Yamazaki Haruka, Ohnishi Yasuo, Horinouchi Sueharu
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
J Bacteriol. 2003 Feb;185(4):1273-83. doi: 10.1128/JB.185.4.1273-1283.2003.
A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) triggers morphological development and secondary metabolism in Streptomyces griseus. A transcriptional activator (AdpA) in the A-factor regulatory cascade switches on a number of genes required for both processes. AdBS11 was identified in a library of the DNA fragments that are bound by AdpA and mapped upstream of ssgA, which is essential for septum formation in aerial hyphae. Gel mobility shift assays and DNase I footprinting revealed three AdpA-binding sites at nucleotide positions about -235 (site 1), -110 (site 2), and +60 (site 3) with respect to the transcriptional start point, p1, of ssgA. ssgA had two transcriptional start points, one starting at 124 nucleotides (p1) and the other starting at 79 nucleotides (p2) upstream of the start codon of ssgA. Of the three binding sites, only sites 1 and 2 were required for transcriptional activation of p1 and p2 by AdpA. The transcriptional switch on of ssgA required the extracytoplasmic function sigma factor, sigma(AdsA), in addition to AdpA. However, it was unlikely that sigma(AdsA) recognized the two ssgA promoters, since their -35 and -10 sequences were not similar to the promoter sequence motifs recognized by sigma(BldN), a sigma(AdsA) homologue of Streptomyces coelicolor A3(2). An ssgA disruptant formed aerial hyphae, but did not form spores, irrespective of the carbon source of the medium, which indicated that ssgA is a member of the whi genes. Transcriptional analysis of ssfR, located just upstream of ssgA and encoding an IclR-type transcriptional regulator, suggested that no read-through from ssfR into ssgA occurred, and ssgA was transcribed in the absence of ssfR. ssgA was thus found to be controlled by AdpA and not by SsfR to a detectable extent. SsfR appeared to regulate spore septum formation independently of SsgA or through interaction with SsgA in some unknown way, because an ssfR disruptant also showed a whi phenotype.
A因子（2-异辛酰基-3R-羟甲基-γ-丁内酯）可触发灰色链霉菌的形态发育和次级代谢。A因子调控级联中的转录激活因子（AdpA）开启了这两个过程所需的许多基因。AdBS11是在与AdpA结合的DNA片段文库中鉴定出来的，并定位在ssgA的上游，ssgA对于气生菌丝中隔膜的形成至关重要。凝胶迁移率变动分析和DNase I足迹分析揭示了相对于ssgA的转录起始点p1，在核苷酸位置约-235（位点1）、-110（位点2）和+60（位点3）处有三个AdpA结合位点。ssgA有两个转录起始点，一个从ssgA起始密码子上游124个核苷酸处开始（p1），另一个从79个核苷酸处开始（p2）。在这三个结合位点中，AdpA对p1和p2的转录激活仅需要位点1和位点2。ssgA的转录开启除了需要AdpA外，还需要胞外功能σ因子σ(AdsA)。然而，σ(AdsA)不太可能识别ssgA的两个启动子，因为它们的-35和-10序列与天蓝色链霉菌A3(2)的σ(AdsA)同源物σ(BldN)识别的启动子序列基序不相似。一个ssgA缺失突变体形成了气生菌丝，但无论培养基的碳源如何都不形成孢子，这表明ssgA是whi基因家族的成员。对位于ssgA上游且编码IclR型转录调节因子的ssfR的转录分析表明，没有发生从ssfR到ssgA的通读，并且在没有ssfR的情况下ssgA也被转录。因此发现ssgA受AdpA控制，而在可检测的程度上不受SsfR控制。SsfR似乎独立于SsgA或通过以某种未知方式与SsgA相互作用来调节孢子隔膜的形成，因为一个ssfR缺失突变体也表现出whi表型。
