Macromolecular Synthesis in Bacillus subtilis During Development of the Competent State

116

161

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.

Section: Discussionsupporting

confidence: 88%

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

Haijema¹,

Hahn²,

Haynes³

et al. 2001

116

161

“…The most suggestive comparison may be with a recent report that when the initiation of chromosome replication is specifically blocked in E. coli, cells become elongated, with centrally located nucleoids (Bernander et aL, 1995). mecA inactivation may be responsible for the DNA replication arrest of competent cells It has been shown that competent cells of B. subtilis are essentially inactive with respect to DNA replication (Dooley et aL, 1971;Dubnau and Cirigliano, 1973), and that chromosome replication in these cells is arrested at or near the replication terminus (McCarthy and Nester, 1967). Reversal of this block occurs with a delay, resulting in a prolonged lag before competent cells resume growth (McCarthy and Nester, 1967;Nester and Stocker, 1963).…”

Section: Inappropriate Comk Synthesis Causes An Abnormality In Nucleosupporting

confidence: 57%

“…These cells are physiologically distinct; they are more buoyant than the non-competent cells and can be separated from the latter on density gradients (Hadden and Nester, 1968;Haseltine-Cahn and Fox, 1968). Additionally, they are blocked in DNA replication (Dooley et aL, 1971;Dubnau and Cirigliano, 1973), and when resuspended in fresh medium, exhibit a prolonged lag period before the resumption of growth (Nester and Stocker, 1963). As in the case of other globally regulated states, especially those that can arrest growth, such as sporulation, SOS and heat shock, a mechanism must exist to return the system to the uninduced state (Neihardt, 1987).…”

Section: Introductionmentioning

confidence: 99%

Inactivation of mecA prevents recovery from the competent state and interferes with cell division and the partitioning of nucleoids in Bacillus subtilis

Hahn¹,

Bylund

Haines

et al. 1995

The development of genetic competence in Bacillus subtilis requires the synthesis of ComK, a transcription factor, which is normally produced as a culture enters the stationary phase. This synthesis is known to be regulated in part by the protein MecA. Loss-of-function mutations in mecA result in overexpression of ComK and its appearance early during exponential growth. We show here that mecA inactivation also causes a loss of colony-forming ability, especially during stationary phase. This loss is accompanied by the appearance of cells in which normal nucleoid separation has failed to occur. Renografin gradient fractionation of mecA cultures grown to competence reveals that nearly 100% of the cells band at the low buoyant density characteristic of competent cells, and that this low density is competence-related. The loss of viability, the low buoyant density and the nucleoid separation defect, are all comK-dependent. The loss of viability can be reversed by even the transient introduction of mecA+. It is proposed that these effects of ComK overexpression are related to the DNA replication arrest normally exhibited by the competent cell fraction and that MecA is needed to reverse this arrest and to permit escape from the competent state. The shift of nearly 100% of the cells to light buoyant density in a mecA mutant culture strongly suggests that the MecA protein is a regulator of the cell-type-specific expression of competence.

“…Competence and sporulation may be viewed as the penultimate and ultimate responses to stress. They must be tightly regulated, probably because competent and sporulating cells are non-dividing (Dooley et al, 1971;B. J. Haijema, J. Hahn, D. Dubnau, unpublished observations).…”

Section: Introductionmentioning

confidence: 99%

Characterization of ylbF, a new gene involved in competence development and sporulation in Bacillus subtilis

Tortosa¹,

Albano²,

Dubnau³

2000

SummaryWe used mini Tn10 transposition to generate a library of Bacillus subtilis insertion mutants, with the goal of identifying and characterizing new competence genes. Two new regulatory genes were identified in our screen: ypuN (also known as rsiX, the anti-s X factor) and ylbF. The disruption of ylbF leads to a dramatic decrease in the expression of comK, encoding the competence transcription factor. Our data show that ylbF positively controls ComK at a post-transcriptional level. It has been reported previously that ComK is degraded in vivo and in vitro by a multimeric protein complex composed of ClpP, ClpC and MecA. This proteolysis is inhibited by the ComS peptide. We show that both the overexpression of comS and the inactivation of mecA individually suffice to bypass the competence phenotype of the ylbF mutation. This mutation does not seem to alter the cellular concentrations of MecA or ClpP, and we propose a role for YlbF in modulating the translation, stability or activity of ComS. In addition to its role in competence, ylbF also appears to regulate sporulation by acting before stage II.