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，例如以运动性为特征的探索性生活方式、固着生活方式（生物膜形成）、遗传能力的表达或孢子形成。虽然后一种生活方式是指后细胞的特征，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，作为主调节器控制两种生活方式。它的活性是通过与其拮抗剂SinI和SlrR的抑制性相互作用来控制的，SlrR的表达反过来又在负反馈回路中被SinR抑制。该调节系统符合开关的标准。我们发现一种新的磷酸二酯酶YmdB是B群体内的hepatocellular基因表达和异质性所必需的。枯草杆菌。通过这个项目，我们希望解开B中生物膜和运动基因的YmdB和YmdB基因表达之间的分子联系。枯草杆菌。该项目基于以下假设：我们迄今为止的结果与YmdB是具有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涉及SinR蛋白的翻译或稳定性。ymdB突变体中SinR数量的增加导致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调节子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生物膜开关的影响，我们将确定这些蛋白在群体和特定亚群中的绝对量，这些亚群已根据其生物膜和运动基因表达模式进行分类。此外，我们将研究一种SinR突变蛋白，通过生物化学和结构分析恢复ymdB突变体的p53基因表达。从这两种方法中获得的数据将输入到开关的建模中。为了研究YmdB影响P2P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，将评估RNA-Seq数据，以了解单核苷酸分辨率下YmdB的靶点。通过RNA-Seq和蛋白质组分析，我们期望能够鉴定YmdB的直接靶点。最后，我们旨在研究YmdB与其靶分子之间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在表型异质性和生物膜形成和B中运动性之间的转换中的分子机制。枯草杆菌。

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.