Entry into stationary phase in Bacillus subtilis is linked not only to a redirection of the gene expression program but also to posttranslational events such as protein degradation. Using 35 S-labeled methionine pulse-chase labeling and two-dimensional polyacrylamide gel electrophoresis we monitored the intracellular proteolysis pattern during glucose starvation. Approximately 200 protein spots diminished in the wild-type cells during an 8-h time course. The degradation rate of at least 80 proteins was significantly reduced in clpP, clpC, and clpX mutant strains. Enzymes of amino acid and nucleotide metabolism were overrepresented among these Clp substrate candidates. Notably, several first-committed-step enzymes for biosynthesis of aromatic and branched-chain amino acids, cell wall precursors, purines, and pyrimidines appeared as putative Clp substrates. Radioimmunoprecipitation demonstrated GlmS, IlvB, PurF, and PyrB to be novel ClpCP targets. Our data imply that Clp proteases down-regulate central metabolic pathways upon entry into a nongrowing state and thus contribute to the adaptation to nutrient starvation. Proteins that are obviously nonfunctional, unprotected, or even "unemployed" seem to be recognized and proteolyzed by Clp proteases when the resources for growth become limited.

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Clp-controlled proteolysis in Bacillus subtilis seems to play a substantial role, particularly under stress conditions. Calibrated Western blot analyses were used to estimate the approximate numbers of heat-inducible Clp molecules within a single cell. According to these numbers, the different Clp ATPases do not seem to compete for the proteolytic subunit ClpP. Coimmunoprecipitation experiments revealed the predicted specific ClpX-ClpP, ClpC-ClpP, and ClpE-ClpP interactions. ClpE and ClpX are rapidly degraded in wild-type cells during permanent heat stress but remained almost stable in a clpP mutant, suggesting ClpP-dependent degradation. In particular, ClpCP appeared to be involved in the degradation of the short-lived ClpE ATPase, indicating a negative "autoregulatory" circuit for this particular Clp ATPase at the posttranslational level. Analysis of the half-life of stress-inducible clp mRNAs during exponential growth and heat shock revealed precise regulation of the synthesis of each Clp protein at the posttranscriptional level as well to meet the needs of B. subtilis.

SummaryThe carboxyvinyl transfer from phosphoenolpyruvate to UDP-N -acetylglucosamine is the first committed step in the pathway of peptidoglycan formation. This crucial reaction for bacterial cell growth is catalysed by the MurA enzymes. Gram-negative bacteria carry one murA gene, whereas in a subgroup of Gram-positive bacteria two separate paralogues, MurAA and MurAB, exist. This study provides evidence that in the Gram-positive bacterium Bacillus subtilis , the MurAA protein is specifically degraded by the ClpCP protease. This Clp-dependent degradation is especially enhanced upon entry into stationary phase, thus ensuring an immediate growth arrest due to stalled murein biosynthesis. The MurAA protein can therefore be addressed as a target of Clp-dependent regulatory proteolysis such as the transcriptional regulators CtsR, ComK, Spx in B. subtilis , CtrA in Caulobacter crescentus or RpoS in Escherichia coli . Taking into account all other known regulatory targets of ATP-dependent proteases, MurAA of B. subtilis represents the first example of a metabolic enzyme which is a unique regulatory substrate of Clpdependent proteolysis. Its function as a regulatory metabolic checkpoint resembles that of homoserine trans -succinylase (MetA) in E. coli which is similarly ATP-dependently degraded.

SummaryThe availability of zinc was shown to have a marked influence on the biosynthesis of actinorhodin in Streptomyces coelicolor A3(2). Production of actinorhodin and undecylprodigiosin was abolished when a novel pleiotropic regulatory gene, absC, was deleted, but only when zinc concentrations were low. AbsC was shown to control expression of the gene cluster encoding production of coelibactin, an uncharacterized non-ribosomally synthesized peptide with predicted siderophore-like activity, and the observed defect in antibiotic production was found to result from elevated expression of this gene cluster. Promoter regions in the coelibactin cluster contain predicted binding motifs for the zinc-responsive regulator Zur, and dual regulation of coelibactin expression by zur and absC was demonstrated using strains engineered to contain deletions in either or both of these genes. An AbsC binding site was identified in a divergent promoter region within the coelibactin biosynthetic gene cluster, adjacent to a putative Zur binding site. Repression of the coelibactin gene cluster by both AbsC and Zur appears to be required to maintain appropriate intracellular levels of zinc in S. coelicolor.