Mowa Mohube B, Warner Digby F, Kaplan Gilla, Kana Bavesh D, Mizrahi Valerie
MRC/NHLS/WITS Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for Biomedical TB Research, School of Pathology, University of the Witwatersrand, National Health Laboratory Service, Johannesburg 2000, South Africa.
J Bacteriol. 2009 Feb;191(3):985-95. doi: 10.1128/JB.01409-08. Epub 2008 Nov 21.
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.
核糖核苷酸还原酶（RNRs）对所有活细胞都至关重要，因为它们为DNA合成和修复提供脱氧核糖核苷酸（dNTPs）。在结核分枝杆菌中，由nrdE和nrdF2编码的亚基组成的Ib类RNR对体外生长至关重要。有趣的是，这种专性人类病原体的基因组还包含nrdF1（Rv1981c）和nrdB（Rv0233）基因，分别编码一个替代的Ib类RNR小（R2）亚基和一个推定的Ic类RNR R2亚基。然而，这些亚基在结核分枝杆菌致病过程中在dNTP供应中的作用尚不清楚。在本研究中，我们证明nrdF1和nrdB在体外暴露于各种应激后对结核分枝杆菌的生长和存活不是必需的，此外，在小鼠体内生长和存活也不需要这两个基因。这些观察结果表明在所测试的条件下替代R2亚基没有特殊作用。通过构建结核分枝杆菌和耻垢分枝杆菌的nrdR缺陷突变体，我们确定编码必需的Ib类RNR亚基的基因受到NrdR型阻遏物的特异性调控。此外，耻垢分枝杆菌mc(2)155缺失包括nrdHIE和nrdF2重复序列的56-kb染色体区域的菌株，以及仅保留一个nrdF2拷贝的突变体，由于靶标水平降低，对I类RNR抑制剂羟基脲高度敏感。总之，我们的观察结果确定了分枝杆菌dNTP供应中的一个潜在脆弱性，从而为将Ib类RNR作为药物发现靶点提供了令人信服的理由。
