ABSTRACTListeria monocytogenesσBand positive regulatory factor A (PrfA) are pleiotropic transcriptional regulators that coregulate a subset of virulence genes. A positive regulatory role for σBinprfAtranscription has been well established; therefore, observations of increased virulence gene expression and hemolytic activity in a ΔsigBstrain initially appeared paradoxical. To test the hypothesis thatL. monocytogenesσBcontributes to a regulatory network critical for appropriate repression as well as induction of virulence gene expression, genome-wide transcript profiling and follow-up quantitative reverse transcriptase PCR (qRT-PCR), reporter fusion, and phenotypic experiments were conducted usingL. monocytogenes prfA*,prfA* ΔsigB, ΔprfA, and ΔprfAΔsigBstrains. Genome-wide transcript profiling and qRT-PCR showed that in the presence of active PrfA (PrfA*), σBis responsible for reduced expression of the PrfA regulon. σB-dependent modulation of PrfA regulon expression reduced the cytotoxic effects of a PrfA* strain in HepG2 cells, highlighting the functional importance of regulatory interactions between PrfA and σB. The emerging model of the role of σBin regulating overall PrfA activity includes a switch from transcriptional activation at the P2prfApromoter (e.g., in extracellular bacteria when PrfA activity is low) to posttranscriptional downregulation of PrfA regulon expression (e.g., in intracellular bacteria when PrfA activity is high).