Sue David, Boor Kathryn J, Wiedmann Martin
Department of Food Science, Cornell University, 412 Stocking Hall, Ithaca, NY 14853, USA.
Microbiology (Reading). 2003 Nov;149(Pt 11):3247-3256. doi: 10.1099/mic.0.26526-0.
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.
单核细胞增生李斯特菌是一种食源性病原体，能够在包括低pH值和高渗透压在内的多种环境条件下存活并生长。替代西格玛因子西格玛B有助于单核细胞增生李斯特菌在极端条件下生存。本研究的目的是鉴定并确认单核细胞增生李斯特菌中特定的西格玛B依赖性基因，并表征它们在各种应激条件下的表达模式。基于每个基因上游存在预测的西格玛B依赖性启动子序列，opuCA、lmo1421和bsh被鉴定为假定的西格玛B依赖性基因。opuCA和lmo1421分别编码已知的和假定的相容性溶质转运蛋白，bsh编码一种共轭胆汁盐水解酶（BSH）。报告基因融合和半定量RT-PCR技术用于确认这些应激反应基因的西格玛B依赖性调控，并确定它们对环境应激的表达模式。RT-PCR表明，相对于野生型细胞中的水平，单核细胞增生李斯特菌缺失西格玛B细胞在稳定期时opuCA、lmo1421和bsh的转录水平降低。此外，单核细胞增生李斯特菌缺失西格玛B菌株中的BSH活性被消除。RT-PCR证实了opuCA的生长阶段依赖性表达，在稳定期细胞中表达水平最高。通过RT-PCR测定，单核细胞增生李斯特菌野生型菌株在暴露于0.5 M KCl 10分钟和15分钟后，opuCA表达分别有两倍和三倍的诱导，lmo1421表达分别有七倍和五倍的诱导，这表明指数期单核细胞增生李斯特菌在暴露于盐胁迫时西格玛B活性迅速诱导。单拷贝染色体opuCA- Gus报告基因融合也显示，指数期细胞暴露于增加的盐浓度（0.5 M NaCl或0.5 M KCl）后，opuCA表达有显著诱导。结合最近表明opuCA和bsh在单核细胞增生李斯特菌毒力中起作用的研究结果，此处呈现的数据进一步证明了特定的西格玛B介导的对单核细胞增生李斯特菌环境应激抗性和宿主内存活的贡献。
