Phosphate effect on filipin production and morphological differentiation in <i>Streptomyces filipinensis</i> and the role of the PhoP transcription factor

The biosynthesis of the antifungal filipin in Streptomyces filipinensis is very sensitive to phosphate regulation. Concentrations as low as 2.5 mM block filipin production. This effect is, at least in part, produced by repression of the transcription of most filipin biosynthetic genes. The role of the two-component PhoRP system in this process was investigated. The phoRP system of S. filipinensis was cloned and transcriptionally characterised. PhoP binds to two PHO boxes present in one of its two promoters. Filipin production was greatly increased in ΔphoP and ΔphoRP mutants, in agreement with a higher transcription of the fil genes, and the effect of phosphate repression on the antibiotic production of these strains was significantly reduced. No PhoP binding was observed by electrophoretic mobility gel shift assays (EMSAs) with the promoter regions of the fil gene cluster thus suggesting an indirect effect of mutations. Binding assays with cell-free extracts from the wild-type and mutant strains on fil genes promoters revealed retardation bands in the parental strain that were absent in the mutants, thus suggesting that binding of the putative transcriptional regulator or regulators controlled by PhoP was PhoP dependent. Noteworthy, PhoP or PhoRP deletion also produced a dramatic decrease in sporulation ability, thus indicating a clear relationship between the phosphate starvation response mediated by PhoP and the sporulation process in S. filipinensis. This effect was overcome upon gene complementation, but also by phosphate addition, thus suggesting that alternative pathways take control in the absence of PhoRP.菲律宾链霉菌（Streptomyces filipinensis）中抗真菌菲律宾菌素（filipin）的生物合成对磷酸盐调控极为敏感，低至2.5 mM的磷酸盐浓度即可阻断其产生。该效应至少部分源于对绝大多数菲律宾菌素生物合成基因的转录抑制。本研究针对双组分PhoRP系统（two-component PhoRP system）在该过程中的作用展开了探究：我们克隆了菲律宾链霉菌的phoRP系统并对其进行转录特征分析，发现PhoP蛋白可结合其两个启动子之一中存在的两个PHO框（PHO boxes）。ΔphoP与ΔphoRP突变株的菲律宾菌素产量显著提升，这与fil基因簇的转录水平上调相一致，且磷酸盐对这些菌株抗生素合成的抑制效应也显著减弱。通过电泳迁移率变动分析（electrophoretic mobility gel shift assays, EMSAs）未检测到PhoP与fil基因簇启动子区域的结合，这表明突变体的效应是间接的。针对野生型与突变株的无细胞提取物开展的fil基因启动子结合实验显示，亲本菌株中出现了迁移阻滞条带，而突变株中则无此类条带，这提示受PhoP调控的推定转录调控因子的结合依赖于PhoP。值得注意的是，PhoP或PhoRP的缺失还会导致产孢能力大幅下降，这表明菲律宾链霉菌中由PhoP介导的磷酸盐饥饿应答与产孢过程之间存在明确关联。该效应可通过基因互补（gene complementation）得以恢复，同时添加磷酸盐也可逆转这一现象，这提示在PhoRP缺失的情况下，存在其他调控通路发挥主导作用。