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

AmfS自动调节肽对链霉菌形态分化和次生代谢的控制

基本信息

批准号：
15380066
负责人：
UEDA Kenji
金额：
$ 3.01万
依托单位：
Nihon University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

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.

amf基因簇编码一个可能的分泌系统的肽形态，AmfS，诱导气生菌丝体形成在灰色链霉菌。在这里，我们研究了转录控制机制的启动子前amfT（PamfT）指导amfTSBA操纵子的转录。高分辨率的S1分析映射的转录起始点在31个核苷酸上游的翻译起始密码子的amfT。低分辨率分析表明，PamfT在野生型中受到发育调控，在amfR突变体中完全被废除。PamfT的-35区域包含BldD结合的共有序列，BldD是天蓝色链霉菌A3中形态和生理发育的多效性负调节因子（2）。克隆的灰色链霉菌bldD基因座与天蓝色链霉菌的bldD基因座具有很高的序列相似性。在野生型和A因子缺陷突变体中，BldD的转录都是组成型的，这表明BldD的调节作用不依赖于A因子。凝胶阻滞试验表明，纯化的BldD和AmfR重组蛋白特异性结合PamfT。在野生型细胞中过量产生BldD赋予秃头表型（在气生生长和链霉素生产中有缺陷），并引起PamfT活性的显著抑制。amfT缺失突变体也表现出秃表型，但PamfT活性不受影响。bldD过量生产的野生型菌株和amfT突变体都不能在交叉饲养试验中诱导amfS突变体的气生生长，这表明这些菌株在活性AmfS肽的生产中有缺陷。总体结果表明，两个独立的监管机构，AmfR和BldD，控制PamfT活性，通过直接结合，以确定转录水平的AMF操纵子负责生产和分泌的AmfS肽，诱导勃起的气生菌丝在灰色链霉菌。