DistinctclpPGenes Control Specific Adaptive Responses inBacillus thuringiensis

Abstract

ClpP and ClpC are subunits of the Clp ATP-dependent protease, which is ubiquitous among prokaryotic and eukaryotic organisms. The role of these proteins in stress tolerance, stationary-phase adaptive responses, and virulence in many bacterial species has been demonstrated. Based on the amino acid sequences of the Bacillus subtilis clpC and clpP genes, we identified one clpC gene and two clpP genes (designated clpP1 and clpP2) in Bacillus thuringiensis. Predicted proteins ClpP1 and ClpP2 have approximately 88 and 67% amino acid sequence identity with ClpP of B. subtilis, respectively. Inactivation of clpC in B. thuringiensis impaired sporulation efficiency. The clpP1 and clpP2 mutants were both slightly susceptible to salt stress, whereas disruption of clpP2 negatively affected sporulation and abolished motility. Virulence of the clp mutants was assessed by injecting bacteria into the hemocoel of Bombyx mori larvae. The clpP1 mutant displayed attenuated virulence, which appeared to be related to its inability to grow at low temperature (25°C), suggesting an essential role for ClpP1 in tolerance of low temperature. Microscopic examination of clpP1 mutant cells grown at 25°C showed altered bacterial division, with cells remaining attached after septum formation. Analysis of lacZ transcriptional fusions showed that clpP1 was expressed at 25 and 37°C during the entire growth cycle. In contrast, clpP2 was expressed at 37°C but not at 25°C, suggesting that ClpP2 cannot compensate for the absence of ClpP1 in the clpP1 mutant cells at low temperature. Our study demonstrates that ClpP1 and ClpP2 control distinct cellular regulatory pathways in B. thuringiensis.

PrcR, a PucR-type transcriptional activator, is essential for proline utilization and mediates proline-responsive expression of the proline utilization operon putBCP in Bacillus subtilis

“…Disruption of the chromosomal prcR gene by integration of plasmid pRN5101-derived pGS2047 through a Campbell-like single-crossover recombination was performed as described previously (Fedhila et al, 2002). The integration plasmid was introduced into B. subtilis cells by the protoplast method (Chang & Cohen, 1979).…”

“…To construct plasmid pGS2047, a DNA fragment carrying an internal region close to the N terminus of prcR and flanked by HindIII and BamHI sites was amplified by PCR and cloned between HindIII and BamHI sites of the thermosensitive plasmid pRN5101 (Fedhila et al, 2002). To construct plasmids pGS2090, pGS2097 and pGS2098, DNA fragments carrying various lengths of the regulatory region plus the N-terminal coding region of putB and flanked by EcoRI and BamHI sites were amplified by PCR and cloned individually between EcoRI and BamHI sites of the integrative promoter probe vector pDL (Yuan & Wong, 1995).…”

Transcriptional regulation of the l-lactate permease gene lutP by the LutR repressor of Bacillus subtilis RO-NN-1

“…Disruption of the chromosomal lutR or lutP gene by integration of plasmid pGS2471 or pGS2614 through a Campbell-like single-crossover recombination was performed as described previously (Fedhila et al, 2002). The integration plasmid was introduced into competent B. subtilis cells by the method described previously (Contente & Dubnau, 1979).…”

“…To construct plasmid pGS2471 for the disruption of lutR, a DNA fragment (+36 to +307 relative to the translational start site of lutR) was amplified by PCR with the primer pair A961 plus A968, and then cloned between HindIII and BamHI sites of the thermosensitive plasmid pRN5101 (Fedhila et al, 2002). To construct plasmid pGS2614 for the disruption of lutP, a DNA fragment (+46 to +352 relative to the translational start site of lutP) was amplified by PCR with the primer pair B098 plus B099, and then cloned between HindIII and BamHI sites of the thermosensitive plasmid pRN5101.…”

Expressions of alkaline phosphatase genes during phosphate starvation are under positive influences of multiple cell wall hydrolase genes in <i>Bacillus subtilis</i>

“…The phoA-bgaB fusion was also integrated at the amyE loci of the lytD mutant BNB12 and the lytF mutant BM1713 to generate the strain BM1691 and BM1719, respectively. Construction of the lytF mutant BM1713 by integration of the plasmid pGS2136 onto the chromosome through a single-crossover recombination was performed as described previously (Fedhila et al, 2002). The pRN5101-based plasmid pGS2136 was constructed by PCR and the primer pair A284 plus A285.…”