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.Listeria monocytogenes is a gram-positive, facultative, intracellular pathogen responsible for listeriosis, a serious infection with a mortality rate of up to 30%, mainly among pregnant women, their fetuses, and immunocompromised persons. Listeriosis is characterized by gastroenteritis and fetoplacental and central nervous system infection, resulting in spontaneous abortion, neonatal death, septicemia, and meningitis (58). L. monocytogenes is widespread in nature and is predominantly transferred to humans by ingestion of contaminated foods (13). The bacterium can invade and multiply inside a wide range of phagocytic and nonphagocytic mammalian cells and has served as an important model system for studying the mechanisms of both intracellular parasitism and host cell biology (7,40,41).A great deal of effort has been invested in isolating L. monocytogenes virulence genes and understanding mechanisms of pathogenesis (reviewed in reference 58). However, our knowledge of the basic physiology of this important pathogen is very limited, making it difficult to gain a comprehensive understanding of its pathogenesis. There has been increasing evidence that regulation of carbon metabolic pathways plays a critical role in the virulence of pathogenic bacteria. Regulation of the Salmonella enterica virulence operon spv is controlled by cyclic AMP-cyclic AMP receptor protein complex, the major catabolite regulator, as well as by the positive regulator SpvR (38). Similarly, Escherichia coli STb enterotoxin production is repressed by glucose, and repression is relieved by addition of cyclic AMP (4). Leukotoxin production in Actinobacillus actinomycetemcomitan...

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.

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.Listeria monocytogenes is a gram-positive food-borne bacterial parasite of mammals that causes listeriosis, an infection with a 30% mortality rate in susceptible humans that is characterized by fetoplacental and central nervous system infections and gastroenteritis (38). The risk group for listeriosis includes pregnant women, neonates, the elderly, and immunocompromised adults. Listeria infection has been an important model system for the study of host-pathogen interactions and mechanisms of intracellular parasitism (38). This bacterium is an intracellular pathogen that induces its own uptake by nonphagocytic cells and spreads from cell to cell via actinbased motility (4). Even though L. monocytogenes is able to grow intracellularly in a variety of mammalian cells, it is a facultative pathogen that can adapt to saprophytic growth on decaying soil vegetation (28). It is therefore interesting to examine how this bacterium senses the environment in order to regulate expression of its virulence determinants.Previous researchers have thoroughly investigated the molecular determinants of L. monocytogenes pathogenesis. The genes that encode all the currently known virulence factors are positively regulated by the transcriptional activator PrfA (3). These genes are repressed, however, when L. monocytogenes is grown in the presence of fermentable sugars (28). This carbon source-mediated repression of virulence genes does not involve CcpA, the global regulator of catabolite control in many gram-positive bacteria (2, 16, 36), but instead is due to effects of sugar metabolism on PrfA activity. Rapidly metabolized carbon sources alter the phosphorylation state of components of the phosphoenolpyruvate-dependent phosphotransferase system; one or more of these components appear to inhibit PrfA (17,27). Given that there is some uncertainty about the mechanisms that couple utilization of carbon sources and expression of...