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Abstract
Listeria monocytogenes is a food-borne pathogen that can persist and grow under a wide variety of environmental conditions including low pH and high osmolarity. The alternative sigma factor σ B contributes to L. monocytogenes survival under extreme conditions. The purpose of this study was to identify and confirm specific σ B-dependent genes in L. monocytogenes and to characterize their expression patterns under various stress conditions. opuCA, lmo1421 and bsh were identified as putative σ B-dependent genes based on the presence of a predicted σ B-dependent promoter sequence upstream of each gene. opuCA and lmo1421 encode known and putative compatible solute transporter proteins, respectively, and bsh encodes a conjugated bile salt hydrolase (BSH). Reporter fusions and semi-quantitative RT-PCR techniques were used to confirm σ B-dependent regulation of these stress-response genes and to determine their expression patterns in response to environmental stresses. RT-PCR demonstrated that opuCA, lmo1421 and bsh transcript levels are reduced in stationary-phase L. monocytogenes ΔsigB cells relative to levels present in wild-type cells. Furthermore, BSH activity is abolished in a L. monocytogenes ΔsigB strain. RT-PCR confirmed growth-phase-dependent expression of opuCA, with highest levels of expression in stationary-phase cells. The L. monocytogenes wild-type strain exhibited two- and threefold induction of opuCA expression and seven- and fivefold induction of lmo1421 expression following 10 and 15 min exposure to 0·5 M KCl, respectively, as determined by RT-PCR, suggesting rapid induction of σ B activity in exponential-phase L. monocytogenes upon exposure to salt stress. Single-copy chromosomal opuCA–gus reporter fusions also showed significant induction of opuCA expression following exposure of exponential-phase cells to increased salt concentrations (0·5 M NaCl or 0·5 M KCl). In conjunction with recent findings that indicate a role for opuCA and bsh in L. monocytogenes virulence, the data presented here provide further evidence of specific σ B-mediated contributions to both environmental stress resistance and intra-host survival in L. monocytogenes.
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