Novel Two-Component System MacRS Is a Pleiotropic Regulator That Controls Multiple Morphogenic Membrane Protein Genes in Streptomyces coelicolor
Novel Two-Component System MacRS Is a Pleiotropic Regulator That Controls Multiple Morphogenic Membrane Protein Genes in Streptomyces coelicolor
复制标题
新型双组分系统 MacRS 是一种多效性调节剂，可控制天蓝色链霉菌中的多个形态发生膜蛋白基因
DOI：
10.1128/aem.02178-18
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发表时间：
2019-02-01
影响因子：
4.4
通讯作者：
Pang, Xiuhua
中科院分区：
文献类型：
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作者：
Liu, Meng;Zhang, Peipei;Pang, Xiuhua
与天蓝色链霉菌的大多数注释的双组分系统（TCS）一样，TCS SCO 2120/2121的功能尚不清楚。根据我们的研究结果，我们已经指定了这个TCS MacRS，用于形态发生和放线菌紫素调节器/传感器。我们的研究表明，无论是单或双突变的MacRS在很大程度上阻止放线菌紫素的生产，但提高气生菌丝体的形成。染色质免疫沉淀（ChIP）测序，使用S.表达MacR-Flag融合蛋白的coelicolor菌株，鉴定了MacR的体内靶标，并且这些靶标的DNA酶I足迹法揭示了MacR结合的共有序列，TGAGTACnnGTACTCA，其含有两个7-bp反向重复序列。一个全基因组搜索发现的网站相同或高度相似的六个基因编码推定的膜蛋白或脂蛋白的上游这个共识序列。这些预测的网站被证实为MacR结合位点的DNA酶I足迹和电泳迁移率变动测定在体外和ChIP定量PCR在体内，和转录分析表明，MacR显着影响这些靶基因的表达。其中三个基因，SCO 6728，SCO 4924和SCO 4011，破坏，显着加速气生菌丝体的形成，表明它们的基因产物是新的形态发生因子。双杂交试验表明，这三种蛋白质，我们命名为形态发生膜蛋白A（MmpA; SCO 6728），MmpB（SCO 4924），和MmpC（SCO 4011），相互作用，并与推定的膜蛋白和MacR目标SCO 4225。值得注意的是，SAV 6081/82和SVEN 1780/81是来自S.阿维菌素和S.委内瑞拉，分别可以替代MacRS，表明功能保护。我们的发现揭示了MacRS在链霉菌细胞形态发生和次级代谢中的作用。重要意义TCS通过改变基因表达帮助细菌适应环境胁迫。然而，大多数TCS在链霉菌模式菌株S. coelicolor未知。我们研究了之前未表征的MacRS TCS，并鉴定了DNA结合蛋白MacR的核心DNA识别序列，即两个七个核苷酸的反向重复序列。我们进一步发现，MacR直接控制一组膜蛋白，包括MmpA-C，这是新的形态发生因子，延迟气生菌丝体的形成。我们还发现，这些膜蛋白相互作用，其他链霉菌物种有保守的MacRS同源物。我们的研究结果表明MacRS在形态发生和/或其他膜相关活动中的保守作用。此外，我们的研究表明，MacRS的影响，虽然间接，生产的签名代谢产物放线菌紫素，进一步表明，MacRS及其同系物的功能作为新的多效性的调控系统在链霉菌。
As with most annotated two-component systems (TCSs) of Streptomyces coelicolor, the function of TCS SCO2120/2121 was unknown. Based on our findings, we have designated this TCS MacRS, for morphogenesis and actinorhodin regulator/sensor. Our study indicated that either single or double mutation of MacRS largely blocked production of actinorhodin but enhanced formation of aerial mycelium. Chromatin immunoprecipitation (ChIP) sequencing, using an S. coelicolor strain expressing MacR-Flag fusion protein, identified in vivo targets of MacR, and DNase I footprinting of these targets revealed a consensus sequence for MacR binding, TGAGTACnnGTACTCA, containing two 7-bp inverted repeats. A genome-wide search revealed sites identical or highly similar to this consensus sequence upstream of six genes encoding putative membrane proteins or lipoproteins. These predicted sites were confirmed as MacR binding sites by DNase I footprinting and electrophoretic mobility shift assays in vitro and by ChIP-quantitative PCR in vivo, and transcriptional analyses demonstrated that MacR significantly impacts expression of these target genes. Disruption of three of these genes, SCO6728, SCO4924, and sco4011, markedly accelerated aerial mycelium formation, indicating that their gene products are novel morphogenic factors. Two-hybrid assays indicated that these three proteins, which we have named morphogenic membrane protein A (MmpA; SCO6728), MmpB (SCO4924), and MmpC (SCO4011), interact with one another and with the putative membrane protein and MacR target SCO4225. Notably, SAV6081/82 and SVEN1780/81, homologs of MacRS TCS from S. avermitilis and S. venezuelae, respectively, can substitute for MacRS, indicating functional conservation. Our findings reveal a role for MacRS in cellular morphogenesis and secondary metabolism in Streptomyces.IMPORTANCE TCSs help bacteria adapt to environmental stresses by altering gene expression. However, the roles and corresponding regulatory mechanisms of most TCSs in the Streptomyces model strain S. coelicolor are unknown. We investigated the previously uncharacterized MacRS TCS and identified the core DNA recognition sequence, two seven-nucleotide inverted repeats, for the DNA-binding protein MacR. We further found that MacR directly controls a group of membrane proteins, including MmpA-C, which are novel morphogenic factors that delay formation of aerial mycelium. We also discovered that these membrane proteins interact with one another and that other Streptomyces species have conserved MacRS homologs. Our findings suggest a conserved role for MacRS in morphogenesis and/or other membrane-associated activities. Additionally, our study showed that MacRS impacts, albeit indirectly, the production of the signature metabolite actinorhodin, further suggesting that MacRS and its homologs function as novel pleiotropic regulatory systems in Streptomyces.