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

我们在人类医学中使用的大约四分之三的抗生素来自土壤细菌和真菌的天然产物。我们称这些天然产物为专门的代谢产物，因为它们具有专门的功能，通常是在竞争激烈的土壤环境中杀死其他细菌和真菌。这些分子对蠕虫、昆虫甚至植物也是有毒的，许多分子已被用于医学治疗寄生虫感染，并在农业中用作除草剂或杀虫剂。最大的专门代谢物生产者是链霉菌属细菌，它们生产约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专门代谢产物的产生知之甚少。这一点很重要，因为当我们在实验室中培养它们时，它们只产生大约10%的专门代谢产物。我们通过对它们细胞中所有DNA的测序知道了这一点，这表明它们有制造更多DNA的指令和能力。如果我们能够理解它们是如何控制其生产的，我们应该能够设计菌株，以在所有超过600种已知的链霉菌中生产所有专门的代谢产物，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剂的一部分，我们表征了一种称为MtrA的DNA结合蛋白，它存在于所有链霉菌属物种中。MtrA控制迄今为止测试的所有链霉菌菌株中的抗生素生产，并且是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，这些蛋白质通常是通过与启动子DNA结合来控制基因表达的转录因子。MtrA的DNA结合活性由MtrB控制，MtrB是一种传感器激酶，其跨越细胞膜并感测细胞外的信号，然后磷酸化并激活细胞内的MtrA。MtrA与天蓝色链霉菌和S.委内瑞拉。MtrA还结合其他转录因子，这在细菌中是不常见的，并结合参与产生专门的代谢物的酶，据我们所知，这在任何其他细菌转录因子之前从未被描述过。在这个例子中，我们将使用S。委内瑞拉作为一个模型来验证通过MtrA调节抗生素生产，因为它通过与转运蛋白基因和酶Cm 1 S结合来控制氯霉素生产，所述酶Cm 1 S催化生物合成途径中的最后一步。看来抗生素的生物合成不是从头开始的。相反，我们假设它就像一把上膛的枪，前体被制造出来，但最后一步被MtrA阻断。当MtrA被关闭时，就像扣动扳机一样-最后一步发生，活性抗生素被制造，转运基因被表达，活性化合物从细胞中输出。在这个项目中，我们将测试这个“上膛的枪”的假设，其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控的看法。MtrA还与一个密切相关的反应调节因子Vnz 13500结合，该因子在链霉菌中是保守的，可能也被MtrB激活，这表明它在链霉菌中的功能是复杂的。委内瑞拉。为了测试这些功能是否在其他链霉菌中是保守的，我们将使用远亲的S。也是实验上容易处理的。我们和其他研究小组公布的数据表明，S。coelicolor MtrAB和SCO 3008（其Vnz 13500同源物）参与控制其抗生素十一烷基灵菌红素的产生，我们将测试它是否在转录和翻译后水平上都这样做。

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.