Control of bistable gene expression in Bacillus subtilis by the transcription factor SinR and the phosphodiesterase YmdB

转录因子 SinR 和磷酸二酯酶 YmdB 控制枯草芽孢杆菌中的双稳态基因表达

• 批准号: 276691051
• 负责人: Professor Dr. Jörg Stülke
• 金额: --
• 依托单位: Abteilung für Allgemeine Mikrobiologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/276691051?language=en
• 关键词: Control; bistable; gene; expression; Bacillus

Cells of the Gram-positive soil bacterium Bacillus subtilis are capable of making a choice between different lifestyles, such as the explorative lifestyle that is characterized by motility, the sessile lifestyle (biofilm formation), the expression of genetic competence or sporulation. While the latter lifestyles are characteristic for post-exponential cells, growing cell have to choose between motility and biofilm formation; and the two lifestyles are mutually exclusive. The SinR protein is a transcription factor that controls both lifestyles as a master regulator. Its activity is controlled by inhibitory interactions with its antagonists SinI and SlrR, the expression of SlrR in turn is repressed in a negative feedback loop by SinR. This regulatory system meets the criteria for a bistable switch. We have discovered that a novel phosphodiesterase, YmdB, is required for bistable gene expression and heterogeneity within the population of B. subtilis. With this project, we want to unravel the molecular link between the YmdB and bistable gene expression of biofilm and motility genes in B. subtilis. The project is based on the following hypothesis: The results we have so far are compatible with the idea that YmdB is a phosphodiesterase with an RNase activity. One of the substrates of YmdB activity must be implicated in the translation or stability of the SinR protein. The increased amounts of SinR in the ymdB mutant result in an imbalance in the concentrations of SinR and its antagonists, SinI and SlrR. This is accompanied by permanent repression of biofilm genes and constitutive expression of motility genes of the SigD regulon. To study the effect of YmdB on the bistable switch that is made up of SinR and its antagonist proteins we will determine the absolute quantities of these proteins in populations and in specific sub-populations that have been sorted according to their mode of biofilm and motility gene expression. Moreover, we will study a SinR mutant protein that restores bistable gene expression to the ymdB mutant by biochemical and structural analysis. The data obtained from both approaches will feed into the modelling of the bistable switch. To study the molecular mechanism by which YmdB affects bistable gene expression, we will first determine the stabilities of the SinR protein and of the sinR mRNA in the wild type and the ymdB mutant. Moreover, RNA-Seq data will be evaluated to get insights into the target of YmdB at single nucleotide resolution. From the RNA-Seq and the proteome analysis, we expect to be able to identify the direct target of YmdB. Finally, we aim at studying the molecular details of the interaction between YmdB and its target molecule. We are confident that the proposed program will allow us to identify the molecular mechanism for the implication of YmdB in phenotypic heterogeneity and in the switch between biofilm formation and motility in B. subtilis.

革兰氏阳性土壤细菌枯草芽孢杆菌的细胞能够在不同的生活方式之间做出选择，例如以运动性为特征的探索生活方式，无根生活方式（生物膜形成），表达遗传能力或产孢。后一种生活方式是后指数细胞的特征，生长细胞必须在运动和生物膜形成之间做出选择；这两种生活方式是相互排斥的。SinR蛋白是一种转录因子，作为主要调节器控制两种生活方式。其活性受其拮抗剂SinI和SlrR的抑制相互作用控制，SlrR的表达反过来又被SinR在负反馈回路中抑制。这个调节系统符合双稳态开关的标准。我们发现一种新的磷酸二酯酶YmdB是枯草芽孢杆菌群体内双稳态基因表达和异质性所必需的。通过本项目，我们希望揭示枯草芽孢杆菌中YmdB与生物膜和运动基因双稳态基因表达之间的分子联系。该项目基于以下假设：我们目前的结果与YmdB是一种具有rna酶活性的磷酸二酯酶的想法是一致的。YmdB活性的底物之一必须与SinR蛋白的翻译或稳定性有关。ymdB突变体中SinR量的增加导致SinR及其拮抗剂SinI和SlrR浓度的不平衡。这伴随着生物膜基因的永久抑制和SigD调控的运动基因的组成性表达。为了研究YmdB对由SinR及其拮抗剂蛋白组成的双稳态开关的影响，我们将根据它们的生物膜模式和运动基因表达来确定这些蛋白在种群和特定亚种群中的绝对数量。此外，我们将通过生化和结构分析来研究一种能够恢复ymdB突变体双稳态基因表达的SinR突变蛋白。从这两种方法获得的数据将用于双稳态开关的建模。为了研究YmdB影响双稳态基因表达的分子机制，我们将首先确定野生型和YmdB突变体中SinR蛋白和SinR mRNA的稳定性。此外，将对RNA-Seq数据进行评估，以在单核苷酸分辨率下深入了解YmdB的靶标。通过RNA-Seq和蛋白质组分析，我们希望能够确定YmdB的直接靶点。最后，我们旨在研究YmdB与其靶分子相互作用的分子细节。我们相信，所提出的程序将使我们能够确定YmdB在枯草芽孢杆菌表型异质性和生物膜形成和运动之间切换的分子机制。