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

项目摘要/摘要 水平基因转移是微生物进化的驱动力。它在很大程度上是由移动基因介导的 分子，包括病毒、接合质粒、整合和接合分子(ICE；又称接合 转座子)，许多细菌基因组包含几个可移动的遗传元件，包括冰和温带噬菌体。 结合素是众所周知的促进抗生素耐药性、毒力、 共生、新陈代谢功能等等。冰之所以被首先发现，是因为它们赋予了这些表型中的一些。 然而，绝大多数冰块赋予细菌的潜在表型尚不清楚。现在很明显，冰 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冰的表型，而且一些 这些表型包括冰和细菌病毒之间的功能相互作用。同样明显的是，一些冰， 当被激活时，会导致宿主细菌的生长停滞。 尽管冰的普遍性和重要性，但我们对这些要素的理解存在重大缺陷， 尤其是在革兰氏阳性细菌中。值得注意的是，人们对冰与其宿主细胞之间的相互作用知之甚少。 包括共存的病毒，以及冰对它们的细菌宿主的适应性的影响，特别是在机械上 水平。此外，关于由ICE编码的功能和由主机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冰在不同物种中发挥作用的宿主范围和效率。 ICE通常存在于宿主基因组中，并且传递功能通常不会在大量的 一个群体中的大多数细胞。然而，一小部分细胞(通常为~1%或更少)包含活性ICE 这就是表达接合所需的基因。对总体人群的分析通常不足以检测效果 在具有活动ICE的少数细胞上。取而代之的是，对活跃的 需要亚群来确定由激活的ICE引起的表型。通过以下方式可视化这些细胞 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 用于监测基因表达和修复和重组蛋白在细胞内位置的变化 对移动遗传因素、DNA损伤(和其他扰动)造成的压力的反应。 我们的工作将继续集中在精选冰的生命周期上，包括来自革兰氏阳性的ICEBs1和Tn916 细菌。在单个细胞和小亚群中可视化事件将使我们能够回答以前困难的或 对ICE生命周期及其对宿主细胞的影响的未研究的基本问题。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冰川和 噬菌体，特别是这些过程如何影响可移动遗传元件的生命周期，以及可移动遗传元件如何 影响这些进程。我们的发现应该与许多细菌物种的生物学相关，特别是关于 在不同环境中生长的细菌之间的基因转移，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.