Role of RNA polymerase in bacterial differentiation

描述（由申请人提供）：该项目的目标是揭示枯草芽孢杆菌孢子形成和基质产生的发育程序的基因控制机制，并区分它们。孢子的形成传统上被认为是自由活细胞的行为，但我们现在了解到分化也发生在结构的背景下，多细胞群落（生物膜）由基质生成细胞链和孢子形成细胞组成。事实上，控制进入孢子形成的机制与控制基质产生的机制密切交织在一起。这一建议解决了我们对孢子形成和多细胞化的理解中的重要空白，有四个具体目标：(1)我们将通过两种传感器组氨酸激酶识别触发孢子形成和多细胞化的自然环境信号。(2)我们将使用一种新设计的微流体装置实时观察细胞命运在浮游状态和基质产生状态之间的转换。我们将确定双负反馈回路如何控制开关，以及细胞如何区分孢子形成和基质产生的不同命运。(3)我们将确定d -氨基酸在生物膜生命周期后期是如何产生的，以及它们是如何触发生物膜分解的。我们将确定控制负责d-氨基酸产生的消旋酶基因表达的调控机制，d-氨基酸如何被纳入肽聚糖以及它们如何触发基质淀粉样纤维成分的释放。(4)我们将确定控制细胞分化为孢子的调控事件的完整级联，包括控制从一个sigma因子转换到另一个sigma因子的机制。了解d -氨基酸如何导致生物膜解体将为阻断致病菌形成生物膜的策略提供信息。此外，革兰氏阳性菌的主要模式生物枯草芽孢杆菌的基因控制研究已经并将继续为相关致病菌（如葡萄球菌、肠球菌和炭疽芽孢杆菌）的分子生物学提供基本见解。

DESCRIPTION (provided by applicant): The objectives of the project are to uncover the mechanisms of gene control that turn on, and distinguish between, developmental programs of spore formation and matrix production by Bacillus subtilis. Spore formation has been traditionally viewed as a behavior of free-living cells, but we now understand that differentiation also occurs in the context of structured, multicellular communities (biofilms) consisting of chains of matrix-producing cells as well as spore-forming cells. Indeed, the mechanisms that govern entry into sporulation are intimately interwoven with those that govern matrix production. This proposal addresses important gaps in our understanding of sporulation and multicellularity with four specific aims: (1) We will identify the natural environmental signals that trigger spore formation and multicellularity by two sensor histidine kinases. (2) We will visualize cell fate switching between planktonic and matrix-producing states in real time using a newly devised microfluidic device. We will determine how a double-negative feedback loop controls switching and how cells discriminate between alternative fates of spore formation and matrix production. (3) We will determine how D-amino acids are produced late in the life cycle of the biofilm and how they trigger biofilm disassembly. We will determine the regulatory mechanisms that control the expression of the racemase genes that are responsible for D-amino acid production, how D-amino acids are incorporated into the peptidoglycan and how they trigger the release of an amyloid-fiber component of the matrix. (4) We will determine the full cascade of regulatory events that govern the differentiation of a cell into a spore, including the mechanisms that govern switching from one sigma factor to another. Understanding how D-amino acids cause biofilm disassembly will inform strategies for blocking biofilm formation by pathogenic bacteria. Also, research into gene control by B. subtilis, the principal model organism for Gram-positive bacteria, has provided, and will continue to provide, fundamental insights into the molecular biology of related, pathogenic bacteria, such as Staphylococcus, Enterococcus and B. anthracis.