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

项目总结 许多接合质粒携带抗生素耐药基因，并可在远距离转移 细菌出现的频率很高。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，其中几个还 携带超广谱内酰胺酶和碳青霉烯酶基因。这种可移动的电阻器构成了一种 细菌感染治疗的全球性威胁，但对质粒的调节知之甚少 转移是这一威胁的基础。成功的DNA转移需要生产和组装 接合机制的许多组件，它们被组织成15-30 kb的长操纵子。 这些操纵子的表达，特别是在异源宿主中的表达，会带来巨大的适应成本； 基因组编码的H-NS和其他核样相关蛋白(NAP)沉默 外源基因，是维持某些质粒所必需的。必须解除沉默 接合作用发生，以及干扰H-NS与启动子和 已经描述了使能转录起始。然而，长时间异种基因的表达 操纵子在伸长过程中经常被终止因子Rho沉默，Rho作用于 与H-NS和延伸因子NusG一致，并由操纵子特定的NusG反沉默 并列动物。许多接合质粒编码H-NS和NusG同源物，这是我们提出的 包括DNA转移的关断开关。我们将使用R6K，一种来自 为了测试这一想法，我们已经知道R6K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 它的靶点，并探讨H-NX的募集机制。在目标2中，我们将测试 ACTx，编码在R6K转移操纵子内的NusG准对数，激活共轭和 促进RNA的合成。我们推测在临床上发现的H-NX/ACTx对 质粒共同进化，以确保其常驻质粒的成功传播，以及 相关的耐药性决定因素，通过细菌种群。

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.