Post-initiation control of conjugation by plasmid-encoded H-NS and NusG homologs

通过质粒编码的 H-NS 和 NusG 同源物进行缀合的启动后控制

• 批准号: 10301108
• 负责人: IRINA ARTSIMOVITCH
• 金额: $ 22.21万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-09 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10301108
• 关键词: Agriculture; Alleles; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Adhesins; Bacterial Infections; Binding; Carbapenems; Cell Wall; Centers for Disease Control and Prevention (U.S.); Clinical; Colistin; Collection; Complex; DNA; DNA-Directed RNA Polymerase; Data; Distant; Elements; Elongation Factor; Enterobacteriaceae; Escherichia coli; Evolution; F Factor; Family; Food; Frequencies; Gene Expression Regulation; Gene Silencing; Genes; Genome; Histones; Homologous Gene; Horizontal Gene Transfer; Hospitals; Human; In Vitro; Infection; Klebsiella pneumoniae; Lactamase; Learning; Maintenance; Mediating; Modeling; Operon; Pathogenicity Island; Plasmids; Polymyxins; Population; Prevalence; Process; Production; Prophages; Proteins; RNA; RNA chemical synthesis; Regulation; Reporting; Resistance; Resort; Rho Factor; Risk Factors; Sampling; Shapes; Site; Source; Structural Protein; Structure; Testing; Toxin; Transcription Elongation; Transcription Initiation; Treatment outcome; Virulence; Work; antibiotic resistant infections; capsule; carbapenemase; colistin resistance; cost; crosslink; exhaust; fitness; helicase; human pathogen; in vivo; insight; interest; member; mutant; novel; paralogous gene; premature; promoter; recruit; resistance gene; rho; termination factor; transcription factor; transcription factor S-II; transcription termination

PROJECT SUMMARY Many conjugative plasmids carry antibiotic-resistance genes and can transfer between distant bacteria at high frequencies. Plasmids carrying colistin-resistance mcr alleles have been found on five continents in agricultural, environmental, and clinical samples, and several of these also carry extended-spectrum-lactamase and carbapenemase genes. This mobile resistome poses a global threat to treatment of bacterial infections, but little is known about the regulation of plasmid transfer that underlies this threat. Successful DNA transfer requires production and assembly of many components of the conjugation machinery, which are organized into long 15-30 kb operons. Expression of these operons, particularly in a heterologous host, imposes significant fitness costs; genome-encoded H-NS and other nucleoid-associated proteins (NAPs) silence expression of foreign genes and are required for maintenance of some plasmids. Silencing must be relieved for conjugation to happen, and counter-silencers that interfere with H-NS binding to promoters and enable transcription initiation have been described. However, the expression of long xenogeneic operons is frequently silenced during elongation by the termination factor Rho, which acts in concert with H-NS and the elongation factor NusG, and counter-silenced by operon-specific NusG paralogs. Many conjugative plasmids encode H-NS and NusG homologs, which we propose comprise an off/on switch of DNA transfer. We will use R6K, a model conjugative plasmid from Escherichia coli, to test this idea; we already know that R6K-encoded H-NX inhibits conjugation. In Aim 1, we will test the effects of cellular NAPs, NusG and Rho on silencing by H-NX, determine its target sites, and investigate the mechanism of H-NX recruitment. In Aim 2, we will test whether ActX, a NusG paralog encoded within the R6K transfer operon, activates conjugation and promotes processive RNA synthesis. We hypothesize that H-NX/ActX pairs found on clinical plasmids have co-evolved to assure successful dissemination of their resident plasmids, and associated resistance determinants, through bacterial populations.

项目摘要 许多接合性质粒携带抗病基因，并能在远缘种间转移， 高频率的细菌。已发现携带粘菌素抗性mcr等位基因的质粒 在五大洲的农业，环境和临床样本中，其中一些还 携带超广谱内酰胺酶和碳青霉烯酶基因。这种移动的阻力构成了 对细菌感染治疗的全球性威胁，但对质粒的调控知之甚少 这一威胁背后的转移。成功的DNA转移需要生产和组装 接合机制的许多组分，其被组织成长的15-30 kb操纵子。 这些操纵子的表达，特别是在异源宿主中的表达，造成显著的适应性成本; 基因组编码的H-NS和其他核相关蛋白（NAPs）沉默表达 外源基因，并且是维持某些质粒所必需的。必须解除沉默， 和干扰H-NS与启动子结合的反沉默剂， 已经描述了启动转录。然而，长异种的表达 操纵子在延伸过程中经常被终止因子Rho沉默， 与H-NS和延伸因子NusG一致，并被操纵子特异性NusG反沉默 旁系亲属许多接合质粒编码H-NS和NusG同源物，我们提出， 包括DNA转移的开关。我们将使用R6 K，一种来自 我们已经知道R6 K编码的H-NX抑制接合。 在目标1中，我们将测试细胞NAP、NusG和Rho对H-NX沉默的影响，确定细胞NAP、NusG和Rho对H-NX沉默的影响。 其靶位点，并研究H-NX募集的机制。在目标2中，我们将测试 ActX是在R6 K转移操纵子内编码的NusG parasites，其激活缀合并 促进RNA合成。我们假设临床上发现的H-NX/ActX对 质粒共同进化以确保其固有质粒的成功传播， 相关的耐药决定因素，通过细菌种群。