ABSTRACTRho is a hexameric molecular motor that functions as a conserved transcription terminator in the majority of bacterial species and is a potential drug target. Psu is a bacteriophage P4 capsid protein that inhibitsEscherichia coliRho by obstructing its ATPase and translocase activities. In this study, we explored the anti-Rho activity of Psu for Rho proteins from different pathogens. Sequence alignment and homology modeling of Rho proteins from pathogenic bacteria revealed the conserved nature of the Psu-interacting regions in all these proteins. We chose Rho proteins from various pathogens, includingMycobacterium smegmatis,Mycobacterium bovis,Mycobacterium tuberculosis,Xanthomonas campestris,Xanthomonas oryzae,Corynebacterium glutamicum,Vibrio cholerae,Salmonella enterica, andPseudomonas syringae. The purified recombinant Rho proteins of these organisms showed variable rates of ATP hydrolysis on poly(rC) as the substrate and were capable of releasing RNA from theE. colitranscription elongation complexes. Psu was capable of inhibiting these two functions of all these Rho proteins.In vivopulldown assays revealed direct binding of Psu with many of these Rho proteins.In vivoexpression ofpsuinduced killing ofM. smegmatis,M. bovis,X. campestris, andE. coliexpressingS. entericaRho indicating Psu-induced inhibition of Rho proteins of these strains under physiological conditions. We propose that the “universal” inhibitory function of the Psu protein against the Rho proteins from both Gram-negative and Gram-positive bacteria could be useful for designing peptides with antimicrobial functions and that these peptides could contribute to synergistic antibiotic treatment of the pathogens by compromising the Rho functions.IMPORTANCEBacteriophage-derived protein factors modulating different bacterial processes could be converted into unique antimicrobial agents. Bacteriophage P4 capsid protein Psu is an inhibitor of theE. colitranscription terminator Rho. Here we show that apart from antagonizingE. coliRho, Psu is able to inhibit Rho proteins from various phylogenetically unrelated Gram-negative and Gram-positive pathogens. Upon binding to these Rho proteins, Psu inhibited them by affecting their ATPase and RNA release functions. The expression of Psuin vivokills various pathogens, such asMycobacteriumandXanthomonasspecies. Hence, Psu could be useful for identifying peptide sequences with anti-Rho activities and might constitute part of synergistic antibiotic treatment against pathogens.
The moonlighting function of bacteriophage P4 capsid protein, Psu, as a transcription antiterminator