Bacillus subtilis diacylglycerol kinase (DgkA) enhances efficient sporulation

Abstract

The sn-1,2-diacylglycerol kinase homologue gene, dgkA, is a sporulation gene indispensable for the maintenance of spore stability and viability in Bacillus subtilis. After 6 h of growth in resuspension medium, the endospore morphology of the dgkA mutant by standard phase-contrast microscopy was normal; however, after 9 h, the endospores appeared mostly dark by phase-contrast microscopy, suggesting a defect in the spores. Moreover, electron microscopic studies revealed an abnormal cortex structure in mutant endospores 6 h after the onset of sporulation, an indication of cortex degeneration. In addition, a significant decrease in the dipicolinic acid content of mutant spores was observed. We also found that dgkA is expressed mainly during the vegetative phase. It seems likely that either the DgkA produced during growth prepares the cell for an essential step in sporulation or the enzyme persists into sporulation and performs an essential function.

FIG. 1.

Expression pattern of dgkA-lacZ. The activity of β-galactosidase was determined in Difco sporulation medium with 5 mM IPTG. Time zero is the point of initiation of the stationary phase. β-Galactosidase activity was determined as previously described by the method of Miller (14) using o-nitrophenyl-β-D-galactopyranoside as the substrate. The specific activity of the enzyme was expressed in nanomoles of substrate (o-nitrophenyl-β-d-galactopyranoside) hydrolyzed per milligram per minute. Symbols: open square and open circle, wild-type strain 168 supplemented with and without 5 mM IPTG, respectively; solid square and solid circle, strain DGKAd (dgkA-lacZ) with and without 5 mM IPTG, respectively.

FIG. 2.

Phase-contrast microscopy of dgkA mutant cells are shown. Typical phase-contrast micrographs for the cells of wild-type B. subtilis 168 and mutant DGKAd at T₆ (A and C) and T₉ (B and D), respectively, are shown. Cells were grown and allowed to sporulate at 37°C in Difco sporulation medium.

FIG. 3.

Electron micrographs of endospore-forming B. subtilis cells at T₆. (A) Wild-type strain 168 with normal cortex; (B) dgkA mutant exhibiting defective cortex structure. Abbreviations: OC, outer coat; IC, inner coat; Ct, cortex.

FIG. 4.

DPA accumulation in the dgkA mutant. The time (in hours) after initiation of sporulation is shown on the x axis. Cells were harvested by centrifuging (10,000 × g 2 min) 1.5 ml of culture prior to assay. DPA was assayed by resuspending the sampled pellet in 1 ml of sterile distilled water, boiling it for 20 min, and then cooling it for 15 min on ice after which it was centrifuged at 6,800 × g for 2 min. A portion of the supernatant (600 μl) was reacted with 200 μl of solution containing 25 mg of l-cysteine, 0.31 g of FeSO₄ · 7H₂O, 80 mg of (NH₄)₂SO₄, and 25 ml of 50 mM sodium acetate (pH 4.6, adjusted with acetic acid). The DPA content was determined as a measure of the optical density at 440 nm (OD₄₄₀). Symbols: diamond, wild-type strain 168; square, dgkA mutant.