Listeria monocytogenes σBRegulates Stress Response and Virulence Functions

Abstract

While the stress-responsive alternative sigma factor σ B has been identified in different species of Bacillus,Listeria, and Staphylococcus, theσ B regulon has been extensively characterized only in B. subtilis.We combined biocomputing and microarray-based strategies to identify σ B-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidateσ B-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted σ B-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and Δ sigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significantσ B-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting σ B-dependent expression, 54 were preceded by a sequence resembling the σ B promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the σ B-dependent nature of a subset of eight selected promoter regions. Notably, theσ B-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g.,gadB,ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g.,inlA,inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, σ B also contributes to regulation of virulence gene expression in L. monocytogenes.These findings strongly suggest thatσ B contributes to L. monocytogenes gene expression during infection.