Wu L F, Mandrand-Berthelot M A
Biochimie. 1986 Jan;68(1):167-79. doi: 10.1016/s0300-9084(86)81081-1.
The Mu dl (ApR lac) bacteriophage was used to generate mutants of Escherichia coli which were defective in formate hydrogenlyase. Three mutants were chosen for further analysis: they lacked hydrogenase (hydrogen: benzyl viologen oxidoreductase) activity, but produced normal levels of fumarate reductase activity and two- to three-fold reduced levels of benzyl viologen (BV)-dependent formate dehydrogenase activity. Two of them (hydC) were shown to contain about 4-fold reduced amounts of formate hydrogenlyase and fumarate-dependent H2 uptake activities. The third one (hydD) was totally devoid of both activities. Their insertion sites were located at 77 min on the E. coli map. Subdivision of these mutants into two classes was subsequently based on the restoration capacity of hydrogenase activity with high concentration of nickel in the growth media. Addition of 500 microM NiCl2 led to a complete recovery of hydrogenase activity, and to the concomitant restoration of normal BV-linked formate dehydrogenase, formate hydrogenlyase and fumarate-dependent H2 uptake activities in the hydC mutants. The hydD mutant was insensitive to the effect of nickel. Expression of the lac operon in hydC and hydD mutants was induced by anaerobiosis. It was not increased by the addition of formate under anaerobic conditions. The presence of nitrate resulted in slightly reduced beta-galactosidase activities in the hydC mutants, whereas those found in the hydD mutant reached only one third of the level obtained in its absence. Fumarate had no effect on both classes. Moreover, in contrast to the hydD locus, the hydC::Mu dl fusions were found to be dependent upon the positive control exerted by the nirR gene product and were totally repressed by an excess of nickel. In addition, the low levels of overall hydrogenase-dependent activities found in a nirR strain were also relieved by the presence of nickel. Our results strongly suggest that the pleiotropic regulatory gene nirR is essential for the expression of a gene (hydC) involved in either transport or processing of nickel in the cell, whose alteration leads to a loss of hydrogenase activity.
利用Mu dl（ApR lac）噬菌体产生了甲酸氢化酶有缺陷的大肠杆菌突变体。选择了三个突变体进行进一步分析：它们缺乏氢化酶（氢：苄基紫精氧化还原酶）活性，但延胡索酸还原酶活性水平正常，且依赖苄基紫精（BV）的甲酸脱氢酶活性水平降低了两到三倍。其中两个（hydC）显示甲酸氢化酶和依赖延胡索酸的H2摄取活性降低了约4倍。第三个（hydD）则完全没有这两种活性。它们的插入位点位于大肠杆菌染色体图谱的77分钟处。随后根据生长培养基中高浓度镍对氢化酶活性的恢复能力，将这些突变体分为两类。添加500μM NiCl2可使氢化酶活性完全恢复，并使hydC突变体中正常的BV连接的甲酸脱氢酶、甲酸氢化酶和依赖延胡索酸的H2摄取活性同时恢复。hydD突变体对镍的作用不敏感。hydC和hydD突变体中lac操纵子的表达由厌氧诱导。在厌氧条件下添加甲酸不会使其增加。硝酸盐的存在导致hydC突变体中β-半乳糖苷酶活性略有降低，而在hydD突变体中发现的β-半乳糖苷酶活性仅为无硝酸盐时的三分之一。延胡索酸对这两类突变体均无影响。此外，与hydD基因座相反，发现hydC::Mu dl融合依赖于nirR基因产物施加的正调控，并被过量的镍完全抑制。此外，nirR菌株中发现的低水平总体氢化酶依赖性活性也因镍的存在而得到缓解。我们的结果强烈表明，多效调节基因nirR对于细胞中参与镍转运或加工的基因（hydC）的表达至关重要，该基因的改变导致氢化酶活性丧失。
