Ribonucleotide reduction in Mycobacterium tuberculosis: function and expression of genes encoding class Ib and class II ribonucleotide reductases.
Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses a class Ib ribonucleotide reductase (RNR), encoded by the nrdE and nrdF2 genes, in addition to a putative class II RNR, encoded by nrdZ. In this study we probed the relative contributions of these RNRs to the growth and persistence of M. tuberculosis. We found that targeted knockout of the nrdF2 gene could be achieved only in the presence of a complementing allele, confirming that this gene is essential under normal, in vitro growth conditions. This observation also implied that the alternate class Ib small subunit encoded by the nrdF1 gene is unable to substitute for nrdF2 and that the class II RNR, NrdZ, cannot substitute for the class Ib enzyme, NrdEF2. Conversely, a DeltanrdZ null mutant of M. tuberculosis was readily obtained by allelic exchange mutagenesis. Quantification of levels of nrdE, nrdF2, nrdF1, and nrdZ gene expression by real-time, quantitative reverse transcription-PCR with molecular beacons by using mRNA from aerobic and O(2)-limited cultures showed that nrdZ was significantly induced under microaerophilic conditions, in contrast to the other genes, whose expression was reduced by O(2) restriction. However, survival of the DeltanrdZ mutant strain was not impaired under hypoxic conditions in vitro. Moreover, the lungs of B6D2/F(1) mice infected with the DeltanrdZ mutant had bacterial loads comparable to those of lungs infected with the parental wild-type strain, which argues against the hypothesis that nrdZ plays a significant role in the virulence of M. tuberculosis in this mouse model.
School of Pathology of the National Health Laboratory Service and Department of Molecular Medicine and Hematology, University of the Witwatersrand Medical School, Johannesburg 2000, South Africa.
Stephanie S
SS
Dawes
Digby F
DF
Warner
Liana
L
Tsenova
Juliano
J
Timm
John D
JD
McKinney
Gilla
G
Kaplan
Harvey
H
Rubin
Valerie
V
Mizrahi
D000818:Animals; D005260:Female; D006918:Hydroxyurea / Q000494:pharmacology; D051379:Mice; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D010084:Oxidation-Reduction; D012329:RNA, Bacterial / Q000032:analysis; D012264:Ribonucleotide Reductases / Q000145:classification / Q000235:genetics* / Q000502:physiology; D012265:Ribonucleotides / Q000378:metabolism
D012329:RNA, Bacterial; D012265:Ribonucleotides; D012264:Ribonucleotide Reductases; D006918:Hydroxyurea