Turonova Hana, Haddad Nabila, Hernould Mathieu, Chevret Didier, Pazlarova Jarmila, Tresse Odile
Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, University of Chemistry and TechnologyPrague, Czechia.
SECALIM UMR1014, Institut National de la Recherche AgronomiqueNantes, France.
Front Microbiol. 2017 May 18;8:913. doi: 10.3389/fmicb.2017.00913. eCollection 2017.
has been reported as a major cause of bacterial food-borne enteritides in developed countries during the last decade. Despite its fastidious growth requirements, including low level of oxygen and high level of CO, this pathogen is able to persist in the environment without permanent loss of its viability and virulence. As is not able to multiply outside a host, the cells spend significant amount of time in stationary phase of growth. The entry into the stationary phase is often correlated to resistance to various stresses in bacteria. The switching between exponential and stationary phases is frequently mediated by the regulator sigma S (RpoS). However, this factor is absent in and molecular mechanisms responsible for transition of cells to the stationary phase remain elusive. In this work, proteomic profiles of cells from exponential and stationary phases were compared using 2-D electrophoresis (2DE) fingerprinting combined with mass spectrometry analysis and qRT-PCR. The identified proteins, whose expression differed between the two phases, are mostly involved in protein biosynthesis, carbon metabolism, stress response and motility. Altered expression was observed also in the pleiotropic regulator CosR that was over-expressed during stationary phase. A shift between transcript and protein level evolution of CosR throughout the growth of was observed using qRT-PCR and (2DE). From these data, we hypothesized that CosR could undergo a negative autoregulation in stationary phase. A consensus sequence resulting from promoter sequence alignment of genes potentially regulated by CosR, including its own upstream region, among strains is proposed. To verify experimentally the potential autoregulation of CosR at the DNA level, electrophoretic mobility shift assay was performed with DNA fragments of CosR promoter region and rCosR. Different migration pattern of the promoter fragments indicates the binding capacity of CosR, suggesting its auto-regulation potential.
在过去十年中，它已被报道为发达国家细菌性食源性肠炎的主要病因。尽管其生长要求苛刻，包括低氧水平和高二氧化碳水平，但这种病原体能够在环境中持续存在，而不会永久丧失其活力和毒力。由于它无法在宿主外繁殖，细胞在生长的稳定期会花费大量时间。进入稳定期通常与细菌对各种压力的抗性相关。指数期和稳定期之间的转换通常由调节因子σS（RpoS）介导。然而，在该病原体中不存在这种因子，细胞向稳定期转变的分子机制仍然难以捉摸。在这项工作中，使用二维电泳（2DE）指纹图谱结合质谱分析和qRT-PCR比较了指数期和稳定期细胞的蛋白质组学图谱。鉴定出的在两个阶段表达不同的蛋白质大多参与蛋白质生物合成、碳代谢、应激反应和运动性。在多效调节因子CosR中也观察到表达改变，其在稳定期过度表达。使用qRT-PCR和（2DE）观察到CosR在整个生长过程中转录本和蛋白质水平演变之间的转变。根据这些数据，我们假设CosR在稳定期可能经历负自调节。提出了在该病原体菌株中由CosR潜在调控的基因启动子序列比对产生的共有序列，包括其自身的上游区域。为了在DNA水平上通过实验验证CosR的潜在自调节，用CosR启动子区域和rCosR的DNA片段进行了电泳迁移率变动分析。启动子片段的不同迁移模式表明CosR的结合能力，表明其自调节潜力。
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