Chaudhuri A, Krasna A I
Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons, Columbia University, New York, NY 10032.
J Gen Microbiol. 1990 Jun;136(6):1153-60. doi: 10.1099/00221287-136-6-1153.
DNA fragments from Proteus vulgaris and Chromatium vinosum were isolated which restored hydrogenase activities in both hydA and hydB mutant strains of Escherichia coli. The hydA and hydB genes, which map near minute 59 of the genome map, 17 kb distant from each other, are not structural hydrogenase genes, but mutation in either of these genes leads to failure to synthesize any of the hydrogenase isoenzymes. The smallest DNA fragments which restored hydrogenase activity to both E. coli mutant strains were 4.7 kb from C. vinosum and 2.3 kb from P. vulgaris. These fragments were cleaved into smaller fragments which did not complement either of the E. coli mutations. The cloned heterologous genes also restored formate hydrogenlyase activity but they did not restore activity in hydE, hupA or hupB mutant strains of E. coli. The cloned genes, on plasmids, did not lead to the synthesis of proteins of sufficient size to be the hydrogenase catalytic subunit. The hydrogenase proteins synthesized by hydA and hydB mutant strains of E. coli transformed by cloned genes from P. vulgaris and C. vinosum were shown by isoelectric and immunological methods to be E. coli hydrogenase. Thus, these genes are not hydrogenase structural genes.
从普通变形杆菌和嗜硫红假单胞菌中分离出的DNA片段，可恢复大肠杆菌hydA和hydB突变株中的氢化酶活性。hydA和hydB基因位于基因组图谱的59分钟附近，彼此相距17 kb，它们不是氢化酶结构基因，但其中任何一个基因发生突变都会导致无法合成任何一种氢化酶同工酶。能使两种大肠杆菌突变株恢复氢化酶活性的最小DNA片段，来自嗜硫红假单胞菌的是4.7 kb，来自普通变形杆菌的是2.3 kb。这些片段被切割成更小的片段，这些小片段不能互补任何一种大肠杆菌突变。克隆的异源基因也恢复了甲酸氢化酶活性，但它们没有恢复大肠杆菌hydE、hupA或hupB突变株中的活性。质粒上的克隆基因不会导致合成足够大小的蛋白质以成为氢化酶催化亚基。通过等电和免疫方法显示，由来自普通变形杆菌和嗜硫红假单胞菌的克隆基因转化的大肠杆菌hydA和hydB突变株合成的氢化酶蛋白是大肠杆菌氢化酶。因此，这些基因不是氢化酶结构基因。
