Listeria monocytogenes σ B Regulates Stress Response and Virulence Functions

Abstract

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.