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Cross-Resistance of Escherichia coli RNA Polymerases Conferring Rifampin Resistance to Different Antibiotics

Ming Xu,^1, Yan Ning Zhou,¹ Beth P. Goldstein,^2, and Ding Jun Jin^1,3*

Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute-Frederick, National Institutes of Health, Frederick,³ Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland,¹ Lepetit Research Center, Gerenzano, Varese, Italy²

Received 4 December 2004/ Accepted 14 January 2005

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 Rif^r 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 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 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.

Present address: Nanjing University of Science and Technology, Nanjing, People's Republic of China.

Present address: Vicuron Pharmaceuticals, King of Prussia, PA 19406.