Yamazaki H, Ohnishi Y, Horinouchi S
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
J Bacteriol. 2000 Aug;182(16):4596-605. doi: 10.1128/JB.182.16.4596-4605.2000.
A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) at an extremely low concentration triggers streptomycin production and aerial mycelium formation in Streptomyces griseus. A-factor induces the expression of an A-factor-dependent transcriptional activator, AdpA, essential for both morphological and physiological differentiation by binding to the A-factor receptor protein ArpA, which has bound and repressed the adpA promoter, and dissociating it from the promoter. Nine DNA fragments that were specifically recognized and bound by histidine-tagged AdpA were isolated by cycles of a gel mobility shift-PCR method. One of them was located in front of a gene encoding an extracytoplasmic function sigma factor belonging to a subgroup of the primary sigma(70) family. The cloned gene was named AdpA-dependent sigma factor gene (adsA), and the gene product was named sigma(AdsA). Transcription of adsA depended on A-factor and AdpA, since adsA was transcribed at a very low and constant level in an A-factor-deficient mutant strain or in an adpA-disrupted strain. Consistent with this, transcription of adsA was greatly enhanced at or near the timing of aerial hyphae formation, as determined by low-resolution S1 nuclease mapping. High-resolution S1 mapping determined the transcriptional start point 82 nucleotides upstream of the translational start codon. DNase I footprinting showed that AdpA bound both strands symmetrically between the transcriptional start point and the translational start codon; AdpA protected the antisense strand from positions +7 to +41 with respect to the transcriptional start point and the sense strand from positions +12 to +46. A weak palindrome was found in the AdpA-binding site. The unusual position bound by AdpA as a transcriptional activator, in relation to the promoter, suggested the presence of a mechanism by which AdpA activates transcription of adsA in some unknown way. Disruption of the chromosomal adsA gene resulted in loss of aerial hyphae formation but not streptomycin or yellow pigment production, indicating that sigma(AdsA) is involved only in morphological development and not in secondary metabolic function. The presence of a single copy in each of the Streptomyces species examined by Southern hybridization suggests a common role in morphogenesis in this genus.
极低浓度的A因子（2-异辛酰基-3R-羟甲基-γ-丁内酯）可触发灰色链霉菌产生链霉素并形成气生菌丝体。A因子通过与已结合并抑制adpA启动子的A因子受体蛋白ArpA结合，并使其从启动子上解离，从而诱导A因子依赖性转录激活因子AdpA的表达，AdpA对形态和生理分化至关重要。通过凝胶迁移率变动-PCR方法循环分离出九个被组氨酸标签化的AdpA特异性识别并结合的DNA片段。其中一个位于编码属于主要σ（70）家族一个亚组的胞外功能σ因子的基因之前。克隆的基因被命名为AdpA依赖性σ因子基因（adsA），基因产物被命名为σ（AdsA）。adsA的转录依赖于A因子和AdpA，因为在A因子缺陷突变株或adpA破坏株中，adsA以非常低且恒定的水平转录。与此一致的是，如低分辨率S1核酸酶图谱分析所确定的，在气生菌丝形成的时间点或接近该时间点时，adsA的转录大大增强。高分辨率S1图谱确定了转录起始点在翻译起始密码子上游82个核苷酸处。DNase I足迹分析表明，AdpA在转录起始点和翻译起始密码子之间对称地结合两条链；AdpA保护反义链相对于转录起始点从+7到+41的位置，以及有义链从+12到+46的位置。在AdpA结合位点发现了一个弱回文序列。AdpA作为转录激活因子与启动子相关的异常结合位置表明存在一种机制，通过该机制AdpA以某种未知方式激活adsA的转录。染色体adsA基因的破坏导致气生菌丝形成丧失，但不影响链霉素或黄色色素的产生，表明σ（AdsA）仅参与形态发育，而不参与次级代谢功能。通过Southern杂交检测的每个链霉菌物种中均存在单拷贝，这表明其在该属的形态发生中具有共同作用。
