ABSTRACT
A comprehensive set of bicyclomycin-resistant mutants of transcription termination protein Rho has been characterized in Escherichia coli by in vivo and in vitro assays. Several of the mutant Rho proteins have functional defects. Strains with either the L208R or the S266A mutation in the bacterial chromosome have a higher intracellular concentration of the Rho protein than strains containing a wild-type copy of the rho gene. Strains carrying the L187R, L208R or S266A mutations in the chromosome also have a mutant phenotype; a plasmid-located arabinose promoter is constitutively de-repressed in these strains. The L208R and S266A mutant strains also have a rate of growth defect. When the S266A mutation is located on a high-copy plasmid, the mutant grows more slowly than a wild-type strain. In contrast to the majority of the bicyclomycin-resistant mutants, these two mutants show clear phenotypic differences from wild-type cells. These differences are also seen in vitro. In vitro transcription termination by RhoL208R and RhoS266A is defective at the lambda tR1 terminator, but can be enhanced by NusG. These functionally defective Rho mutations have been located near the putative catalytic site on a model of Rho based on the F1-ATPase. This indicates that this region of the Rho molecule is crucial for Rho function. The crucial region overlaps the putative bicyclomycin-binding site, suggesting an explanation for the efficacy of bicyclomycin as an antibiotic.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Escherichia coli/genetics , Rho Factor/genetics , Drug Resistance, Microbial , Escherichia coli/growth & development , Escherichia coli/metabolism , Genes, Reporter , Models, Molecular , Mutation , Phenotype , Plasmids , Rho Factor/analysisABSTRACT
A total of 38 bicyclomycin-resistant mutants of Escherichia coli transcription termination protein Rho have been isolated. The locations of their mutations identify the ATP-binding region as the functional domain inhibited by bicyclomycin. Strains containing the S266C, S266A and L208R Rho mutations are very resistant to bicyclomycin in vivo. In a similar way, the mutant Rho proteins containing these mutations are very resistant to bicyclomycin in vitro. These data suggest that Ser266 and Leu208 might make direct contact with the antibiotic. These two residues are close to each other in the tertiary structure of a model of Rho based on the alpha and beta subunits of the F(1) ATPase, supporting the validity of the model. The strain containing the G337S Rho mutation also has high bicyclomycin resistance, and the proximity of L208, S266 and G337 in the quaternary structure of the Rho model has enabled a candidate bicyclomycin-binding pocket to be delineated. As a whole, the bicyclomycin sensitivities of the mutants are consistent with the locations of their respective mutations in the model of Rho based on the F(1) ATPase, therefore supporting the emerging consensus model of Rho structure.
