Summary In Bacillus subtilis, the competence transcription factor ComK activates its own transcription as well as the transcription of genes that encode DNA transport proteins. ComK is expressed in about 10% of the cells in a culture grown to competence. Using DNA microarrays representing ≈ 95% of the protein‐coding open reading frames in B. subtilis, we compared the expression profiles of wild‐type and comK strains, as well as of a mecA mutant (which produces active ComK in all the cells of the population) and a comK mecA double mutant. In these comparisons, we identified at least 165 genes that are upregulated by ComK and relatively few that are downregulated. The use of reporter fusions has confirmed these results for several genes. Many of the ComK‐regulated genes are organized in clusters or operons, and 23 of these clusters are preceded by apparent ComK‐box promoter motifs. In addition to those required for DNA uptake, other genes that are upregulated in the presence of ComK are probably involved in DNA repair and in the uptake and utilization of nutritional sources. From this and previous work, we conclude that the ComK regulon defines a growth‐arrested state, distinct from sporulation, of which competence for genetic transformation is but one notable feature. We suggest that this is a unique adaptation to stress and that it be termed the ‘K‐state’.

A set of competence (com) mutants of Bacillus subtiL was constructed by using Tn917lacZ as a mutagen. In about half of the mutants, the promoterless lacZ element on the transposon was placed under cOntrol of putative comr promoters. Expression of the mutant crom genes was studied by using the ,Bgalactosidase tag. Two of the mutant genes (those represented by com-124 and com-138) were expressed early in the growth cycte in all of the media tested and were not dependent on the spoOA or spoOH product for expression. The remaing mutants, which represented a minimum of four additional genes, expresed "-galactosidase in stationary phae during the period in which competence developed. We conclude that expression of com genes is probably regulated trnscriptionally and in a growth stage-speific manner. Expression of these genes was aLo depenideht on growth in competence medium and, like 6ompetence development, required the presence of glucose and was dependent on the spoOH products. The dependence on the spoOA gene product was partialy byjsed by the abrB703 mutation. These effects were qualitatively equivalent to those on competence devilopment. The latter was dependent on spoOA and spo0H, and the spoOA dependency was partially suppressed by abri703. Several of the mutants were still capable of resolution into light and heavy buoyant denity cell ftions when grown in competence mium. All of these expressd -galac to a greater extent In the Ulight showing that expression of these com genes was cell type specific. Development of competeiice was not mardly affected by ntutations in spoOB, spoOE, spoOF, spoOJ, or sigB, the structural gene for cr. galactosidase specific activity was higher in the Renografin light buoyant density fraction than in the heavy fraction. Development of genetic competence in MATERIALS AND METHODSStrains and strain construction. The strains used are listed in Table 1. The com mutations were moved into different genetic backgrounds by selection for resistance to erythromycin (5 pg/ml) or erythromycin (5 FLg/ml) plus lincomycin (25 ,g/mil). spo0 markers were moved by congression. spoOA mutants were routinely checkbd for protease and antibiotic production to confirm the presence or absence of abr partial suppressor mutations. The sigB mutation (2)

The Gram-positive, rod-forming bacterium Bacillus subtilis efficiently binds and internalizes transforming DNA. The localization of several competence proteins, required for DNA uptake, has been studied using fluorescence microscopy. At least three proteins (ComGA, ComFA, and YwpH) are preferentially associated with the cell poles and appear to colocalize. This association is dynamic; the proteins accumulate at the poles as transformability develops and then delocalize as transformability wanes. DNA binding and uptake also occur preferentially at the cell poles, as shown using fluorescent DNA and in single-molecule experiments with laser tweezers. In addition to the prominent polar sites, the competence proteins also localize as foci in association with the lateral cell membrane, but this distribution does not exhibit the same temporal changes as the polar accumulation. The results suggest the regulated assembly and disassembly of a DNA-uptake machine at the cell poles.

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.

comR (pnpA) is a newly identified gene in Bacillus subtilis that is necessary for the expression of late competence genes. Transformability of a comR (pnpA) mutant is 1-5% of that seen in comR+ strains. Cloning and sequencing identified ComR as polynucleotide phosphorylase (PNPase). The PNPase amino acid sequence has 50% identity and 67% similarity with the Escherichia coli enzyme. Enzymatic assays show that this is the only PNPase activity in B. subtilis. comR (pnpA) is necessary for comG-lacZ and comK-lacZ expression, but this requirement is bypassed by a mecA disruption. In B. subtilis, the loss of PNPase has little effect on expression from a fusion of the srfA promoter directly to lacZ, but is necessary for normal expression from certain srfA-lacZ fusions that include portions of the normal srfA transcript. When a srfA-lacZ translational fusion is tested in isogenic pnpA+ and pnpA derivatives of E. coli, lower expression is seen in the pnpA mutant. Since expression from lacZ fusions to comA, sinR, and mecA appeared similar in the B. subtilis pnpA and pnpA+ strains, the loss of PNPase does not have a strong general effect on gene expression. These results suggest that PNPase may be necessary for modification of the srfA transcript in order to activate translation or stabilize the transcript, and that this may be necessary for competence development. This is the first evidence of post-transcriptional effects on the development of competence in B. subtilis.