ClpP ofBacillus subtilisis required for competence development, motility, degradative enzyme synthesis, growth at high temperature and sporulation

Abstract

SummaryThe 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 ⌬clpP mutant was constructed and shown to display a pleiotropic phenotype, including a deficiency in both sporulation initiation and competence for DNA uptake. The ⌬clpP 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 spoIIA and spoIIG operons, encoding the F and 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 ⌬clpP mutant. MecA has been characterized previously as a negative regulator of comK expression, whose overproduction inhibits both sporulation and competence development. Expression of a mecAЈ-ЈlacZ translational fusion is shown to be increased in the ⌬clpP 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 ⌬clpP mutant.

MecA, an adaptor protein necessary for ClpC chaperone activity

“…However, it was already observed in vivo and in vitro that MecA is also a ClpCP substrate (23,44). We observed in vitro that the presence of our model substrates also stabilize MecA (data not shown; Fig.…”

Signal Peptide Peptidase- and ClpP-like Proteins of Bacillus subtilis Required for Efficient Translocation and Processing of Secretory Proteins

“…First, TepA might be involved in the regulation of post-exponential growth phase-specific processes, like ClpP of B. subtilis, which was recently shown to have pleiotropic effects on protein secretion, the development of competence for DNA binding and uptake, and sporulation. Most likely, these effects of ClpP are due to the degradation of regulatory proteins in the cytosol (47). Notably, TepA seems to be specific for protein secretion, and unlike mutations in clpP, the tepA mutation did not result in a filamentous cell morphology and impaired growth at high temperature (48°C).…”

The ClpXP Protease Is Responsible for the Degradation of the Epsilon Antidote to the Zeta Toxin of the Streptococcal pSM19035 Plasmid

“…The B. subtilis mutant strains used in this study were separately transformed with the pBT233-1 and pBT233-1S plasmids that contain the erythromycin resistance that is conferred by derivatives of the pSM19035 plasmid. Because the B. subtilis ⌬clpP mutant QB4916 is deficient in DNA uptake (34), to obtain the proper strains, the plasmids containing YB886 pBT233-1 and YB886 pBT233-1S were transformed with chromosomal DNA from QB4916 (Table 1). All resultant strains were tested for plasmid stability as described under "Experimental Procedures."…”