ComK-induced cell death is reversed by upregulating the SigB or Spx pathway in Bacillus subtilis

Natural competence of bacteria is an important tool for researchers to rapidly engineer genetic mutations. In Bacillus subtilis, the master regulator for competence is ComK, which is typically only expressed during stationary phase, allowing cells to uptake DNA and incorporate it into the genome. Here, we engineered cells to overexpress comK in exponential phase using inducible promoters, thereby inducing competence without going through stationary phase. We observed that overexpressing comK in exponentially growing cells inhibited DNA replication and cell division, leading to low DNA content, elongated cells, and eventually cell death. To rescue comK-induced cell death, we isolated suppressors of comK overexpression using both spontaneous suppression and a transposon-based method (transposon sequencing). From both methods, the suppressors upregulated either the sigma B (SigB) or Spx pathway, which are known to be activated in response to stress. Importantly, while upregulating SigB or Spx suppressed cell death, it also reduced competence by lowering ComK protein levels. Altogether, our results collectively show that during competence, normal cellular functions such as DNA replication and cell division are inhibited. While cells can upregulate stress responses to facilitate these normal cellular functions, these processes inadvertently lower ComK levels and inhibit competence. Our study highlights the sophisticated cellular regulatory systems that specifically direct cell growth, competence, or stress management.IMPORTANCENaturally competent bacteria, such as Bacillus subtilis, take up DNA to use it as nutrients and generate new genotypes. Researchers exploit natural competence to manipulate bacterial genomes. While all B. subtilis cells in a population are capable of being competent, only a small portion of cells do so. To synchronously induce competence in a cell population, we overexpressed the master competence regulator comK. We found that competence is associated with inhibited cellular functions, such as cell division and DNA replication. Suppressor mutant screens revealed that increasing expression of either stress regulator sigma B or Spx restores cell division and DNA replication functions, but in turn inhibits competence. Our study emphasizes the complex regulatory networks that balance cell growth, competence, and stress responses.