Ribonucleotide reduction in Mycobacterium tuberculosis:: Function and expression of genes encoding class Ib and class II ribonucleotide reductases
Ribonucleotide reduction in Mycobacterium tuberculosis:: Function and expression of genes encoding class Ib and class II ribonucleotide reductases
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DOI：
10.1128/iai.71.11.6124-6131.2003
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发表时间：
2003-11-01
影响因子：
3.1
通讯作者：
Mizrahi, V
中科院分区：
文献类型：
--
作者：
Dawes, SS;Warner, DF;Mizrahi, V
结核分枝杆菌是结核病的病原体，除了nrdZ编码的II类核糖核苷酸还原酶外，还含有由nrdE和nrdF2基因编码的Ib类核糖核苷酸还原酶(RNR)。在这项研究中，我们探讨了这些RNRs对结核分枝杆菌的生长和持久性的相对贡献。我们发现，只有在存在互补等位基因的情况下，才能实现对nrdF2基因的定向敲除，这证实了该基因在正常的体外生长条件下是必不可少的。这也表明nrdF1基因编码的Ib类小亚基不能替代nrdF2，II类RNR NrdZ不能替代Ib类酶NrdEF2。相反，通过等位基因交换突变，可以很容易地获得结核分枝杆菌DeltanrdZ零突变。用分子信标实时定量逆转录-聚合酶链式反应检测了nrdE、nrdF2、nrdF1和nrdZ基因的表达水平，结果表明，nrdZ基因在微好氧条件下被显著诱导，而其他基因在氧气限制条件下表达降低。然而，DeltanrdZ突变株在体外低氧条件下的存活没有受到影响。此外，感染DeltanrdZ突变体的B6D2/F-1小鼠的肺的细菌载量与感染亲本野生型菌株的肺的细菌量相当，这驳斥了nrdZ在该小鼠模型中对结核分枝杆菌毒力起重要作用的假设。
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.