CcpC-Dependent Regulation of Citrate Synthase Gene Expression in Listeria monocytogenes

Genetic content of lacZ fusions integrated at the L. monocytogenes int′-′comK locus. The regions fused to lacZ and used for assays of gene expression in L. monocytogenes are indicated below the gene map. The arrows above the gene map indicate the annealing sites and orientations of primers used for RT-PCR experiments. See the legend to Fig. 1 for additional details.

RT-PCR analysis of the effect of ccpA mutation on citZ expression. RNA was extracted from wild-type, ccpC mutant, ccpA mutant, or ccpC ccpA double-mutant cells growing exponentially in BHI medium. Primer OMM88 was used for RT, and OMM88 and OMM87 were the primers used in PCR with the cDNA. The upper panels show the results for the citZ transcript (two different amounts of RNA and two different numbers of PCR cycles). The lower panels show the results for two different amounts of rRNA used as a control. The intensities of the bands were quantified using ImageQuant software. In two independent experiments, the average values for the citZ transcript relative to the wild type, normalized to rRNA, were 0.9 ± 0.1 for the ccpA mutant, 10 ± 2 for the ccpC mutant, and 9 ± 3 for the ccpA ccpC mutant.

Primer extension analysis to map the tsp for the lmo1568 gene. Three oligonucleotide primers that were labeled with ³²P at the 5′ end and were complementary to lacZ mRNA (see Materials and Methods) were annealed to total cellular RNA that was extracted from wild-type strain EGD-e and ccpC null mutant cells carrying a lmo1568-lacZ fusion and growing exponentially in BHI medium and were extended by using deoxynucleoside triphosphates and reverse transcriptase. The DNA products were separated by electrophoresis on a 6% polyacrylamide-7 M urea gel, and their mobilities were compared with the mobilities of dideoxynucleotide sequencing ladders (lanes A, C, G, and T) generated using two of the same primers used for primer extension and the relevant cloned DNA as the template. (The length of the third reverse transcript was extrapolated from its mobility relative to the mobilities of DNA sequences having known lengths generated with the other primers.) The left and right lanes in each set show products obtained with RNA from the wild-type (WT) and ccpC mutant strains, respectively. The following primers were used: for the left panel, OMM070; for the middle panel, OMM071; and for the right panel, OBB102. The arrows indicate the positions of the primer extension products. The sequence of the lmo1568 promoter region is shown below the gels. The consensus recognition sequences for CcpC are indicated by uppercase letters. The putative −35 and −10 regions and transcription start site (+1) of the deduced P2 promoter are also indicated. The start codon (ATG) is italicized.

RT-PCR analysis of read-through transcription from the P1 promoter into lmo1568. RNA was extracted from cells growing exponentially in BHI medium. Lanes 1, 3, 5, 7, 9, and 11 contained RT-PCR products from the wild-type strain, and lanes 2, 4, 6, 8, 10, and 12 contained RT-PCR products from the ccpC null strain. For analysis of read-through transcription from P1, the primer used for RT was OMM64; the PCR was primed with OMM50 and OMM64 (Fig. 2). One microgram of RNA was used for RT, and 10% of the product was used as a template for the PCR. To assay for rRNA as a normalization factor, 50 ng of RNA was used for cDNA synthesis and 10% of the product was used as a template for a PCR. The numbers of PCR cycles are indicated below the gels. The band intensities for the ccpC mutant strain RNA normalized to rRNA and relative to the wild-type intensities (wt) for the experiment shown were determined by using densitometry and ImageQuant software and are also indicated below the gels. In three independent experiments, the average value for the read-through transcript normalized to rRNA and relative to the wild-type value was 3.5 ± 0.5 for the ccpC mutant.

RT-PCR analysis of read-through transcription from citZ into the citC gene and regulation of citC by CcpC. RNA was extracted from wild-type and ccpC mutant cells growing exponentially in BHI medium. To assay for citZ-citC read-through transcription or for citC transcription, 500 ng of RNA was used for RT and 10% of the product was used as a template for a PCR. To assay for rRNA as a normalization factor, 50 ng of RNA was used for cDNA synthesis and 10% of the product was used as a template for PCR. Data from two independent experiments (experiments 1 and 2) are shown. Each pair of lanes shows the results obtained with RNA from wild-type cells (lanes a) and from ccpC mutant cells (lanes b). The numbers of PCR cycles are indicated below the gels. Band intensities were quantified by using densitometry and ImageQuant software. (A) Read-through transcription from citZ into the downstream citC gene. The primers used were OMM140 and OMM98 (Fig. 2). In two independent experiments, the average amount of read-through transcript (normalized to the amount of rRNA) was 2.5- ± 0.5-fold larger in the ccpC mutant than in the wild-type strain. (B) Transcripts of the citC gene. The primers used were OMM97 and OMM98 (Fig. 2). In two independent experiments, the amount of citC transcript (normalized to the amount of rRNA) was 3- ± 0.2-fold larger in the ccpC mutant than in the wild-type strain.

Gel mobility shift assay of the interaction of L. monocytogenes CcpC with the P1 and P2 promoters. CcpC-His₆ was incubated with ³²P-labeled DNA fragments containing either the lmo1568 (left panel) or lmo1569 (right panel) promoter region. The concentrations of CcpC-His₆ used in the reactions were as follows: for lmo1568, 0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, and 1,000 nM (lanes 1 to 10, respectively); and for lmo1569, 0, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, and 1,000 nM (lanes 1 to 9, respectively). See Materials and Methods for details.

DNase I footprinting assay of the interaction between CcpC-His₆ and the P2 promoter region. A ³²P-labeled DNA fragment corresponding to the lmo1568 promoter region was incubated with various amounts of CcpC-His₆ prior to DNase I digestion. The concentrations of CcpC-His₆ (in nM) used in the reactions are indicated above the lanes. The vertical bar indicates the region protected from DNase I digestion by CcpC-His₆. In the sequence of the lmo1568 promoter region shown on the right, the region protected by CcpC-His₆ from DNase I digestion (positions −9 to −61 with respect to the transcription start site, as deduced from other experiments in which a DNA sequence ladder was generated) is indicated by bold type. The consensus recognition sequences for CcpC are indicated by uppercase letters. The transcription start site is indicated by a single uppercase T, and the −10 and −35 regions are also indicated. The start codon of lmo1568 is indicated by italics.

Effect of medium composition on expression of a lacZ fusion in L. monocytogenes. Strains LMM16 (ccpC ⁺) (gray bars) and LMM21 (ΔccpC::spc) (black bars), both of which carried an lmo1568-lacZ fusion, were grown at 37°C in BHI medium supplemented with 0.5% glucose, 1.6 mg glutamine per ml, or 0.5% citrate or with combinations of these supplements. β-Galactosidase activity was measured using samples taken at various points during the exponential phase of growth. The values are the averages of two experiments.