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

原核生物复制、细胞周期、基因表达和水平基因转移的机制和调控，重点关注枯草芽孢杆菌

• 批准号: 10792219
• 负责人: ALAN D GROSSMAN
• 金额: $ 15.05万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2027-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10792219
• 关键词: Affect; Antibiotic Resistance; Bacillus subtilis; Bacteria; Bacterial Genome; Bacteriophages; Biological Assay; Biology; Cell Cycle; Cells; Cellular Stress; Chromosomes; DNA Damage; DNA biosynthesis; Development; Elements; Environment; Event; Evolution; Fluorescence; Gene Expression; Gene Expression Profiling; Gene Transfer; Genes; Genetic Recombination; Genome; Gram-Positive Bacteria; Growth; Horizontal Gene Transfer; Human Microbiome; Individual; Life Cycle Stages; Location; Measurement; Mediating; Metabolic; Microscopy; Mobile Genetic Elements; Organism; Phenotype; Plasmids; Population; Prevalence; Process; Prokaryotic Cells; Proteins; Regulation; Stress; Symbiosis; Virulence; Virus; Visualization; Work; biological adaptation to stress; driving force; fitness; microbial; repaired; response; single cell analysis; tool; trait

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.

项目概要/摘要 水平基因转移（HGT）是微生物进化的驱动力。它主要是由移动遗传介导的 元件，包括病毒、接合质粒以及整合和接合元件（ICE；又名接合元件） 转座子），许多细菌基因组含有多种可移动遗传元件，包括 ICE 和温带噬菌体。 接合元件是众所周知的试剂，有助于抗生素抗性、毒力、 共生、代谢功能等等。 ICE 首次被发现是因为它们赋予了其中一些表型。 然而，绝大多数 ICE 赋予细菌的潜在表型尚不清楚。现在很明显，ICE 可以赋予有益的表型，远远超出一些最初表征的 ICE，并且一些 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，会导致宿主细菌生长停滞。 尽管 ICE 很普遍且很重要，但我们对这些要素的理解仍存在重大缺陷， 尤其是革兰氏阳性菌。值得注意的是，人们对 ICE 与其宿主细胞之间的相互作用知之甚少 包括共存病毒，以及 ICE 对细菌宿主健康的影响，特别是在机械方面 等级。此外，人们对 ICE 编码的功能与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 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表达在大量的基因组中。 群体中的大多数细胞。然而，一小部分细胞（通常约为 1% 或更少）含有活性 ICE 即表达接合所需的基因。对大量人群的检测通常不足以检测效果 在少量具有活跃 ICE 的细胞上。相反，对活性中的单个细胞进行分析和测量 需要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 可用于监测基因表达以及修复和重组蛋白的细胞位置变化 对由移动遗传元件、DNA 损伤（和其他扰动）引起的压力的反应。 我们的工作将继续关注革兰氏阳性菌的精选 ICE 的生命周期，包括 ICEBs1 和 Tn916 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或 ICE 生命周期的基本问题及其对宿主细胞的影响尚未被研究。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 和 噬菌体，特别是这些过程如何影响移动遗传元件的生命周期以及移动遗传元件如何 影响这些过程。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（包括人类微生物组）中生长的细菌之间进行基因转移。