Cloning and characterization of the A-factor receptor gene from Streptomyces griseus
Cloning and characterization of the A-factor receptor gene from Streptomyces griseus
复制标题
灰色链霉菌 A 因子受体基因的克隆和表征
DOI：
10.1128/jb.177.21.6083-6092.1995
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发表时间：
1995
影响因子：
3.2
通讯作者：
S. Horinouchi
中科院分区：
文献类型：
--
作者：
Hiroyasu Onaka;Noriko Ando;T. Nihira;Yasuhiro Yamada;Teruhiko Beppu;S. Horinouchi
A因子（2-异辛酰-3R-羟甲基-γ-丁内酯）及其特异性受体蛋白控制灰色链霉菌中链霉素的产生、链霉素抗性和气生菌丝体的形成。从S. griseus IFO 13350.为了制备用于通过PCR方法克隆arpA的寡核苷酸引物，测定了ArpA和赖氨酰内肽酶产生的片段的NH 2-末端氨基酸序列。当在大肠杆菌中在T7启动子的控制下表达时，以这种方式克隆的arpA基因指导具有A因子特异性结合活性的蛋白质的合成。因此，arpA基因的结论是编码一个276个氨基酸的蛋白质与计算的分子量为29.1 kDa，通过核苷酸测序确定。用ArpA的同源二聚体观察到A因子结合活性。ArpA的NH 2端含有一个α-螺旋-转角-α-螺旋的DNA结合基序，与许多DNA结合蛋白具有很高的相似性，这表明ArpA通过直接与某个关键基因结合而发挥其对各种表型的调控功能。虽然一个突变株缺陷的ArpA蛋白和A-因子生产过量生产链霉素，并形成气生菌丝体和孢子早于野生型菌株，因为阻遏物样行为的ArpA，引入ArpA到这个突变体废除同时其链霉素生产和气生菌丝体的形成。所有这些数据是一致的想法，ArpA作为一个阻遏型调节剂的次级代谢产物的形成和形态发生在早期生长阶段和A-因子在一定的临界细胞内浓度释放去阻遏，从而导致继发性代谢和气生菌丝体形成的开始。链霉菌属中ArpA样蛋白的存在，如PCR所揭示的，与A-因子样化合物的存在一起，表明在该属的许多物种中存在类似于A-因子系统的激素控制。
A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) and its specific receptor protein control streptomycin production, streptomycin resistance, and aerial mycelium formation in Streptomyces griseus. The A-factor receptor protein (ArpA) was purified from a cell lysate of S. griseus IFO 13350. The NH2-terminal amino acid sequences of ArpA and lysyl endopeptidase-generated fragments were determined for the purpose of preparing oligonucleotide primers for cloning arpA by the PCR method. The arpA gene cloned in this way directed the synthesis of a protein having A-factor-specific binding activity when expressed in Escherichia coli under the control of the T7 promoter. The arpA gene was thus concluded to encode a 276-amino-acid protein with a calculated molecular mass of 29.1 kDa, as determined by nucleotide sequencing. The A-factor-binding activity was observed with a homodimer of ArpA. The NH2-terminal portion of ArpA contained an alpha-helix-turn-alpha-helix DNA-binding motif that showed great similarity to those of many DNA-binding proteins, which suggests that it exerts its regulatory function for the various phenotypes by directly binding to a certain key gene(s). Although a mutant strain deficient in both the ArpA protein and A-factor production overproduces streptomycin and forms aerial mycelium and spores earlier than the wild-type strain because of repressor-like behavior of ArpA, introduction of arpA into this mutant abolished simultaneously its streptomycin production and aerial mycelium formation. All of these data are consistent with the idea that ArpA acts as a repressor-type regulator for secondary metabolite formation and morphogenesis during the early growth phase and A-factor at a certain critical intracellular concentration releases the derepression, thus leading to the onset of secondary metabolism and aerial mycelium formation. The presence of ArpA-like proteins among Streptomyces spp., as revealed by PCR, together with the presence of A-factor-like compounds, suggests that a hormonal control similar to the A-factor system exists in many species of this genus.