Function and regulation of class I ribonucleotide reductase-encoding genes in mycobacteria

Ribonucleotide reductases (RNRs) are crucial to all living cells, since they provide deoxyribonucleotides (dNTPs) for DNA synthesis and repair. In Mycobacterium tuberculosis, a class Ib RNR comprising nrdE- and nrdF2-encoded subunits is essential for growth in vitro. Interestingly, the genome of this obligate human pathogen also contains the nrdF1 (Rv1981c) and nrdB (Rv0233) genes, encoding an alternate class Ib RNR small (R2) subunit and a putative class Ic RNR R2 subunit, respectively. However, the role(s) of these subunits in dNTP provision during M. tuberculosis pathogenesis is unknown. In this study, we demonstrate that nrdF1 and nrdB are dispensable for the growth and survival of M. tuberculosis after exposure to various stresses in vitro and, further, that neither gene is required for growth and survival in mice. These observations argue against a specialist role for the alternate R2 subunits under the conditions tested. Through the construction of nrdR-deficient mutants of M. tuberculosis and Mycobacterium smegmatis, we establish that the genes encoding the essential class Ib RNR subunits are specifically regulated by an NrdR-type repressor. Moreover, a strain of M. smegmatis mc(2)155 lacking the 56-kb chromosomal region, which includes duplicates of nrdHIE and nrdF2, and a mutant retaining only one copy of nrdF2 are shown to be hypersensitive to the class I RNR inhibitor hydroxyurea as a result of depleted levels of the target. Together, our observations identify a potential vulnerability in dNTP provision in mycobacteria and thereby offer a compelling rationale for pursuing the class Ib RNR as a target for drug discovery.