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CcpC-Dependent Regulation of citB and lmo0847 in Listeria monocytogenes

Hyun-Jin Kim, Meghna Mittal, and Abraham L. Sonenshein^*

Department of Molecular Biology and Microbiology, Tufts University School of Medicine and Graduate Program in Molecular Microbiology, Sackler School of Graduate Biomedical Sciences, 136 Harrison Avenue, Boston, Massachusetts 02111

Received 15 October 2004/ Accepted 11 October 2005

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.

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