Kazmierczak Mark J, Wiedmann Martin, Boor Kathryn J
Department of Food Science, Cornell University, Ithaca, 413 Stocking Hall, NY 14853, USA.
Microbiology (Reading). 2006 Jun;152(Pt 6):1827-1838. doi: 10.1099/mic.0.28758-0.
Listeria monocytogenes sigmaB and PrfA are pleiotropic regulators of stress response and virulence gene expression. Quantitative RT-PCR (qRT-PCR) was used to measure transcript levels of sigmaB- and PrfA-dependent genes in exponential-phase L. monocytogenes wild-type and DeltasigB strains as well as in bacteria exposed to environmental stresses (0.3 M NaCl or growth to stationary phase) or present in the vacuole or cytosol of human intestinal epithelial cells. Stationary-phase or NaCl-exposed L. monocytogenes showed sigmaB-dependent increases in opuCA (10- and 17-fold higher, respectively) and gadA transcript levels (77- and 14-fold higher, respectively) as compared to non-stressed, exponential-phase bacteria. While PrfA activity, as reflected by plcA transcript levels, was up to 95-fold higher in intracellular L. monocytogenes as compared to non-stressed bacteria, sigmaB activity was only slightly higher in intracellular than in non-stressed bacteria. Increased plcA transcript levels, which were similar in both host cell vacuole and cytosol, were associated with increases in both prfA expression and PrfA activity. qRT-PCR assays were designed to measure expression of prfA from each of its three promoter regions. Under all conditions, readthrough transcription from the upstream plcA promoter was very low. The relative contribution to total prfA transcription from the sigmaA-dependent P1prfA promoter ranged from approximately 17 % to 30 %, while the contribution of the P2prfA region, which appears to be transcribed by both sigmaA and sigmaB, ranged from approximately 70 % to 82 % of total prfA transcript levels. In summary (i) sigmaB is primarily activated during environmental stress and does not contribute to PrfA activation in intracellular L. monocytogenes and (ii) the partially sigmaB-dependent P2prfA promoter region contributes the majority of prfA transcripts in both intra- and extracellular bacteria.
单核细胞增生李斯特菌的σB和PrfA是应激反应和毒力基因表达的多效性调节因子。使用定量逆转录聚合酶链反应（qRT-PCR）来测量指数期单核细胞增生李斯特菌野生型和ΔsigB菌株中σB和PrfA依赖性基因的转录水平，以及暴露于环境应激（0.3 M NaCl或生长至稳定期）的细菌或存在于人类肠道上皮细胞液泡或细胞质中的细菌中的转录水平。与未受应激的指数期细菌相比，处于稳定期或暴露于NaCl的单核细胞增生李斯特菌显示opuCA转录水平分别升高10倍和17倍，gadA转录水平分别升高77倍和14倍，呈现出σB依赖性增加。虽然与未受应激的细菌相比，细胞内单核细胞增生李斯特菌中plcA转录水平所反映的PrfA活性高达95倍，但细胞内σB活性仅比未受应激的细菌略高。宿主细胞液泡和细胞质中plcA转录水平的增加相似，这与prfA表达和PrfA活性的增加相关。设计qRT-PCR测定法来测量prfA从其三个启动子区域各自的表达。在所有条件下，来自上游plcA启动子的通读转录非常低。σA依赖性P1prfA启动子对总prfA转录的相对贡献约为17%至30%，而似乎由σA和σB共同转录的P2prfA区域的贡献占总prfA转录水平的约70%至82%。总之，（i）σB主要在环境应激期间被激活，对细胞内单核细胞增生李斯特菌中的PrfA激活没有贡献；（ii）部分依赖σB的P2prfA启动子区域在细胞内和细胞外细菌中贡献了大部分prfA转录本。
