Contribution of rpoB2 RNA polymerase beta subunit gene to rifampin resistance in Nocardia species

Abstract

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.

FIG. 1.

Distribution of rpoB duplication among Nocardia strains. Total DNAs extracted from N. asteroides IFM 0319^T (lane 1), N. asteroides IFM 10159 (lane 2), N. asteroides IFM 10162 (lane 3), N. brasiliensis IFM 0236^T (lane 4), N. brasiliensis IFM 0406 (lane 5), N. brasiliensis IFM 10132 (lane 6), N. brasiliensis IFM 10160 (lane 7), N. farcinica IFM 0284^T (lane 8), N. farcinica IFM 10125 (lane 9), and N. farcinica IFM 10152 (lane 10) were digested with BamHI and probed with a 437-bp fragment containing the C-terminal region of rpoB. The probe was prepared from the total DNA of N. farcinica IFM 10152 by PCR using the primers NFrpoBF and NFrpoBR.

FIG. 2.

Alignment of the RIF-binding regions of RNAP β subunits among N. farcinica IFM 10152 (RpoB and RpoB2), N. asteroides IFM 0319^T (Nast), and M. tuberculosis H37Rv (Mtub). Amino acid substitutions are represented in reverse color. RIF-binding regions (clusters I and II) are boxed.

FIG. 3.

Restriction maps of pNV1.2 and pNVrpoB2. See text for details.

FIG. 4.

Construction of the ΔrpoB2 mutant. A. Strategy for making an in-frame, unmarked deletion of rpoB2. See text for details. Δ indicates a deletion allele. The ScaI half-site generated after ligation of a 4.9-kb ScaI-SphI fragment containing rpoB2 to pUC19 digested with HincII and SphI is shown in parentheses. B. Southern hybridization analysis of a ΔrpoB2 mutant. NotI-digested total DNAs extracted from the wild-type strain (lane 1) and a ΔrpoB2 mutant (lane 2) were probed with a 0.6-kb EcoRI fragment containing nfa46450 (short black bar in panel A).