Mechanisms and regulation of replication, the cell cycle, gene expression, and horizontal gene transfer in prokaryotes, focusing on Bacillus subtilis

项目总结/摘要 水平基因转移（Horizontal gene transfer，HGT）是微生物进化的重要驱动力。它在很大程度上是由移动的遗传 元件，包括病毒、接合质粒、整合和接合元件（ICE; aka conjugative 转座子），并且许多细菌基因组含有几个移动的遗传元件，包括ICE和温带细菌。 接合元件是公知的因子，其有助于抗生素抗性、毒力、 共生、代谢功能等等。ICE最初被发现是因为它们赋予了这些表型中的一些。 然而，绝大多数ICE赋予细菌的潜在表型尚不清楚。现在很清楚，ICE 可以赋予有益的表型，这些表型远远超出了一些最初表征的ICE的表型， 这些表型涉及ICE和细菌病毒之间的功能相互作用。同样明显的是，一些ICE， 当被激活时，会导致宿主细菌的生长停滞。 尽管ICE的普遍性和重要性，但我们对这些元素的理解存在重大缺陷， 尤其是革兰氏阳性菌。值得注意的是，对ICE与其宿主细胞之间的相互作用知之甚少 包括与共存的病毒，以及ICE对其细菌宿主的适应性的影响，特别是在一种机制上， 水平此外，人们对ICE编码的功能和宿主编码的功能之间的相互作用知之甚少， 以及这些相互作用如何影响ICE在不同物种中发挥作用的宿主范围和效率。 ICE通常整合在宿主基因组中，并且转移功能通常不在大量的细胞中表达。 一个群体中的大多数细胞。然而，一小部分细胞亚群（通常约1%或更少）含有活性ICE。 表达接合所需的基因。对大量种群的分析往往不足以检测效应 在少数有ICE的细胞上。相反，对活性细胞中的单个细胞的测定和测量， 亚群来确定由激活的ICE引起的表型。这些细胞的可视化， 荧光和延时显微镜是这些分析的关键工具。这些类型的单细胞分析也是 可用于监测基因表达以及修复和重组蛋白在细胞中位置的变化， 对由移动的遗传因素、DNA损伤（和其他扰动）引起的应激的反应。 我们的工作将继续集中在选择ICE的生命周期，包括ICEBs 1和Tn 916，从革兰氏阳性 细菌可视化单细胞和小亚群中的事件将使我们能够回答以前困难的问题， ICE生命周期及其对宿主细胞的影响的基础性未研究的问题。我们在染色体方面的专业知识 动力学、DNA复制、应激反应和微生物发育与我们对ICE的研究很好地吻合， 特别是这些过程如何影响移动的遗传因子的生命周期，以及移动的遗传因子 影响这些进程。我们的发现应该与许多细菌物种的生物学有关，特别是关于 在不同环境中生长的细菌之间转移基因，包括人类微生物组。

Project Summary/Abstract Horizontal gene transfer (HGT) is a driving force in microbial evolution. It is largely mediated by mobile genetic elements, including viruses, conjugative plasmids, and integrative and conjugative elements (ICEs; aka conjugative transposons), and many bacterial genomes contain several mobile genetic elements, including ICEs and temperate phages. Conjugative elements are well known agents that contribute to the spread of genes for antibiotic resistances, virulence, symbiosis, metabolic functions, and more. ICEs were first discovered because they confer some of these phenotypes. However, potential phenotypes conferred to bacteria by the vast majority of ICEs are not known. It is now clear that ICEs can confer beneficial phenotypes that extend well beyond those of some of the initially characterized ICEs, and that some of these phenotypes involve functional interactions between ICEs and bacterial viruses. It is also apparent that some ICEs, when activated, cause growth arrest of their host bacteria. Despite the prevalence and importance of ICEs, there are major deficiencies in our understanding of these elements, especially in Gram-positive bacteria. Notably, little is known about the interactions between ICEs and their host cells including with co-resident viruses, and the effects ICEs have on fitness of their bacterial hosts, especially at a mechanistic level. Furthermore, little is known about the interactions between functions encoded by ICEs and those encoded by hosts, and how these interactions influence the host range and efficiencies with which ICEs function in different species. ICEs typically reside integrated in the host genome, and the transfer functions are generally not expressed in the vast majority of cells in a population. However, a small subpopulation of cells (typically ~1% or less) contain an active ICE that is expressing genes needed for conjugation. Assays of bulk populations are often not sufficient for detecting effects on the small number of cells with an active ICE. Instead, assays and measurements of individual cells in the active subpopulation are required to determine phenotypes caused by an activated ICE. Visualization of these cells by fluorescence and time-lapse microscopy is a critical tool for these analyses. These types of single-cell analyses are also useful for monitoring gene expression and the change in the cellular location of repair and recombination proteins in response to stresses caused by mobile genetic elements, DNA damage (and other perturbations). Our work will continue to focus on the lifecycle of select ICEs, including ICEBs1 and Tn916, from Gram-positive bacteria. Visualizing events in single cells and small subpopulations will allow us to answer previously difficult or unstudied problems fundamental to the ICE lifecycle and their effects on host cells. Our expertise in chromosome dynamics, DNA replication, stress responses, and microbial development dovetails nicely with our studies of ICEs and phages, notably how these processes affect the lifecycles of mobile genetic elements and how mobile genetic elements affect these processes. Our findings should be relevant to the biology of many bacterial species, especially regarding the transfer of genes between bacteria growing in different environments, including the human microbiome.