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

amf基因簇编码一个可能的分泌系统的肽形态原，AmfS，诱导气生菌丝形成在灰色链霉菌。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S1分析将转录起点定位在amfT翻译起始密码子上游31个核苷酸处。低分辨率分析显示PamfT在野生型中受到发育调节，而在amfR突变体中完全消失。PamfT的-35区包含与BldD结合的一致序列(2)，BldD是链霉菌A3形态和生理发育的多效负调节因子。克隆得到的灰褐色稻瘟病菌的bldD位点与颜色稻瘟病菌具有较高的序列相似性。在野生型和a因子缺失突变体中均存在bldD的转录，这表明bldD的调控作用不依赖于a因子。凝胶阻滞实验显示纯化的BldD和AmfR重组蛋白特异性结合PamfT。野生型细胞中BldD的过量产生导致秃发表型（空中生长和链霉素产生缺陷），并导致PamfT活性明显抑制。amft缺失突变体也表现出秃发表型，但PamfT活性不受影响。在交叉饲养试验中，产生bld过量的野生型菌株和amfT突变体都不能诱导amfS突变体在空中生长，这表明这些菌株在产生活性amfS肽方面存在缺陷。结果表明，两个独立的调控因子AmfR和BldD通过直接结合来控制PamfT的活性，从而决定了负责产生和分泌AmfS肽的amf操纵子的转录水平，从而诱导稻瘟病菌气生菌丝的勃起。

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.