BFS2823 without IPTG (□) or BFS2823 with 1 mM IPTG (✚) in E-medium or Spizizen medium. BFS2823 expresses cpgA from a pspac promoter. (B) Morphology defect of CpgA-depleted cells. Phase-contrast micrographs of strain 168 (wild type) and CpgA-depleted cells (BFS2823) grown either in E-medium or Spizizen medium, sampled at and optical density (OD) of 0.4, are shown.

FIG. 2.

EM sections of CpgA-depleted cells throughout the growth phase. Strain 168 (A) and strain BFS2823 (B to D), both grown in E-medium without IPTG, are shown. EM sections (samples were fixed with 2.5% glutaraldehyde in cacodylate buffer and stained with 1% uranyl acetate, and ultrathin sections were contrasted with lead acetate) were prepared as described previously (26) and are shown at different stages of the growth phase. (A) Mid-exponential phase, OD = 0.4; (B) OD = 0.4; (C) OD = 0.8; (D) stationary phase. In contrast to strain 168, a variety of misshapen cells with some abnormal division planes are visible throughout the growth phase with depleted cells. BFS2823 with IPTG or a derivative of BFS2823, ectopically expressing cpgA from a xyl promoter, produces largely normal cells (data not shown).

FIG. 3.

EM sections of CpgA-depleted cells reveal nonuniform deposition of cell wall. (A) Wild-type strain 168; (B) BFS2823 grown without IPTG in E-medium at 37°C, with nonuniform deposition of cell wall (arrows) and multiple aberrant division planes in a minority of cells.

FIG. 4.

HPLC analysis of the muropeptides in peptidoglycan from depleted and nondepleted strains. (A) Strain 168; (B) BFS2823 without IPTG; (C) BFS2823 with IPTG. Purified peptidoglycan (2) was digested with cellosyl N-acetylmuramidase, and the resulting muropeptides were reduced as described previously (3). The muropeptide mixtures were applied to a 5-μm Vydac C₁₈ reversed-phase column and eluted as described previously (3) a, main monomer; b, main dimer.