Insights into the function of the WhiB-like protein of mycobacteriophage TM4-a transcriptional inhibitor of WhiB2
Insights into the function of the WhiB-like protein of mycobacteriophage TM4-a transcriptional inhibitor of WhiB2
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DOI：
10.1111/j.1365-2958.2010.07235.x
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发表时间：
2010-08-01
影响因子：
3.6
通讯作者：
Hartmann, Pia
中科院分区：
文献类型：
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作者：
Rybniker, Jan;Nowag, Angela;Hartmann, Pia
已知放线菌的 WhiB 样蛋白可协调铁硫 (Fe-S) 簇，并被认为在许多重要的细菌过程中具有调节功能。对分枝杆菌噬菌体基因组序列的系统测定揭示了这些病毒中存在几种whB样基因。在这里，我们重点关注分枝杆菌噬菌体 TM4 的 WhiB 样蛋白，WhiBTM4。我们提供的证据表明这种病毒蛋白能够协调 Fe-S 簇。从新鲜纯化和重构的 WhiBTM4 获得的紫外-可见吸收光谱与氧敏感的 [2Fe-2S] 簇的存在一致。 WhiBTM4 在分枝杆菌宿主中的表达导致类似于 WhiB2 敲除表型的隔膜阻碍，而 WhiBTM4 的基础表达导致重复感染排除。噬菌体感染过程中 mRNA 水平的定量表明，whiBTM4 是一种高度转录的早期噬菌体基因，也是 WhiB2 的显性负调节因子。引人注目的是，结核分枝杆菌的 apo-WhiB2 和 apo-WhiBTM4 都能够结合到 whiB2 基因上游的保守启动子区域，表明 WhiB2 调节其自身的合成，而在 WhiBTM4 存在的情况下，该合成会受到抑制。因此，我们提供了大量证据支持病毒和细菌 WhiB 蛋白是重要的含有 Fe-S 且具有 DNA 结合能力的转录调节因子的假设。
WhiB-like proteins of actinomycetes are known to co-ordinate iron-sulfur (Fe-S) clusters and are believed to have regulatory functions in many essential bacterial processes. The systematic determination of the genome sequences of mycobacteriophages has revealed the presence of several whiB-like genes in these viruses. Here we focussed on the WhiB-like protein of mycobacteriophage TM4, WhiBTM4. We provide evidence that this viral protein is capable of co-ordinating a Fe-S cluster. The UV-visible absorption spectra obtained from freshly purified and reconstituted WhiBTM4 were consistent with the presence of an oxygen sensitive [2Fe-2S] cluster. Expression of WhiBTM4 in the mycobacterial host led to hindered septation resembling a WhiB2 knockout phenotype whereas basal expression of WhiBTM4 led to superinfection exclusion. The quantification of mRNA-levels during phage infection showed that whiBTM4 is a highly transcribed early phage gene and a dominant negative regulator of WhiB2. Strikingly, both apo-WhiB2 of Mycobacterium tuberculosis and apo-WhiBTM4 were capable of binding to the conserved promoter region upstream of the whiB2 gene indicating that WhiB2 regulates its own synthesis which is inhibited in the presence of WhiBTM4. Thus, we provide substantial evidence supporting the hypothesis of viral and bacterial WhiB proteins being important Fe-S containing transcriptional regulators with DNA-binding capability.