Sigma(B)-dependent expression patterns of compatible solute transporter genes opuCA and lmo1421 and the conjugated bile salt hydrolase gene bsh in Listeria monocytogenes.
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 sigma(B) contributes to L. monocytogenes survival under extreme conditions. The purpose of this study was to identify and confirm specific sigma(B)-dependent genes in L. monocytogenes and to characterize their expression patterns under various stress conditions. opuCA, lmo1421 and bsh were identified as putative sigma(B)-dependent genes based on the presence of a predicted sigma(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 sigma(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 deltasigB cells relative to levels present in wild-type cells. Furthermore, BSH activity is abolished in a L. monocytogenes deltasigB 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 sigma(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 sigma(B)-mediated contributions to both environmental stress resistance and intra-host survival in L. monocytogenes.
Department of Food Science, Cornell University, 412 Stocking Hall, Ithaca, NY 14853, USA.
David
D
Sue
Department of Food Science, Cornell University, 412 Stocking Hall, Ithaca, NY 14853, USA.
Kathryn J
KJ
Boor
Department of Food Science, Cornell University, 412 Stocking Hall, Ithaca, NY 14853, USA.
Martin
M
Wiedmann
D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D000581:Amidohydrolases / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D017930:Genes, Reporter; D008089:Listeria monocytogenes / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012808:Sigma Factor / Q000235:genetics*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D017931:DNA Primers; C480946:Lmo1421 protein, Listeria monocytogenes; C119750:OpuC protein, bacteria; D011993:Recombinant Fusion Proteins; C480945:SigB protein, Listeria monocytogenes; D012808:Sigma Factor; D000581:Amidohydrolases; C023154:choloylglycine hydrolase