A homolog of CcpA mediates catabolite control in Listeria monocytogenes but not carbon source regulation of virulence genes

Abstract

Readily utilizable sugars down-regulate virulence gene expression in Listeria monocytogenes, which has led to the proposal that this regulation may be an aspect of global catabolite regulation (CR). We recently demonstrated that the metabolic enzyme alpha-glucosidase is under CR in L. monocytogenes. Here, we report the cloning and characterization from L. monocytogenes of an apparent ortholog of ccpA, which encodes an important mediator of CR in several low-G+C-content gram-positive bacteria. L. monocytogenes ccpA (ccpALm) is predicted to encode a 335-amino-acid protein with nearly 65% identity to the gene product of Bacillus subtilis ccpA (ccpABs). Southern blot analysis with a probe derived from ccpALm revealed a single strongly hybridizing band and also a second band of much lower intensity, suggesting that there may be other closely related sequences in the L. monocytogenes chromosome, as is the case in B. subtilis. Disruption of ccpALm resulted in the inability of the mutant to grow on glucose-containing minimal medium or increase its growth rate in the presence of preferred sugars, and it completely eliminated CR of alpha-glucosidase activity in liquid medium. However, alpha-glucosidase activity was only partially relieved from CR on solid medium. These results suggest that ccpA is an important element of carbon source regulation in L. monocytogenes. Nevertheless, utilizable sugars still down-regulate the expression of hly, which encodes the virulence factor hemolysin, in a ccpALm mutant, indicating that CcpA is not involved in carbon source regulation of virulence genes.

FIG. 1

Southern blot analysis of L. monocytogenes chromosomal DNA with a ccpA_Lm probe. Chromosomal DNA (3 μg in each reaction) was digested with six different restriction enzymes (X, XbaI; K, KpnI; P, PstI; H, HindIII; B, BamHI; and E, EcoRI) and subjected to agarose gel electrophoresis. After transfer to membrane, the DNA was probed with a [³²P]dCTP-labeled 615-bp ccpA_Lm fragment.

FIG. 2

Disruption of the gene and rescue of adjacent sequences. (A) A 615-bp internal ccpA fragment (open box) was PCR amplified from the L. monocytogenes chromosome with the primers CcpA-05R and CcpA-06L, cloned into the integrational vector pCON1, and conjugated into L. monocytogenes. (B) Shift of the pCON1-CCPA-containing strain to the non-permissive temperature forced integration of the vector into the chromosome at the ccpA locus, resulting in the truncation of the gene after codon 279 (arrow). (C) Sequences from upstream of the integrated vector were rescued by extracting chromosomal DNA from the mutant, digesting it with XbaI, and cloning it directly in E. coli. bla, β-lactamase gene; cat, chloramphenicol acetyltransferase gene; oriT, mobilization signal from plasmid RP4; pE194 ts, replication origin derived from plasmid pE194ts; ColE1, replication origin derived from pUC18.

FIG. 3

(A) Nucleotide sequence of the ccpA_Lm gene. The deduced amino acid sequence is given in single-letter code below the DNA sequence. The putative start codon is shown in bold type, and the stop codon is indicated by an asterisk. A potential ribosome-binding site (RBS) is underlined. Putative −10 and −35 sequences are double underlined. A CRE-like sequence partially overlapping the putative −35 sequence is indicated by dashed inverted arrows. A possible factor-independent transcription terminator is marked with solid inverted arrows. The site of truncation of the ccpA coding sequence in JB15 is indicated with an arrowhead. (B) Comparison of the CRE-like sequence in the ccpA_Lm promoter region with the CRE consensus sequence (59). Ambiguity codes are as follows: W denotes A or T and N denotes A, C, G, or T. Vertical bars indicate matching bases.

FIG. 4

Alignment of L. monocytogenes CcpA with three other CcpA proteins. The B. subtilis (B_sub; GenBank accession no. 115950), B. megaterium (B_meg; accession no. 1168844), and S. xylosus (S_xyl; accession no. 3023459) CcpA proteins are compared with the deduced L. monocytogenes (L_mon) CcpA sequence. Positions identical in at least two of four sequences are shaded gray. Residues constituting the helix-turn-helix (HTH) domain at the N terminus are marked with a line. The alignment was performed with the Pileup program of the GCG software package.

FIG. 5

Growth of strains 10403S and JB15 (10403S ccpA::pCON1) on defined medium. The wild-type strain 10403S is on the left and JB15 is on the right in both panels. (A) Glucose-containing MM plate showing growth of the two strains after 4 days of incubation at 30°C. (B) Streaks made on BHI agar at the same time as on the plate shown in panel A, after 48-h incubation at 30°C.

FIG. 6

CR of α-glucosidase in strains 10403S and JB15 (10403S ccpA::pCON1) in liquid medium. Cells were grown at 37°C in LB medium buffered with 100 mM MOPS (pH 7.0), supplemented with the indicated sugars (each, 25 mM). Specific activity of α-glucosidase was measured in exponentially growing cells by using p-nitrophenyl-α-d-glucoside as the substrate. Mal, maltose; Glc, glucose; Fru, fructose; Cel, cellobiose; wt, wild type. Each sample was analyzed in triplicate, and the data represent the means and standard errors of the means of three independent experiments.

FIG. 7

(A) Growth rates of L. monocytogenes and levels of expression of hly-gus on different sugars. Strain AML73 was grown in LB medium buffered with 100 mM MOPS (pH 7.4), and doubling times and β-glucuronidase activity were measured in the presence of the indicated sugars (each, 25 mM). The data represent the means of either two (β-glucuronidase activity; each experiment was done in triplicate) or three (doubling times and final cell densities) separate experiments. Standard deviations were less than 10% in all cases. Glc, glucose; Fru, fructose; Cel, cellobiose; Tre, trehalose; Mal, maltose; Suc, sucrose; Gal, galactose. (B) hly-gus expression in JB153 (ccpA::pCON1) and AML73 (wild type). Sugars were added (each at a concentration of 25 mM) to LB medium buffered with 100 mM MOPS (pH 7.4), and β-glucuronidase activity was measured 2 h into stationary phase. Data represent means ± standard errors of the means of three independent experiments. LB, LB medium; Glc, glucose; Cel, cellobiose.