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)

Summary The Rok (YkuW) protein acts as a negative regulator of comK, which encodes the competence transcription factor of Bacillus subtilis. In the absence of Rok, ComK is overproduced, and when excess Rok is present comK transcription is inhibited. Rok acts transcriptionally to repress comK expression but does not affect ComK stability, which is controlled by the MecA switch. Gel‐shift assays show that Rok binds directly to a DNA fragment that contains the comK promoter. SinR and AbrB act negatively on rok transcription, and the inactivation of rok bypasses the positive requirements for sinR and abrB for the expression of comK. Therefore, the dependence of comK expression on SinR and AbrB may be a result of their repression of rok transcription. It has also been shown in vivo that Rok and ComK can indivi‐dually repress rok transcription, and that Rok and ComK bind to the rok promoter in vitro. These interactions establish feedback loops, and the roles of these circuits are discussed.

Rok is a repressor of the transcriptional activator ComK and is therefore an important regulator of competence in Bacillus subtilis (T. T. Hoa, P. Tortosa, M. Albano, and D. Dubnau, Mol. Microbiol. 43:15-26, 2002). To address the wider role of Rok in the physiology of B. subtilis, we have used a combination of transcriptional profiling, gel shift experiments, and the analysis of lacZ fusions. We demonstrate that Rok is a repressor of a family of genes that specify membrane-localized and secreted proteins, including a number of genes that encode products with antibiotic activity. We present evidence for the recent introduction of rok into the B. subtilis-Bacillus licheniformis-Bacilllus amyloliquefaciens group by horizontal transmission.ComK is the major transcription factor driving the development of competence for transformation in Bacillus subtilis (37). rok encodes a direct repressor of comK transcription (18) and thereby plays an important, but incompletely understood, role in the complex regulation that takes place at the promoter of comK. Five proteins are known to bind to PcomK (reviewed in reference 9): ComK itself, DegU, Rok, AbrB, and CodY, the last three of which exert negative effects.The use of a rok-lacZ fusion construct revealed no major change in rok transcription during growth in the population as a whole (18). Given this, it is surprising that the regulation of rok transcription is complex; rok is repressed by Rok itself, by ComK, and by the transition state regulators SinR and AbrB, although only ComK and Rok have been shown to bind directly to Prok (18). As cultures approach the end of exponential growth, the concentrations of active AbrB and SinR decrease (reviewed in references 31 and 32), leading to the expectation that rok transcription would increase. However, since ComK acts negatively on Prok, the increased synthesis of ComK in competent cells after the cessation of exponential growth would tend to place a limit on increased rok transcription. In addition, negative autoregulation at Prok would be expected to maintain a constant time-averaged level of rok expression. Transient changes in the concentration of Rok may play a role in the timing of competence expression, in the selection of which cells will develop competence, and in limiting the final level of comK expression in the competent subpopulation. Although transient changes have not been detected, it may be that our experiments lacked sufficient time resolution to detect these fluctuations. A major unanswered question concerns the possibility that the activity of Rok is somehow regulated, perhaps responding to the presence of an unknown corepressor. To extend our understanding of the role of Rok, we identified additional genes regulated by this protein and asked whether Rok was capable of acting positively or only as a repressor. By transcriptional profiling and the use of lacZ fusions, we have identified at least seven gene clusters that are negatively regulated by rok, including several involved in the production of bacteriocin...

