Nocardia species are gram-positive environmental saprophytes, but some cause the infectious disease nocardiosis. The complete genomic sequence of Nocardia farcinica IFM 10152 has been determined, and analyses indicated the presence of two different RNA polymerase beta subunit genes, rpoB and rpoB2, in the genome (J. Ishikawa, A. Yamashita, Y. Mikami, Y. Hoshino, H. Kurita, K. Hotta, T. Shiba, and M. Hattori, Proc. Natl. Acad. Sci. USA 101:14925-14930, 2004). These genes share 88.8% identity at the nucleotide level. Moreover, comparison of their amino acid sequences with those of other bacterial RpoB proteins suggested that the nocardial RpoB protein is likely to be rifampin (RIF) sensitive, whereas RpoB2 protein contains substitutions at the RIF-binding region that are likely to confer RIF resistance. Southern analysis indicated that rpoB duplication is widespread in Nocardia species and is correlated with the RIF-resistant phenotype. The introduction of rpoB2 by using a newly developed Nocardia-Escherichia coli shuttle plasmid vector and transformation system conferred RIF resistance to Nocardia asteroides IFM 0319T, which has neither RIF resistance nor rpoB duplication. Furthermore, unmarked rpoB2 deletion mutants of N. farcinica IFM 10152 showed no significant resistance to RIF. These results indicated the contribution of rpoB2 to RIF resistance in Nocardia species. Since this is the first example of genetic engineering of the Nocardia genome, we believe that this study, as well as our determination of the N. farcinica genome sequence, will be a landmark in Nocardia genetics.