The Nonmevalonate Pathway of Isoprenoid Biosynthesis in Mycobacterium tuberculosis Is Essential and Transcriptionally Regulated by Dxs
The Nonmevalonate Pathway of Isoprenoid Biosynthesis in Mycobacterium tuberculosis Is Essential and Transcriptionally Regulated by Dxs
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DOI：
10.1128/jb.01402-09
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发表时间：
2010-05-01
影响因子：
3.2
通讯作者：
Parish, Tanya
中科院分区：
文献类型：
--
作者：
Brown, Amanda C.;Eberl, Matthias;Parish, Tanya
结核分枝杆菌通过非甲羟戊酸或DOXP途径合成类异戊二烯。先前的研究表明，该途径中的三种酶（Dxr、IspD和IspF）都是体外生长所必需的。我们证明了关键基因dxs1和gcpE的重要性，证实了该途径的核心重要性，并且合成酶的第二个同源物（dxs2）不能弥补dxs1的缺失。我们观察了过表达Dxr、Dxs1、Dxs2和GcpE对结核分枝杆菌存活和生长的影响。过表达dxs1或dxs2对生长有抑制作用，而过表达dxr或gcpE对生长无抑制作用。毒性可能是，至少部分是由于细胞中丙酮酸的消耗。通过代谢物4-羟基-3-甲基-但-2-烯基焦磷酸的积累来测量，过表达dxs1或gcpE导致通过该途径的通量增加。我们确定了dxr的功能性翻译起始位点和启动子区域，并证明它与gcpE和其他两个基因一起作为多顺反子mRNA的一部分表达。在过表达Dxs1的细胞中可以观察到该操纵子的表达增加，这表明转录控制是由该途径的第一个酶通过未知调节剂影响的。
Mycobacterium tuberculosis synthesizes isoprenoids via the nonmevalonate or DOXP pathway. Previous work demonstrated that three enzymes in the pathway (Dxr, IspD, and IspF) are all required for growth in vitro. We demonstrate the essentiality of the key genes dxs1 and gcpE, confirming that the pathway is of central importance and that the second homolog of the synthase (dxs2) cannot compensate for the loss of dxs1. We looked at the effect of overexpression of Dxr, Dxs1, Dxs2, and GcpE on viability and on growth in M. tuberculosis. Overexpression of dxs1 or dxs2 was inhibitory to growth, whereas overexpression of dxr or gcpE was not. Toxicity is likely to be, at least partially, due to depletion of pyruvate from the cells. Overexpression of dxs1 or gcpE resulted in increased flux through the pathway, as measured by accumulation of the metabolite 4-hydroxy-3-methyl-but-2-enyl pyrophosphate. We identified the functional translational start site and promoter region for dxr and demonstrated that it is expressed as part of a polycistronic mRNA with gcpE and two other genes. Increased expression of this operon was seen in cells overexpressing Dxs1, indicating that transcriptional control is effected by the first enzyme of the pathway via an unknown regulator.