Dual role of CcpC protein in regulation of aconitase gene expression in Listeria monocytogenes and Bacillus subtilis.

The role of the CcpC regulatory protein as a repressor of the genes encoding the tricarboxylic acid branch enzymes of the Krebs cycle (citrate synthase, citZ; aconitase, citB; and isocitrate dehydrogenase, citC) has been established for both Bacillus subtilis and Listeria monocytogenes. In addition, hyperexpression of citB-lacZ reporter constructs in an aconitase null mutant strain has been reported for B. subtilis. We show here that such hyperexpression of citB occurs in L. monocytogenes as well as in B. subtilis and that in both species the hyperexpression is unexpectedly dependent on CcpC. We propose a revision of the existing CcpC-citB regulatory scheme and suggest a mechanism of regulation in which CcpC represses citB expression at low citrate levels and activates citB expression when citrate levels are high.

CcpC-dependent regulation of citrate synthase gene expression in Listeria monocytogenes.

Citrate synthase, the first and rate-limiting enzyme of the tricarboxylic acid branch of the Krebs cycle, was shown to be required for de novo synthesis of glutamate and glutamine in Listeria monocytogenes. The citrate synthase (citZ) gene was found to be part of a complex operon with the upstream genes lmo1569 and lmo1568. The downstream isocitrate dehydrogenase (citC) gene appears to be part of the same operon as well. Two promoters were shown to drive citZ expression, a distal promoter located upstream of lmo1569 and a proximal promoter located upstream of the lmo1568 gene. Transcription of citZ from both promoters was regulated by CcpC by interaction with a single site; assays of transcription in vivo and assays of CcpC binding in vitro revealed that CcpC interacts with and represses the proximal promoter that drives expression of the lmo1568, citZ, and citC genes and, by binding to the same site, prevents read-through transcription from the distal, lmo1569 promoter. Expression of the lmo1568 operon was not affected by the carbon source but was repressed during growth in complex medium by addition of glutamine.

CcpC-dependent regulation of citB and lmo0847 in Listeria monocytogenes.

In Bacillus subtilis, the catabolite control protein C (CcpC) plays a critical role in regulating the genes encoding the enzymes of the tricarboxylic acid branch of the Krebs citric acid cycle. A gene encoding a potential CcpC homolog and two potential target genes were identified in the Listeria monocytogenes genome. In vitro gel mobility shift assays and DNase I footprinting experiments showed that L. monocytogenes CcpC (CcpC(Lm)) interacts with the promoter regions of citB(Lm) (the gene that is likely to encode aconitase) and lmo0847 (encoding a possible glutamine transporter) and that citrate is a specific inhibitor of this interaction. To study in vivo promoter activity, a new lacZ reporter system was developed. This system allows stable integration into the chromosome of a promoter region transcriptionally fused to a promoterless lacZ gene at a nonessential, ectopic locus. Analysis of strains carrying a citB(Lm)-lacZ or lmo0847-lacZ fusion revealed that CcpC(Lm) represses citB(Lm) and lmo0847 in media containing an excess of glucose and glutamine. In addition, regulation of citB(Lm) expression in rich medium was growth phase dependent; during exponential growth phase, expression was very low even in the absence of CcpC(Lm), but a higher level of citB(Lm) expression was induced in stationary phase, suggesting the involvement of another, as yet unidentified regulatory factor.