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 基因簇编码肽形态发生素 AmfS 的可能分泌系统，AmfS 可诱导灰色链霉菌中气生菌丝体的形成。在这里，我们检查了 amfT（PamfT）之前的启动子指导 amfTSBA 操纵子转录的转录控制机制。高分辨率 S1 分析将转录起始点定位在 amfT 翻译起始密码子上游 31 个核苷酸处。低分辨率分析表明，PamfT 在野生型中受到发育调节，而在 amfR 突变体中完全被消除。 PamfT 的 -35 区域包含 BldD 结合的共有序列，BldD 是天蓝色链霉菌 A3(2) 形态和生理发育的多效性负调节因子。 S.griseus 的克隆 bldD 基因座与 S.coelicolor 对应物显示出高度的序列相似性。 bldD 的转录在 S.griseus 的野生型和 A 因子缺陷突变体中均持续发生，这表明 BldD 的调节作用独立于 A 因子。凝胶阻滞测定显示纯化的 BldD 和 AmfR 重组蛋白特异性结合 PamfT。野生型细胞中 BldD 的过量产生导致秃头表型（空中生长和链霉素产生缺陷）并导致 PamfT 活性显着抑制。 amfT 缺失的突变体也表现出秃头表型，但 PamfT 活性未受影响。在交叉饲喂测定中，bldD过量产生的野生型菌株和amfT突变体都不能诱导amfS突变体的空中生长，这表明这些菌株在产生活性AmfS肽方面存在缺陷。总体结果表明，两个独立的调节因子 AmfR 和 BldD 通过直接结合来控制 PamfT 活性，以确定负责产生和分泌 AmfS 肽的 amf 操纵子的转录水平，AmfS 肽诱导 S.griseus 中气生菌丝的竖立。