Ueda Kenji, Takano Hideaki, Nishimoto Madoka, Inaba Hiromi, Beppu Teruhiko
Life Science Research Center, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa 252-8510, Japan.
J Bacteriol. 2005 Jan;187(1):135-42. doi: 10.1128/JB.187.1.135-142.2005.
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基因簇编码一种可能用于肽类形态发生素AmfS的分泌系统，该形态发生素可诱导灰色链霉菌气生菌丝体的形成。在此，我们研究了amfT之前的启动子（PamfT）指导amfTSBA操纵子转录的转录调控机制。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在野生型中受发育调控，而在amfR突变体中完全丧失。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控因子。灰色链霉菌克隆的bldD基因座与天蓝色链霉菌的对应基因座具有高度序列相似性。bldD的转录在灰色链霉菌的野生型和A因子缺陷型突变体中均持续发生，这表明BldD的调控作用独立于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表达BldD会导致光秃表型（气生生长和链霉素产生缺陷），并导致PamfT活性显著抑制。amfT缺失的突变体也表现出光秃表型，但PamfT活性不受影响。在交叉喂养试验中，过量表达bldD的野生型菌株和amfT突变体均无法诱导amfS突变体的气生生长，这表明这些菌株在活性AmfS肽的产生方面存在缺陷。总体结果表明，两个独立的调控因子AmfR和BldD通过直接结合来控制PamfT活性，以确定负责AmfS肽产生和分泌的amf操纵子的转录水平，该肽可诱导灰色链霉菌气生菌丝的直立生长。
