Characterization of an A-Factor-Responsive Repressor for amfR Essential for Onset of Aerial Mycelium Formation in Streptomyces griseus
Characterization of an A-Factor-Responsive Repressor for amfR Essential for Onset of Aerial Mycelium Formation in Streptomyces griseus
复制标题
灰色链霉菌中气生菌丝体形成所必需的 amfR A 因子响应阻遏蛋白的表征
DOI：
10.1128/jb.180.19.5085-5093.1998
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发表时间：
1998
影响因子：
3.2
通讯作者：
S. Horinouchi
中科院分区：
文献类型：
--
作者：
K. Ueda;Chia;Tsutomu Tosaki;H. Shinkawa;T. Beppu;S. Horinouchi
a因子（2-异辛基酰- 3r -羟甲基-γ-丁内酯）在稻褐霉气生菌丝形成过程中起着至关重要的作用。amfR是其中一种基因，当克隆到低拷贝数的质粒上时，可以抑制a因子缺陷突变体的空中菌丝阴性表型。染色体amfR基因的破坏导致气生菌丝形成的完全取消，表明amfR对形态发生的发生至关重要。amfR上游区域的克隆和核苷酸测序预测了一个由orf5、orf4和amfR组成的操纵子。与此观点一致，Northern blotting和S1定位分析表明，这三个基因主要由orf5前面的启动子（PORF5）共转录。此外，PORF5仅在a因子存在时才有活性，表明它依赖于a因子。凝胶迁移位移试验显示，在a因子缺乏突变株的细胞提取物中存在一种能够结合PORF5的蛋白（AdpB），而在野生型菌株中则没有。AdpB被纯化至均匀性，发现与转录起始点相关的- 72至- 44 bp区域特异性结合。纯化的AdpB和从营养菌丝体中分离的RNA聚合酶复合物对PORF5的径流转录分析表明，AdpB以浓度依赖的方式抑制转录。由此可见，作为气生菌丝形成开关的AmfR和作为AmfR抑制因子的AdpB是a因子调控级联的成员，导致形态发生。
ABSTRACT A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) is essential for the initiation of aerial mycelium formation inStreptomyces griseus. amfR is one of the genes which, when cloned on a low-copy-number plasmid, suppresses the aerial mycelium-negative phenotype of an A-factor-deficient mutant of S. griseus. Disruption of the chromosomal amfR gene resulted in complete abolition of aerial mycelium formation, indicating that amfR is essential for the onset of morphogenesis. Cloning and nucleotide sequencing of the region upstream ofamfR predicted an operon consisting of orf5,orf4, and amfR. Consistent with this idea, Northern blotting and S1 mapping analyses suggested that these three genes were cotranscribed mainly by a promoter (PORF5) in front of orf5. Furthermore, PORF5 was active only in the presence of A-factor, indicating that it is A-factor dependent. Gel mobility shift assays showed the presence of a protein (AdpB) able to bind PORF5 in the cell extract from an A-factor-deficient mutant but not from the wild-type strain. AdpB was purified to homogeneity and found to bind specifically to the region from −72 to −44 bp with respect to the transcriptional start point. Runoff transcriptional analysis of PORF5 with purified AdpB and an RNA polymerase complex isolated from vegetative mycelium showed that AdpB repressed the transcription in a concentration-dependent manner. It is thus apparent that AmfR as a switch for aerial mycelium formation and AdpB as a repressor for amfR are members in the A-factor regulatory cascade, leading to morphogenesis.