Raczkowska Adrianna, Trzos Joanna, Lewandowska Olga, Nieckarz Marta, Brzostek Katarzyna
Department of Applied Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw, Warsaw, Poland.
PLoS One. 2015 Apr 20;10(4):e0124248. doi: 10.1371/journal.pone.0124248. eCollection 2015.
OmpR is a transcriptional regulator implicated in the control of various cellular processes and functions in Enterobacteriaceae. This study was undertaken to identify genes comprising the OmpR regulon in the human gastrointestinal pathogen Yersinia enterocolitica. Derivatives of an ompR-negative strain with random transposon insertions creating transcriptional fusions with the reporter gene lacZ were isolated. These were supplied with the wild-type ompR allele in trans and then screened for OmpR-dependent changes in β-galactosidase activity. Using this strategy, five insertions in genes/operons positively regulated by OmpR and two insertions in genes negatively regulated by this protein were identified. Genetic analysis of one of these fusion strains revealed that the gene acrR, encoding transcriptional repressor AcrR is negatively regulated by OmpR. Differential analysis of membrane proteins by SDS-PAGE followed by mass spectrometry identified the protein AcrB, a component of the AcrAB-TolC multidrug efflux pump, as being positively regulated by OmpR. Analysis of the activity of the acrR and acrAB promoters using gfp fusions confirmed their OmpR-dependent repression and activation, respectively. The identification of putative OmpR-binding sites and electrophoretic mobility shift assays confirmed that this regulator binds specifically to both promoter regions with different affinity. Examination of the activity of the acrR and acrAB promoters after the exposure of cells to different chemicals showed that bile salts can act as an OmpR-independent inducer. Taken together, our findings suggest that OmpR positively controls the expression of the AcrAB-TolC efflux pump involved in the adaptive response of Y. enterocolitica O:9 to different chemical stressors, thus conferring an advantage in particular ecological niches.
OmpR是一种转录调节因子，参与调控肠杆菌科细菌的各种细胞过程和功能。本研究旨在鉴定人类胃肠道病原体小肠结肠炎耶尔森菌中构成OmpR调控子的基因。分离出ompR阴性菌株的衍生物，这些衍生物带有随机转座子插入，与报告基因lacZ形成转录融合。这些衍生物通过反式提供野生型ompR等位基因，然后筛选β-半乳糖苷酶活性中OmpR依赖性的变化。使用该策略，鉴定出了在受OmpR正向调控的基因/操纵子中的5个插入以及在受该蛋白负向调控的基因中的2个插入。对其中一个融合菌株的遗传分析表明，编码转录阻遏物AcrR的acrR基因受OmpR负向调控。通过SDS-PAGE对膜蛋白进行差异分析，随后进行质谱分析，鉴定出AcrB蛋白，它是AcrAB-TolC多药外排泵的一个组分，受OmpR正向调控。使用gfp融合分析acrR和acrAB启动子的活性，分别证实了它们受OmpR依赖性的抑制和激活。推定的OmpR结合位点的鉴定和电泳迁移率变动分析证实，该调节因子以不同亲和力特异性结合两个启动子区域。在细胞暴露于不同化学物质后检测acrR和acrAB启动子的活性，结果表明胆盐可作为一种不依赖OmpR的诱导剂。综上所述，我们的研究结果表明，OmpR正向控制AcrAB-TolC外排泵的表达，该外排泵参与小肠结肠炎耶尔森菌O:9对不同化学应激源的适应性反应，从而在特定生态位中赋予其优势。
