Transcription termination factor rho: The site of bicyclomycin inhibition in Escherichia coli

Abstract

Bicyclomycin is a novel, commercially important antibiotic. Information concerning the site of bicyclomycin inhibition in Escherichia coli has been obtained by the production of bicyclomycin resistant mutants by UV irradiation. Selection by growth in the presence of bicyclomycin of a plasmid clone library generated from a highly resistant mutant in recipient antibiotic-sensitive host cells (E. coli strain W3350) has led to the characterization of three different plasmids that confer drug resistance, which contained the gene encoding the transcription termination factor, rho. These mutant rho genes contained single base changes at nucleotide positions 656, 796, and 1009. Preliminary mechanistic information has been obtained by monitoring the polyC-dependent ATPase activity of rho in the absence and presence of bicyclomycin and dihydrobicyclomycin. Addition of bicyclomycin to aqueous solutions containing rho and ATP led to a decrease in the release of inorganic phosphate with an I50 value of 60-70 microM bicyclomycin. This inhibition is comparable to the drug concentration needed to inhibit bacterial growth on plates. No loss of activity was observed when a similar concentration of dihydrobicyclomycin was used in place of bicyclomycin, while use of 10-fold higher concentrations of this derivative led to partial rho inhibition. PolyC-dependent ATPase activity from partially purified rho isolated from the mutant BCMr108 was not inhibited by bicyclomycin at concentrations (200 microM) found to completely inhibit wild-type rho. These cumulative findings are consistent with the notion that bicyclomycin expresses its activity by interfering with the polyC-dependent ATPase activity of rho.

Loss of overproduction of polypeptide release factor 3 influences expression of the tryptophanase operon of Escherichia coli

“…The drug bicyclomycin is a known inhibitor of Rho factor (37). Mutants resistant to the drug were isolated and characterized previously (35).…”

Bicyclomycin sensitivity and resistance affect Rho factor-mediated transcription termination in the tna operon of Escherichia coli

“…Zwiefka et al (24) found that the antibiotic bicyclomycin (bicozamycin), an inhibitor of the growth of several gramnegative bacterial species (24), interferes with the functions of Rho factor, a protein largely responsible for factor-dependent transcription termination (15,16). They performed in vitro analyses demonstrating that bicyclomycin inhibited the poly (C)-stimulated ATPase activity of Escherichia coli Rho factor (24).…”

“…They performed in vitro analyses demonstrating that bicyclomycin inhibited the poly (C)-stimulated ATPase activity of Escherichia coli Rho factor (24). They also isolated bicyclomycin-resistant mutants and showed that these mutants had alterations of Rho factor that conferred resistance to the drug both in vivo and in vitro (24).…”

“…They performed in vitro analyses demonstrating that bicyclomycin inhibited the poly (C)-stimulated ATPase activity of Escherichia coli Rho factor (24). They also isolated bicyclomycin-resistant mutants and showed that these mutants had alterations of Rho factor that conferred resistance to the drug both in vivo and in vitro (24). In other studies of bicyclomycin resistance in E. coli, a gene that conferred resistance to the drug when it was present on a multicopy plasmid was identified (2).…”

Bacterial Transcription as a Target for Antibacterial Drug Development

“…While Rho is essential for viability in many Gram-negative organisms, Gram-positive organisms appear to be less dependent on it, although it is important in preventing transcription of noncoding RNA in the latter and has activity against Micrococcus luteus (165,166). Bicyclomycin is the only known antibiotic to target Rho, and it does not affect RNA or ATP binding to Rho but does inhibit Rho-dependent transcription termination and ATP hydrolysis (167)(168)(169). Substitutions in E. coli Rho residues L208, M219, S266, and G337 conferred resistance to bicyclomycin, suggesting that Rho was the binding target of bicyclomycin (170,171).…”