ClpP of Bacillus subtilis is required for competence development, motility, degradative enzyme synthesis, growth at high temperature and sporulation

Abstract

The nucleotide sequence of the Bacillus subtilis clpP gene was determined. The predicted protein shows very high similarity to members of the ClpP family of proteolytic subunits (68% amino acid sequence identity with that of Escherichia coli). We show that ClpP plays an essential role in stationary phase adaptive responses. Indeed, a ΔclpP mutant was constructed and shown to display a pleiotropic phenotype, including a deficiency in both sporulation initiation and competence for DNA uptake. The ΔclpP mutant has a highly filamentous morphology and appears to be non-motile, as judged by swarm plate assays. Expression of clpP is strongly induced under heat shock conditions, and ClpP is shown to be essential for growth of B. subtilis at high temperature. The role of ClpP in the sporulation and competence regulatory pathways was investigated. ClpP is required for expression of the spollA and spollG operons, encoding the σ(F) and σ(E) sporulation-specific sigma factors. ClpP is also necessary for the expression of the comK gene, encoding a positive transcriptional regulator of competence genes. ComK- dependent transcription of sacB, encoding the exocellular degradative enzyme levansucrase, was found to be abolished in the ΔclpP mutant. MecA has been characterized previously as a negative regulator of comK expression, whose overproduction inhibits both sporulation and competence development. Expression of a mecA'-lacZ translational fusion is shown to be increased in the ΔclpP mutant. We suggest that ClpP is involved in controlling MecA levels in the cell through proteolysis. Increased levels of MecA in the absence of ClpP are at least partly responsible for the observed pleiotropic phenotype of the ΔclpP mutant.