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

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 (Rif(r)) and reduced rifampin binding to 12 RNAPs purified from different rpoB Rifr mutants and a complete cross-resistance among the different rifamycin derivatives. Most Rif(r) RNAPs were sensitive to streptolydigin, although some exhibited weak resistance to this antibiotic. However, 5 out of the 12 Rif(r) 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 3 H-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 Rif(r) 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 Rif(r) pathogenic bacteria.