Cross-resistance of Escherichia coli RNA polymerases conferring rifampin resistance to different antibiotics

Abstract

In this study we further defined the rifampin-binding sites in Escherichia coli RNA polymerase (RNAP) and determined the relationship between rifampin-binding sites and the binding sites of other antibiotics, including two rifamycin derivatives, rifabutin and rifapentine, and streptolydigin and sorangicin A, which are unrelated to rifampin, using a purified in vitro system. We found that there is almost a complete correlation between resistance to rifampin (Rifr) and reduced rifampin binding to 12 RNAPs purified from different rpoB Rifr mutants and a complete cross-resistance among the different rifamycin derivatives. Most Rifr RNAPs were sensitive to streptolydigin, although some exhibited weak resistance to this antibiotic. However, 5 out of the 12 Rifr RNAPs were partially resistant to sorangicin A, and one was completely cross-resistant to sorangicin A, indicating that the binding site(s) for these two antibiotics overlaps. Both rifampin and sorangicin A inhibited the transition step between transcription initiation and elongation; however, longer abortive initiation products were produced in the presence of the latter, indicating that the binding site for sorangicin A is within the rifampin-binding site. Competition experiments of different antibiotics with 3H-labeled rifampin for binding to wild-type RNAP further confirmed that the binding sites for rifampin, rifabutin, rifapentine, and sorangicin A are shared, whereas the binding sites for rifampin and streptolydigin are distinct. Because Rifr mutations are highly conserved in eubacteria, our results indicate that this set of Rif r mutant RNAPs can be used to screen for new antibiotics that will inhibit the growth of Rifr pathogenic bacteria. Copyright © 2005, American Society for Microbiology. All Rights Reserved.