Mechanisms and regulation of horizontal gene transfer by natural transformation in Vibrio cholerae

项目摘要 达利亚实验室研究自然条件下水平基因转移的调节和机制。 以霍乱弧菌为模型系统进行转化(NT)。而这种细菌是引起 对于腹泻病霍乱，我们不寻求研究细菌的发病机制。相反，我们很好地利用了- 已建立的模型系统和我们开发的用于表征NT在生理上的遗传工具 相关背景。NT有助于抗生素耐药性决定因素和毒力因素的快速传播 在细菌病原体中。因此，刻画NT的调节和机制可能会发现新的 与各种临床相关感染作斗争的方法。 NT所需的基因受到严格调控，只有在霍乱弧菌形成生物膜时才能诱导 甲壳类浮游动物在水环境中的几丁质壳。甲壳素生物膜的形成是 对这种兼性病原体在其环境水库中的生存也很重要，并促进 霍乱通过水路传播。弧菌-甲壳素的相互作用可以在实验室环境中很容易地研究，这 为研究NT提供了一个具有生理相关性和高度易操纵性的模型系统。在接下来的五年， 我们将重点抓好三大领域。首先，霍乱弧菌使用称为IV型菌毛的动态表面附属物来 结合到几丁质表面，并为NT摄取DNA。调节动态延伸的机制 然而，这些附属物的回缩特征仍然很差。使用我们帮助开发的工具 在活细胞中标记IV型菌毛，我们将阐述运动ATPase、次要菌毛蛋白和其他环境线索是如何 调节菌毛的动态活动。第二，我们将研究两种膜嵌入的DNA如何结合 转录因子chis和tfos协同激活霍乱弧菌中的几丁质调节子。膜- 嵌入式调节器的研究还不够深入，这项工作将有助于阐明 膜包埋转录因子与可溶性转录因子的DNA结合活性。第三，我们将使用组合 用细胞生物学和遗传学方法研究通过自然转化进行的水平基因转移。也就是说， 我们将讨论甲壳素生物膜内自然转化的时空动力学；以及 邻居对这些动态的捕食。此外，我们还将正式检验长期存在但未经检验的假设 DNA在自然转化过程中的摄取起着重要的营养作用。总之，我们的基础研究 扩展了对许多关键和保守过程(例如PILI、SIGNAL)的基本了解 转导、水平基因转移)，这是不同微生物物种共有的，包括许多病原体。

Project Summary The Dalia lab studies the regulation and mechanisms of horizontal gene transfer by natural transformation (NT) using Vibrio cholerae as a model system. While this bacterium is the causative agent of the diarrheal disease cholera, we do not seek to study bacterial pathogenesis. Instead, we leverage this well- established model system and the genetic tools we have developed to characterize NT in a physiologically relevant context. NT contributes to the rapid spread of antibiotic resistance determinants and virulence factors in bacterial pathogens. Thus, characterizing the regulation and mechanisms of NT may uncover novel approaches to combat diverse clinically relevant infections. The genes required for NT are tightly regulated, and only induced when V. cholerae forms biofilms on the chitinous shells of crustacean zooplankton in the aquatic environment. The formation of chitin biofilms is also important for the survival of this facultative pathogen in its environmental reservoir, and promotes the waterborne transmission of cholera. Vibrio-chitin interactions can be easily studied in a lab setting, which provides a physiologically relevant and highly tractable model system for studying NT. In the next five years, we will focus on three major areas. First, V. cholerae uses dynamic surface appendages called type IV pili to bind to chitinous surfaces and to take up DNA for NT. The mechanisms that regulate the dynamic extension and retraction of these appendages, however, remains poorly characterized. Using tools we helped develop to label type IV pili in live cells, we will address how motor ATPases, minor pilins, and other environmental cues regulate pilus dynamic activity. Second, we will study how two membrane-embedded DNA-binding transcription factors, ChiS and TfoS, coordinate to activate the chitin regulon in V. cholerae. Membrane- embedded regulators are understudied, and this work will help elucidate the dynamics and constraints for the DNA-binding activity of membrane-embedded vs soluble transcription factors. Third, we will use a combination of cell biological and genetic approaches to study horizontal gene transfer by natural transformation. Namely, we will address the spatiotemporal dynamics of natural transformation within chitin biofilms; and the impact of neighbor predation on these dynamics. Also, we will formally test the long-standing, yet untested, hypothesis that DNA uptake during natural transformation plays an important nutritional role. Together, our basic research extends a fundamental understanding of a number of critical and conserved processes (e.g. pili, signal transduction, horizontal gene transfer) that are shared by diverse microbial species, including many pathogens.