SigmaB- and PrfA-dependent transcription of genes previously classified as putative constituents of the Listeria monocytogenes PrfA regulon

Abstract

Mounting evidence suggests that sigma(B) and PrfA coregulate transcription of multiple genes in Listeria monocytogenes, therefore, the relative contributions of sigma(B) and PrfA to transcript levels of genes identified previously as differentially regulated by PrfA were measured. Group I genes are recognized virulence genes that are positively regulated by PrfA; group II genes were reported previously as negatively regulated by PrfA; and multiple group III genes were proposed to be coregulated by sigma(B) and PrfA. Transcript levels for selected genes were measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in L. monocytogenes 10403S as well as in otherwise isogenic DeltasigB, DeltaprfA, and DeltasigBDeltaprfA strains grown under conditions demonstrated to induce either PrfA activity (0.2% activated charcoal) or both PrfA and sigma(B) activity (stationary phase). Although the Group I gene plcA was positively regulated by PrfA, transcript levels for the group II genes lmo0278 and lmo0178 were not affected by the prfA deletion. While the sigB deletion significantly affected transcript levels for the selected group III genes (i.e., lmo0596, lmo0654, bsh, and opuCA), with lower transcript levels in the DeltasigB strains under all conditions tested, transcript levels for these genes were not significantly affected by the prfA deletion. Our results suggest that the regulatory interactions between PrfA and sigma(B) contribute to PrfA's predominant role as a direct regulator of virulence genes critical for invasion and intracellular survival in L. monocytogenes 10403S, while sigma(B) regulates a wider range of virulence and stress response genes.

FIG. 1.

Transcript levels for selected group I, II, and III genes in the L. monocytogenes parent strain 10403S and in isogenic ΔsigB, ΔprfA, and ΔsigBΔprfA strains exposed to either (A) BHI + 0.2% activated charcoal or (B) BHI alone. Normalized log copy numbers shown on the Y-axis represent log-transformed transcript levels for a given target gene normalized to the geometric mean of the transcript levels for the housekeeping genes rpoB and gap. Genes shown on the X-axis are, from left to right, the group I gene (plcA), group II genes (lmo0178, lmo0278), and group III genes (opuCA, bsh, lmo0596, and lmo0654). Data represent mean values from qRT-PCR data collected from three or four independent RNA extractions (except for transcript levels for lmo0654 and lmo0596 from cells grown in BHI + 0.2% charcoal, which are represented by two replicates); error bars represent one standard deviation.

FIG. 2.

Transcript levels for the selected group I, II, and III genes in the L. monocytogenes parent strain 10403S and in isogenic ΔsigB, ΔprfA, and ΔsigB ΔprfA strains grown to either (A) log phase (OD₆₀₀ = 0.4) or (B) stationary phase (OD₆₀₀ = 2.0). Normalized log copy numbers shown on the Y-axis represent log-transformed transcript levels for a given target gene normalized to the geometric mean of the transcript levels for the housekeeping genes rpoB and gap. Genes shown on the X-axis are, from left to right, the group I gene (plcA), group II genes (lmo0178, lmo0278), and group III genes (opuCA, bsh, lmo0596, and lmo0654). Data represent mean values from qRT-PCR data collected from three or four independent RNA extractions; error bars represent one standard deviation. To enable comparisons between transcript levels for a given gene in a given mutant strain, the data presented in Figs. 1 and 2 were also rearranged by strain and are presented in Suppl. Figs. S1 through S3.

FIG. 3.

L. monocytogenes 10403S promoter sequences of confirmed (i.e., plcA, actA, hly) and putative PrfA-regulated genes (i.e. lmo0178, lmo0278, bsh, lmo0596, lmo0654, opuCA). DNA sequences were obtained from the genome sequence for strain 10403S (Listeria monocytogenes Sequencing Project. Broad Institute of Harvard and MIT; http://www.broad.mit.edu). Promoter sequences are underlined and PrfA boxes are italicized. Deviations from the PrfA binding site consensus sequence are shown by lower case letters. The PrfA boxes and σ^A-dependent −10 and −35 sites for plcA, hly, and actA (Mengaud et al., ; Vazquez-Boland et al., 1992); the PrfA boxes for bsh, lmo0178, lmo0278, and lmo0596 (Glaser et al., ; Dussurget et al., 2002); and the σ^B-dependent −10 and −35 sites for bsh, lmo0596, and opuCA (Kazmierczak et al., 2003) have all been reported previously. For bsh, a putative −10 site has been described 19 nt downstream of the PrfA box (Dussurget et al., 2002) however, this promoter has not been confirmed experimentally and, therefore, was not included in this figure. The putative PrfA boxes for lmo0596, lmo0178, and lmo0278 are shown as described by Glaser et al. (2003). The indicated σ^A-dependent −10 promoter site for lmo0178 was mapped by primer extension (Luo et al., 2004).