Regulation of hly expression in Listeria monocytogenes by carbon sources and pH occurs through separate mechanisms mediated by PrfA

Abstract

Expression of the PrfA-controlled virulence gene hly (encoding the pore-forming cytolysin listeriolysin) is under negative regulation by readily metabolized carbon sources in Listeria monocytogenes. However, the hyperhemolytic strain NCTC 7973 exhibits deregulated hly expression in the presence of repressing sugars, raising the possibility that a defect in carbon source regulation is responsible for its anomalous behavior. We show here that the activity of a second glucose-repressed enzyme, alpha-glucosidase, is 10-fold higher in NCTC 7973 than in 10403S. Using hly-gus fusions, we show that the prfA allele from NCTC 7973 causes deregulated hly-gus expression in the presence of sugars in either the wild-type or the NCTC 7973 background, while the 10403S prfA allele restores carbon source regulation. However, the prfA genotype does not affect the regulation of alpha-glucosidase activity by repressing sugars. Of the two mutational differences in PrfA, only a Gly145Ser change is important for regulation of hly-gus. Therefore, NCTC 7973 and 10403S have genetic differences in at least two loci: one in prfA that affects carbon source regulation of virulence genes and another in an unidentified gene(s) that up-regulates alpha-glucosidase activity. We also show that the decrease in pH associated with utilization of sugars negatively regulates hly-gus expression, although sugars can affect hly-gus expression by another mechanism that is independent of pH.

FIG. 1

Catabolite regulation of α-glucosidase in strains 10403S and NCTC 7973. Cells were grown at 37°C in LB medium buffered with 100 mM MOPS (pH 7.0) and supplemented with the indicated sugars at 25 mM. The specific activity of α-glucosidase in exponentially growing cells was measured as described in Materials and Methods and is expressed as nanomoles of product formed minute⁻¹ milligram of protein⁻¹. Mal, maltose; Glc, glucose; Fru, fructose; Cel, cellobiose. Each sample was analyzed in triplicate, and the data represent the means and standard errors of the means for three independent experiments.

FIG. 2

(A) Growth of 10403S and NCTC 7973 in LB medium supplemented with various sugars. Overnight cultures were grown in LB medium buffered with 100 mM MOPS (pH 7.0) and were diluted 1:25 into fresh medium with or without either glucose or cellobiose at 25 mM. The results of one representative experiment are shown. Similar results were obtained in three independent experiments. OD₆₀₀, optical density at 600 nm. (B) Doubling times of 10403S and NCTC 7973 in buffered LB medium with different carbon sources. Data represent means ± standard errors of the means for three independent experiments.

FIG. 3

Alignment of the deduced carboxy-terminal amino acid sequences of PrfA proteins from four L. monocytogenes strains. The substitutions in the PrfA sequence from NCTC 7973 are indicated by dots above the alignment. The numbers to the left of the sequences correspond to the position of the first residue in the full-length protein. The helix-turn-helix motif is boxed (41). The three residues at the carboxy terminus of PrfA_EGD that were found to be different from the published sequence (28) are indicated by asterisks. Identical residues are shown in white letters on a black background, while divergent residues are in black letters on a white background.

FIG. 4

Strategy for construction of strains for prfA allele exchange by integrative replacement. (A) A promoterless, truncated copy of prfA from either 10403S or NCTC 7973 was cloned into the integrational vector pCON1 and conjugated into the host strains. (B) Shifting the strains to the nonpermissive temperature resulted in the integration of the temperature-sensitive vector into the chromosome at the prfA locus. The two nucleotide changes in the prfA sequence in codons 145 and 229 are represented as a cross and a dot, respectively. The integrated copy of prfA was amplified with the primers PrfA1R and PrfA4L, and the PCR product was sequenced to confirm the sequence changes. bla, β-lactamase gene; cat, chloramphenicol acetyltransferase gene; oriT, mobilization signal from plasmid RP4; pE194ts, replication functions derived from plasmid pE194ts; ColE1, replication functions derived from pUC18.

FIG. 5

Effect of exchanging prfA alleles between strains 10403S and NCTC 7973 on the regulation of hly-gus expression by utilizable sugars. Cells were grown in LB medium buffered with 100 mM MOPS (pH 7.4) with or without either glucose or cellobiose at 25 mM. β-Glucuronidase activity was measured as described in Materials and Methods. Data represent means ± standard errors of the means for two independent experiments, each done in triplicate. Glc, glucose; Cel, cellobiose.

FIG. 6

Effect of exchanging prfA alleles between 10403S and NCTC 7973 on the catabolite regulation of α-glucosidase. Cells were grown in LB medium buffered with 100 mM MOPS (pH 7.0) with or without either glucose or cellobiose at 25 mM. Specific activity of α-glucosidase from exponentially growing cells was measured as described in Materials and Methods. Data represent means ± standard errors of the means for three separate assays from each of two independent experiments. Mal, maltose; Glc, glucose; Cel, cellobiose.

FIG. 7

Effect of pH on the expression of hly-gus. Cells were grown in LB medium buffered with either 100 mM MOPS (pH 7.4 to 6.5) or MES (pH 6.0). Samples were collected at 1 h into stationary phase, and β-glucuronidase specific activity in cell lysates was measured. Data represent means ± standard errors of the means for three independent experiments, each done in triplicate.

FIG. 8

Sugars regulate hly-gus expression by a mechanism that is independent of pH. Cells were grown in LB medium (with 100 mM MOPS, pH 7.4) with or without either glucose or cellobiose at 25 mM. Samples were collected at 1 h into stationary phase, and β-glucuronidase specific activity was measured. The starting pH of each culture was 7.4. The number above each bar represents the pH of the culture at the time samples were collected.

FIG. 9

Effect of prfA_10403S and prfA_{NCTC 7973} alleles on pH regulation of hly-gus expression. Culture conditions were as described in the legend to Fig. 7. Results are expressed as nanomoles of p-nitrophenol formed minute⁻¹ milligram of protein⁻¹ and represent the means ± standard errors of the means for two independent experiments, each done in triplicate.