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

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，如以运动性为特征的探索性生活方式、静止的生活方式(生物膜形成)、遗传能力的表达或产孢量。虽然后一种生活方式是指数后细胞的特征，但生长中的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，作为主调节因子控制着这两种生活方式。它的活性受其拮抗剂SINI和SLrR的抑制作用控制，而SLRR的表达又被SINR在负反馈环中抑制。这个监管系统符合双稳开关的标准。我们发现枯草杆菌种群内的双稳基因表达和异质性需要一种新的磷酸二酯酶YmDB。通过这个项目，我们想要解开YmdB与枯草杆菌生物膜双稳基因表达和运动基因之间的分子联系。该项目基于以下假设：到目前为止，我们得到的结果与YmdB是一种具有核糖核酸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。突变株中SINR含量的增加导致SINR及其拮抗剂SINI和SLRR的浓度失衡。伴随而来的是生物膜基因的永久抑制和SigD调节子运动基因的结构性表达。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，我们将测定这些蛋白在种群中的绝对数量，并根据它们的生物被膜和运动基因的表达方式对特定的亚种群进行分类。此外，我们还将通过生化和结构分析，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提供给双稳开关的建模。为了研究YmdB影响双稳基因表达的分子机制，我们将首先确定sinR蛋白和sinR在野生型和ymdB突变体中的稳定性。此外，将对RNA-Seq数据进行评估，以深入了解单核苷酸分辨率下YmdB的目标。从RNA-Seq和蛋白质组分析中，我们有望识别YmdB的直接靶标。最后，我们的目标是研究YmdB与其靶分子相互作用的分子细节。我们相信，拟议的程序将使我们能够确定YmdB在枯草杆菌表型异质性和生物膜形成和运动性之间切换的分子机制。

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.