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.