Ribonucleotide Reduction in Mycobacterium tuberculosis : Function and Expression of Genes Encoding Class Ib and Class II Ribonucleotide Reductases

Abstract

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 ⌬nrdZ 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 ⌬nrdZ mutant strain was not impaired under hypoxic conditions in vitro. Moreover, the lungs of B6D2/F 1 mice infected with the ⌬nrdZ 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.Mycobacterium tuberculosis is a formidable human pathogen that is estimated to infect one-third of the world's population (2). The success of this pathogen is attributable to its remarkable ability to persist for prolonged periods in a clinically latent state from which it is able to reactivate and cause disease. During the course of infection in humans, the tubercle bacillus is likely to encounter environments where there is limited O 2 availability, most notably the fibrous granulomas (9). This notion has underpinned efforts to identify the metabolic changes that occur in M. tuberculosis in response to hypoxia as a means of modeling the changes that may be associated with clinical latency (13, 37, 44). The value of this approach was underscored by a recent report (38) suggesting that during stationary infection in mice, the bacilli may be in a physiological state that approximates the nonreplicating persistence achieved in the widely used model of adaptation of M. tuberculosis to hypoxia in vitro developed by Wayne and coworkers (44,45).An important class of enzymes that includes both O 2 -dependent and O 2 -independent forms is the ribonucleotide reductases (RNRs), which catalyze the reduction of ribonucleotides to deoxyribonucleotides. These enzymes perform an essential role in the cycling of nucleotides in the cell and during replication of the chromosome in all organisms and provide attractive targets for antiproliferative drugs (40) and subunit ...

Biosynthesis and Recycling of Nicotinamide Cofactors in Mycobacterium tuberculosis

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Alternative oxygen‐dependent and oxygen‐independent ribonucleotide reductases in Streptomyces: cross‐regulation and physiological role in response to oxygen limitation

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