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

项目概要 许多接合质粒携带抗生素抗性基因，并且可以在远距离转移 bacteria at high frequencies.已发现携带粘菌素抗性 mcr 等位基因的质粒 五大洲的农业、环境和临床样本，其中几个还 携带广谱内酰胺酶和碳青霉烯酶基因。 This mobile resistome poses a 细菌感染治疗面临全球威胁，但人们对质粒的调控知之甚少 transfer that underlies this threat.成功的 DNA 转移需要生产和组装 接合机制的许多组件，被组织成长 15-30 kb 的操纵子。 这些操纵子的表达，特别是在异源宿主中的表达，会带来巨大的适应性成本； 基因组编码的 H-NS 和其他类核相关蛋白 (NAP) 的沉默表达 外源基因，并且是维持某些质粒所必需的。 Silencing must be relieved for 缀合的发生，以及干扰 H-NS 与启动子结合的反沉默剂 已经描述了启用转录起始。 However, the expression of long xenogeneic 操纵子在延伸过程中经常被终止因子 Rho 沉默，该因子作用于 与 H-NS 和延伸因子 NusG 协同作用，并被操纵子特异性 NusG 反沉默 旁系同源物。许多接合质粒编码 H-NS 和 NusG 同源物，我们建议 comprise an off/on switch of DNA transfer.我们将使用 R6K，一种模型接合质粒 Escherichia coli, to test this idea;我们已经知道 R6K 编码的 H-NX 会抑制缀合。 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 其靶位点，并研究 H-NX 招募机制。 In Aim 2, we will test whether ActX 是 R6K 转移操纵子内编码的 NusG 旁系同源物，可激活缀合并 promotes processive RNA synthesis.我们假设临床上发现的 H-NX/ActX 对 质粒共同进化以确保其驻留质粒的成功传播，并且 通过细菌群体相关的耐药性决定因素。

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.