Role of SpoVG in asymmetric septation in Bacillus subtilis

Abstract

Deletion of the citC gene, coding for isocitrate dehydrogenase, arrests sporulation of Bacillus subtilis at stage I after bipolar localization of the cell division protein FtsZ but before formation of the asymmetric septum. A spontaneous extragenic suppressor mutation that overcame the stage I block was found to map within the spoVG gene. The suppressing mutation and other spoVG loss-of-function mutations enabled citC mutant cells to form asymmetric septa and to activate the forespore-specific sigma factor sigmaF. However, little induction of mother cell-specific, sigmaE-dependent sporulation genes was observed in a citC spoVG double mutant, indicating that there is an additional defect(s) in compartmentalized gene expression in the citC mutant. These other defects could be partially overcome by reducing the synthesis of citrate, by buffering the medium, or by adding excess MnCl2. Overexpression of the spoVG gene in wild-type cells significantly delayed sigmaF activation. Increased expression and stability of SpoVG in citC mutant cells may contribute to the citC mutant phenotype. Inactivation of the spoVG gene caused a population of otherwise wild-type cells to produce a small number of minicells during growth and caused sporulating cells to complete asymmetric septation more rapidly than normal. Unlike the case for inactivation of the cell division inhibitor gene minD, many of these minicells contained DNA and appeared only when the primary sporulation signal transduction pathway, the Spo0A phosphorelay, was active. These results suggest that SpoVG interferes with or is a negative regulator of the pathway leading to asymmetric septation.

FIG. 1

Expression of spoIIQ′-lacZ (a) and spoIID′-lacZ (b) fusions in citC spoVG double mutants. Cells were grown in DS broth and harvested at the indicated times for measurement of β-galactosidase activity. Symbols: ▵, SJB294 (a) and SJB225 (b) (citC⁺); □, SJB295 (a) and SJB229 (b) (ΔcitC); , KMB97 (ΔcitC spoVG2); ○, KMB198 (a) and KMB208 (b) (ΔcitC ΔspoVG); ◊, KMB200 (a) and KMB209 (b) (ΔspoVG). Time zero is defined as the onset of stationary phase.

FIG. 2

spoVG2 is a recessive mutation. Cells carrying a spoIIQ′-lacZ fusion were grown in DS broth and harvested at the indicated times for measurement of β-galactosidase activity. Symbols: ▵, SJB294 (citC⁺); □, SJB295 (ΔcitC); ○, KMB97 (ΔcitC spoVG2); ●, KMB158 (ΔcitC spoVG2 ΔthrC::spoVG⁺). Time zero is defined as the onset of stationary phase.

FIG. 3

Expression of a spoVG′-lacZ fusion in a ΔcitC mutant. (a) Wild-type (SJB78; ▵) and ΔcitC mutant (KMB162; □) strains were grown in DS broth and harvested at the indicated times for measurement of β-galactosidase activity. (b) Samples of wild-type (SJB294) and ΔcitC mutant (SJB295) cultures were harvested at the 1-h intervals after the onset of stationary phase indicated over the gels and disrupted by sonication. Proteins (10 μg) in the crude extract were separated in sodium dodecyl sulfate-polyacrylamide gels and subjected to immunoblot analysis with anti-SpoVG antibodies.

FIG. 4

Expression of spoIIQ′-lacZ in strains harboring the spoVG gene and the spoVG2 allele on a multicopy plasmid. Cells having pHP13 (vector only; ▵), pKM98 (spoVG⁺; ○), or pKM157 (spoVG2; □) were grown in DS broth and harvested at the indicated times for measurement of β-galactosidase activity. Time zero is defined as the onset of stationary phase.

FIG. 5

Thin-section electron microscopic analysis. A sample of a culture of ΔcitC ΔspoVG mutant cells (KMB197) was harvested at T₆ and prepared for transmission electron microscopy as described in Materials and Methods.

FIG. 6

Minicell formation in a ΔspoVG mutant. Cells of strain KMB200 (ΔspoVG) were grown to mid-exponential phase and induced to sporulate by the resuspension method of Sterlini and Mandelstam (65). Ethanol-fixed cells were observed under phase-contrast microscopy (left-hand panels), and nucleoids were visualized by DAPI staining (right-hand panels). (a to c) Exponential-phase cells, showing an anucleate minicell (a), a minicell containing a condensed nucleoid (b), and a disporic cell (c). (d) Cell population of the mutant at 1 h after resuspension. Arrows 1 to 4 indicate anucleate minicells, arrow 5 indicates a sporulating cell at stage 0-I, arrow 6 indicates a sporulating cell at stage II, and arrows 7 and 8 indicate disporic cells.