A series of Tn9l 71ac insertions define the comnG region of the Bacillus subtUis chromosome. comG mutants are deficient in competence and specifically in the binding of exogenous DNA. The genes included in the comG region are first expressed during the transition from the exponential to the stationary growth phase. From nucleotide sequence information, it was concluded that the comG locus contains seven open reading frames (ORFs), several of which overlap at their termini. High-resolution Si nuclease mapping and primer extension were used to identify the 5' terminus of the comG mRNA. The sequence upstream from the comG start site closely resembled the consensus recognition sequence for the major B. subtilis vegetative RNA polymerase holoenzyme. Complementation analysis confirmed that the comG ORF1 protein is required for the ability of competent cultures to resolve into two populations with different cell densities on Renografin (E. R. Squibb & Sons, Princeton, N.J.) gradients, as well as for full expression of comE, another late competence locus. The predicted comG ORF1 protein showed significant similarity to the virB ORF11 protein from Agrobacterium tumefaciens, which is probably involved in T-DNA transfer. The N-terminal sequences of comG ORF3 and, to a lesser extent, the comG ORF4 and ORF5 proteins were similar to a class of pilin proteins from members of the genera Bacteroides, Pseudomonas, Neisseria, and MoraxeeUa. All of the comG proteins except comG ORF1 possessed hydrophobic domains that were potentially capable of spanning the bacterial membrane. It is likely that these proteins are membrane associated, and they may comprise part of the DNA transport machinery. When present in multiple copies, a DNA fragment carrying the comG promoter was capable of inhibiting the development of competence as well as the expression of several late com genes, suggesting a role for a transcriptional activator in the expression of those genes.

We isolated 28 mutants of Bacillus subils deficient in the development of competence by using the transposon Tn917lacZ as a mutagen. The mutant strains were poorly transformable with pld and chromosoinal DNAs but were normally transducible and exhbited wild-tYpe reslstan to DNAagents. The mutations were genetically mapped, and the mutants were chaacterized with respect to their abilities to bind and take up radiolabeled DNA. All (8,14,15,19), and several protein products that appear to be competence specific have been detected (7,9,20). At least some of these products appear to be synthesized at an enhanced rate during development of competence. It therefore seems reasonable to regard competence as a complex developmental system apparently triggered in response to environmental signals. In addition, since spoOA mutants have been reported to be competence deficient (21), it is likely that the competence regulatory system overlaps that which controls the onset of sporulation and the synthesis of several late-growthassociated products. However, very little is known concerning the regulation of competence and the mode of action of competence-specific gene products.To initiate an investigation of the regulation of competence development and to identify and characterize competence-specific gene products, we used the MATERIALS AND METHODSStrains and plasmids. The B. subtilis strains used were listed in Table 1. pBD347 is a 2.3-kilobase multicopy plasmid that confers resistance to chloramphenicol and was derived from pIM13 (13).Media. Solid media were tryptose blood agar base (TBAB; Difco Laboratories) and minimal medium (1) with appropriate growth supplements. Liquid media were VY (25 g of veal infusion [Difco], 5 g of yeast extract [Difco], 1,000 ml of water) and comipetence medium (5) for growth of phage and recipient cultures in transduction experiments or growth to competence. Mitomycin C was added to TBAB to 0.05 ,ug/ml, methyl methanesulfonate was added to 0.4 pl/ml, and acriflavine was added to 25 ,ug/ml. Erythromycin was used for selections at 5 ,ug/ml and for scoring the presence of Tn917 at 50 pLg/ml. In some expernments the presence of Tn917 was verified by using 5 ,ug of erythromycin per ml plus 25 ,ug of lincomycin per ml.Transformation and transuction. Growth of cultures to competence, their storage, preparation of phage PBS1 and AR9 transducing lysates and transduction, isolation of transforming DNA (both radioactive and nonradioactive), and transformation were as previously described (5,

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.

Summary The adapter protein MecA targets the transcription factor ComK for degradation by the ClpC/ClpP proteolytic complex, thereby negatively regulating competence in Bacillus subtilis. Here we show that MecA also decreases the frequency of transitions to the sporulation pathway as well as the expression of eps, which encodes synthesis of the biofilm matrix exopolysaccharide. We present genetic and biophysical evidence that MecA down-regulates eps expression and spore formation by directly interacting with Spo0A. MecA does not target Spo0A for degradation, and apparently does not prevent the phosphorylation of Spo0A. We propose that it inhibits the transcriptional activity of Spo0A~P by direct binding. Thus, in its interaction with Spo0A, MecA differs from its role in the regulation of competence where it targets ComK for degradation. MecA acts as a general buffering protein for development, acting by two distinct mechanisms to regulate inappropriate transitions to energy-intensive pathways.