Post-translation regulation of antibiotic production in Streptomyces: the loaded gun hypothesis.

我们在人类医学中使用的抗生素中，约有四分之三来自土壤细菌和真菌的天然产物。我们称这些天然产物为特殊代谢物，因为它们具有特殊的功能，通常是在竞争激烈的土壤环境中杀死其他细菌和真菌。这些分子也可能对蠕虫、昆虫甚至植物有毒，许多分子已被用于治疗寄生虫感染，并在农业中用作除草剂或杀虫剂。特殊代谢物的最大生产者是链霉菌，它制造了大约50%的已知抗生素。这些细菌对人类非常重要，但我们对它们如何控制其特殊代谢物的产生知之甚少。这很重要，因为当我们在实验室培养它们时，它们只产生大约10%的专门代谢物。我们通过对它们细胞中所有DNA的测序了解到这一点，这表明它们有制造更多DNA的指令和能力。如果我们能够了解它们是如何控制它们的生产的，我们就应该能够设计菌株，在所有已知的600种链霉菌中开启所有特殊代谢物的生产，并发现许多新的和潜在有用的天然产物，包括抗生素。作为我们努力确定抗生素生产的主要调节因子的一部分，我们表征了一种称为MtrA的DNA结合蛋白，这种蛋白在所有链霉菌物种中都有发现。MtrA控制到目前为止已测试的所有链霉菌菌株的抗生素生产，是一种称为双组分系统的信号转导途径的一部分。这些信号系统在细菌中很常见。MtrA是一种反应调节因子，这些蛋白质是典型的转录因子，通过结合启动子DNA来控制基因表达。MtrA的DNA结合活性由MtrB控制，MtrB是一种传感器激酶，它跨越细胞膜，感知细胞外的信号，然后磷酸化并激活细胞内的MtrA。MtrA结合了大约80%的预测生物合成基因簇，这些基因簇用于在colicolice Streptomyces和S. venezuela中产生特殊的代谢物。MtrA还与其他转录因子结合，这在细菌中是不寻常的，并与制造专门代谢物的酶结合，据我们所知，以前从未描述过任何其他细菌转录因子。在本提案中，我们将使用委内瑞拉葡萄球菌作为模型来描述MtrA对抗生素生产的调节，因为它通过与转运基因和cml酶结合来控制氯霉素的生产，cml酶催化生物合成途径的最后一步。抗生素的生物合成似乎不是从无到有的。相反，我们假设它就像一把上了膛的枪，前体被制造出来，但最后一步被MtrA阻止了。当MtrA被关闭时，就像扣动扳机一样——最后一步发生了，活性抗生素被制造出来，运输基因被表达，活性化合物被从细胞中输出。在这个项目中，我们将测试这个“上了膛的枪”假设，结果可能会改变我们对细菌转录因子和抗生素生物合成调节的看法。MtrA还与一种密切相关的反应调节因子Vnz13500结合，该调节因子在链霉菌中保守，可能也被MtrB激活，这表明其在委内瑞拉葡萄球菌中的功能很复杂。为了测试这些功能是否在其他链菌中保守，我们将使用远亲S. coelicolor，这也是实验可处理的。我们和其他研究小组发表的数据表明，S. coelicolor MtrAB和SCO3008（其Vnz13500同系物）参与控制其抗生素十烯基prodigiosin的产生，我们将在转录和翻译后水平上测试它是否这样做。

Around three quarters of the antibiotics we use in human medicine are derived from the natural products of soil bacteria and fungi. We call these natural products specialised metabolites because they have a specialised function, which is usually to kill other bacteria and fungi in the highly competitive soil environment. These molecules can also be toxic to worms, insects and even plants and many have been used in medicine to treat parasite infections and as herbicides or pesticides in agriculture. The biggest producers of specialised metabolites are Streptomyces bacteria which make around 50% of all known antibiotics. These bacteria are incredibly important to humans but we have relatively little understanding of how they control the production of their specialised metabolites. This is important because they only make around 10% of their specialised metabolites when we grow them in the laboratory. We know this from sequencing all the DNA in their cells which shows they have the instructions and capacity to make many more. If we can understand how they control their production we should be able to engineer strains to switch on production of all the specialised metabolites in all of the >600 known Streptomyces species and discover many new and potentially useful natural products, including antibiotics. As part of our efforts to identify the master regulators of antibiotic production, we characterised a DNA binding protein called MtrA which is found in all Streptomyces species. MtrA controls antibiotic production in all the Streptomyces strains that have been tested so far and is part of a signal transduction pathway called a two-component system. These signalling systems are common in bacteria. MtrA is a response regulator, and these proteins are typically transcription factors which control gene expression by binding to promoter DNA. The DNA binding activity of MtrA is controlled by MtrB, a sensor kinase which spans the cell membrane and senses a signal outside the cell and then phosphorylates and activates MtrA inside the cell. MtrA binds to around 80% of the predicted biosynthetic gene clusters for specialised metabolites in Streptomyces coelicolor and S. venezuelae. MtrA also binds to other transcription factors which is unusual in bacteria and to enzymes involved in making specialised metabolites which, to our knowledge, has never been described before for any other bacterial transcription factor. In this proposal we will use S. venezuelae as a model to characterise the regulation of antibiotic production by MtrA since it controls chloramphenicol production by binding to the transporter genes and to the enzyme CmlS, which catalyses the final step in the biosynthetic pathway. It appears that antibiotic biosynthesis does not occur from scratch. Instead we hypothesise that it is like a loaded gun, the precursor is made but the final step is blocked by MtrA. When MtrA is switched off it is like pulling the trigger - the final step occurs, and the active antibiotic is made, the transport genes are expressed and the active compound is exported from the cell. In this project we will test this "loaded gun" hypothesis and the results will likely change the way we think about bacterial transcription factors and the regulation of antibiotic biosynthesis.MtrA also binds to a closely related response regulator called Vnz13500 which is conserved in Streptomyces species and probably also activated by MtrB suggesting its functions are complex in S. venezuelae. To test whether these functions are conserved in other streptomycetes we will use the distantly related S. coelicolor which is also experimentally tractable. Published data from our and other groups suggests S. coelicolor MtrAB and SCO3008 (its Vnz13500 homologue) are involved in controlling production of its antibiotic undecylprodigiosin and we will test if it does this at both the transcriptional and post translational levels.