SUMMARY Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that causes acute and chronic pneumonia, sepsis, urinary tract infections, and surgical-site infections. Its success as a human pathogen is due in large part to its ability to form biofilms, which confer resistance to the host immune system, antibiotics, and other stresses. Biofilms are structured communities held together by an extracellular matrix composed primarily of exopolysaccharides (EPS) encoded in long operons. As all RNA polymerases are prone to premature termination, the expression of long operons commonly relies on dedicated antitermination factors that belong to the universally conserved family of NusG transcription regulators. NusG paralogs bind to the elongating RNA polymerase and enable uninterrupted synthesis of long RNAs in many bacteria, including Bacillus velezensis, Bacteroidetes fragilis, and Escherichia coli. Surprisingly, P. aeruginosa does not encode NusG paralogs, and the scarce available data suggest that its regulation of transcription termination and antitermination is very different from that of E. coli. We hypothesize that many extended P. aeruginosa operons are subject to premature termination by Rho, possibly with the assistance of NusG and histone-like Mva proteins, and that they use unique antitermination mechanisms to ensure complete synthesis of their multi-cistronic RNAs. We propose to investigate the regulation of P. aeruginosa RNA chain elongation, focusing on two EPS operons, pel and psl. This choice is dictated by outsized contributions of these gene clusters to P. aeruginosa pathogenesis and their expected dependence on antitermination to complete the synthesis of 10-19 kb RNA chains. In Aim 1, we will determine if Rho, NusG, and Mva proteins induce premature termination during transcription of the pel and psl operons using in vivo RNA probing and in vitro assays with purified P. aeruginosa RNA polymerase and transcription factors. In Aim 2, we will ident