Characterization of the Bacillus subtilis ywsC gene, involved in gamma-polyglutamic acid production.
Characterization of the Bacillus subtilis ywsC gene, involved in gamma-polyglutamic acid production.
复制标题
枯草芽孢杆菌 ywsC 基因的表征，该基因参与 γ-聚谷氨酸的生产。
作者：
Yuji Urushibata;S. Tokuyama;Y. Tahara
从发酵大豆中分离的枯草芽孢杆菌IFO16449中克隆了生产γ-聚谷氨酸(PGA)所需的基因。所克隆的4.2kb DNA片段有4个开放阅读框，与B.subtlis 168的ywsC和ywtABC基因序列基本一致。Northern杂交分析表明，这四个基因构成了一个操纵子。三个基因ywsC、ywtA和ywtB被干扰，以确定哪个基因在PGA生物合成中发挥核心作用。在Delta ywsC和Delta ywtA菌株中不产生PGA，表明这两个基因对PGA的产生是必需的。为了阐明YwsC蛋白的功能，在Delta ywsC菌株中产生了组氨酸标记的YwsC(YwsC-His)，并用镍-氮三乙酸亲和层析从转化子的溶菌酶处理裂解物中纯化了YwsC-His。Western印迹分析表明，YwsC-His蛋白由44 kDa和33 kDa两个亚基组成，这两个亚基是由ywsC基因的同相重叠编码的。(14)纯化的L-[(14)C]-谷氨酸在三磷酸腺苷和氯化锰存在下，通过酰基磷酸中间体合成了C标记的PGA，表明该基因编码PGA合成酶(EC6.3.2)。
The genes required for gamma-polyglutamic acid (PGA) production were cloned from Bacillus subtilis IFO16449, a strain isolated from fermented soybeans. There were four open reading frames in the cloned 4.2-kb DNA fragment, and they were almost identical to those in the ywsC and ywtABC genes of B. subtlis 168. Northern blot analysis showed that the four genes constitute an operon. Three genes, ywsC, ywtA, and ywtB, were disrupted to determine which gene plays a central role in PGA biosynthesis. No PGA was produced in Delta ywsC and Delta ywtA strains, indicating that both of these genes are essential for PGA production. To clarify the function of the YwsC protein, histidine-tagged YwsC (YwsC-His) was produced in the Delta ywsC strain and purified from the lysozyme-treated lysate of the transformant by Ni-nitrilotriacetic acid affinity chromatography. Western blot analysis revealed that the YwsC-His protein consists of two subunits, the 44-kDa and 33-kDa proteins, which are encoded by in-phase overlapping in the ywsC gene. (14)C-labeled PGA was synthesized by the purified proteins from L-[(14)C]-glutamate in the presence of ATP and MnCl(2), through an acylphosphate intermediate, indicating that the ywsC gene encodes PGA synthetase (EC 6.3.2), a crucial enzyme in PGA biosynthesis.