The control of morphological differentiation and secondary metabolism by AmfS autoregulatory peptide in Streptomyces

在灰色链霉菌中，AMF基因簇编码了一种可能的分泌系统，它可以诱导气生菌丝体的形成。在这里，我们研究了Amft之前的启动子(PamfT)指导amfTSBA操纵子转录的转录控制机制。高分辨率的S1分析将转录起点定位在AMFT翻译起始密码子上游的31个核苷酸。低分辨率分析表明，PamfT在野生型中受到发育调节，在amfR突变体中完全取消。PamfT的-35区含有天蓝色链霉菌BLdD的共同结合序列，BldD是天蓝色链霉菌A3(2)形态和生理发育的多效性负调控因子。克隆的灰链霉菌BldD基因座与天蓝色链霉菌的BldD基因座具有较高的序列相似性。BldD在灰链霉菌野生型和A因子缺陷型突变株中都存在结构性转录，这表明BldD的调节作用不依赖于A因子。凝胶滞留实验表明，纯化的BldD和AmfR重组蛋白能与PamfT特异结合。在野生型细胞中过量生产BldD导致秃顶表型(气生生长和链霉素生产缺陷)，并导致PamfT活性显着抑制。Amft缺失的突变体也表现出秃顶的表型，但PamfT活性没有受到影响。在交叉饲养试验中，BldD高产野生型菌株和amft突变体都不能诱导AMFS突变体的气生生长，这表明这些菌株在生产活性AMFS肽方面存在缺陷。总体结果表明，两个独立的调节子AmfR和BldD通过直接结合来控制PamfT的活性，以确定AMF操纵子的转录水平，该操纵子负责AMFS肽的产生和分泌，AMFS操纵子诱导灰链霉气生菌丝的竖立。

The amf gene cluster encodes a probable secretion system for a peptidic morphogen, AmfS, which induces aerial mycelium formation in Streptomyces griseus. Here we examined the transcriptional control mechanism for the promoter preceding amfT(PamfT) directing the transcription of the amfTSBA operon. High-resolution S1 analysis mapped a transcriptional start point at 31 nucleotides upstream of the translational start codon of amfT. Low-resolution analysis showed that PamfT is developmentally regulated in the wild type and completely abolished in an amfR mutant. The -35 region of PamfT contained the consensus sequence for the binding of BldD, a pleiotropic negative regulator for morphological and physiological development in Streptomyces coelicolor A3(2). The cloned bldD locus of S.griseus showed high sequence similarity to the S.coelicolor counterpart. Transcription of bldD occurred constitutively in both the wild type and an A-factor-deficient mutant of S.griseus, which suggests that the regulatory role of BldD is independent of A-factor. The gel retardation assay revealed that purified BldD and AmfR recombinant proteins specifically bind PamfT. Overproduction of BldD in the wild-type cell conferred a bald phenotype (defective in aerial growth and streptomycin production) and caused marked repression of PamfT activity. An amfT-depleted mutant also showed a bald phenotype but PamfT activity was not affected. Both the bldD-overproducing wild-type strain and the amfT mutant were unable to induce aerial growth of an amfS mutant in a cross-feeding assay, which indicates that these strains are defective in the production of an active AmfS peptide. The results overall suggests that two independent regulators, AmfR and BldD, control PamfT activity via direct binding to determine the transcriptional level of the amf operon responsible for the production and secretion of AmfS peptide, which induces the erection of aerial hyphae in S.griseus.