A ComGA‐dependent checkpoint limits growth during the escape from competence

Abstract

In Bacillus subtilis, competence for transformation develops in 5-10% of the cells in a stationary phase culture. These cells exhibit a prolonged lag in the resumption of growth and cell division during the escape from competence. To better understand the basis of this lag, we have characterized competent cultures microscopically. To distinguish the minority of competent cells, a translational fusion between ComK, the competence transcription factor, and the green fluorescent protein (GFP) was used as a marker. Only 5-10% of the cells in a competent culture were fluorescent, indicating that ComK synthesis is an all or nothing event. To validate the identification of competent cells, we demonstrated the coincident expression of comEA, a late competence gene, and comK-gfp. Competent cells resemble stationary phase cells; the majority are single (not in chains), contain single nucleoids, and rarely contain FtsZ rings. Upon dilution into fresh medium, competent cells maintain this appearance for about 2 h. In contrast, the majority of non-competent cells rapidly resume growth, exhibiting chaining, nuclear division and FtsZ-ring formation. The late competence protein ComGA is required for the competence-related block in chromosome replication and cell division. In the competent cells of a comGA mutant culture, chromosomal replication and FtsZ-ring formation were no longer blocked, although competent comGA mutant cells were abnormal in appearance. It is likely that one role for ComGA is to prevent growth, chromosome replication and cell division until ComK can be eliminated by degradation. A mutation in the ATP-binding site of comGA inactivated the protein for transformation but did not prevent it from inhibiting DNA replication and cell division. The buoyant density difference between competent and non-competent cells depends on the competence-specific growth arrest.

Plasmid-Encoded ComI Inhibits Competence in the Ancestral 3610 Strain of Bacillus subtilis

“…One way in which pBS32 might inhibit competence is by the in- hibition of competence gene expression. In laboratory strains, competence genes are expressed in only a subpopulation (16,17,(38)(39)(40). To examine gene expression of a heterogeneous population, the ancestral, laboratory, and cured strains were fluorescently labeled such that the P comK promoter that directs expression of the ComK transcription factor was fused to YFP (lacA::P comK -YFP), while the ComK-dependent P comGA promoter that directs expression of ComGA, an ATPase required for DNA uptake, was fused to mCherry (thrC::P comGA -mCherry) (41).…”

Genetic characterization of the hdrRM operon: a novel high-cell-density-responsive regulator in Streptococcus mutans

“…Therefore, it is conceivable that there is a regulatory connection between the hdrM phenotypes associated with growth and natural competence. This result would not be surprising, given that competence escape in Bacillus subtilis is linked to cell growth (Hahn et al, 1995;Haijema et al, 2001). In addition, it has been demonstrated that natural competence in S. mutans is greatly enhanced within the biofilm (Li et al, 2001).…”

DivIVA-Mediated Polar Localization of ComN, a Posttranscriptional Regulator of Bacillus subtilis

“…6A). No septal band of GFP-ComN was found in the competent cells, however, probably because divisome assembly is blocked as a result of competence development (13). Superimposing the ComGA-CFP and GFP- Fluorescence micrographs of membrane-stained wild-type (PY79) and comN mutant (FG1076) bacteria.…”