CcpC-Dependent Regulation of Citrate Synthase Gene Expression inListeria monocytogenes

Abstract

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...

Transcriptome Sequencing of Listeria monocytogenes Reveals Major Gene Expression Changes in Response to Lactic Acid Stress Exposure but a Less Pronounced Response to Oxidative Stress

“…Genes annotated as enzymes involved in certain steps of the citric acid cycle (citZ -citrate synthase, citB -aconitase, and citC -isocitrate dehydrogenase) were also upregulated in both 6179 and R479a following exposure to hydrogen peroxide. Two of these genes, citC and citZ, are part of an operon (Mittal et al, 2009) along with the upstream gene (a lmo1568 homolog), which was upregulated as well. However, the relevance of these three citric acid cycle genes to stress response is currently unknown.…”

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

“…Similar citrate-dependent derepression has also been described for the citB gene of L. monocytogenes (Kim et al, 2006). In L. monocytogenes, CcpC represses transcription of citB as well as of citZ and the lmo0847 gene, which encodes a putative glutamine transporter (Kim et al, 2006;Mittal et al, 2009). The organization of the CcpC binding sites in the L. monocytogenes citB regulatory region is almost identical to that in B. subtilis, with a dyad symmetry element centred at position 268 and a half-dyad at positions 228 to 232 (Kim et al, 2006).…”

“…The citZ gene encodes the sole citrate synthase in L. monocytogenes. A previously described insertion-deletion mutation in the citZ gene (Mittal et al, 2009) was utilized; genomic DNA from this strain, LMM33, was introduced into strain LMM12 (citB null) by double crossover recombination, producing strain LMM34. Importantly, the citB citZ double mutant strain exhibited very low levels of citB-lacZ activity compared with the citB single mutant (Fig.…”

“…Citrate-antagonized repression of the B. subtilis citZ, citB and citC genes by CcpC has been described in detail (Jourlin-Castelli et al, 2000;Kim et al, 2003a, b). In the closely related intracellular pathogen Listeria monocytogenes, the citZ and citB genes are regulated by CcpC and CodY, but not by CcpA (Kim et al, 2006;Mittal, 2008;Mittal et al, 2009).…”

Three genes encoding citrate synthases in Saccharopolyspora erythraea are regulated by the global nutrient‐sensing regulators GlnR, DasR, and CRP

“…Furthermore, in other species, CCR serves as a regulatory mechanism via a homologous CRP and regulates CSs. For example, in B. subtilis and Listeria monocytogenes , citZ is controlled by the LysR family members CcpA (Kim et al ., ) and CcpC (Jourlin‐Castelli et al ., ; Mittal et al ., ) respectively. However, the CRP homolog in S. coelicolor has no role in CCR, but play critical roles in spore germination, colony development, and antibiotic production (Derouaux et al ., 2004a,b; Piette et al ., ; Gao et al ., ).…”