Stoker K, Reijnders W N, Oltmann L F, Stouthamer A H
Department of Microbiology, Vrije Universiteit, Amsterdam, The Netherlands.
J Bacteriol. 1989 Aug;171(8):4448-56. doi: 10.1128/jb.171.8.4448-4456.1989.
To isolate genes from Escherichia coli which regulate the labile hydrogenase activity, a plasmid library was used to transform hydL mutants lacking the labile hydrogenase. A single type of gene, designated hydG, was isolated. This gene also partially restored the hydrogenase activity in hydF mutants (which are defective in all hydrogenase isoenzymes), although the low hydrogenase 1 and 2 levels were not induced. Therefore, hydG apparently regulates, specifically, the labile hydrogenase activity. Restoration of this latter activity in hydF mutants was accompanied by a proportional increase of the H2 uptake activity, suggesting a functional relationship. H2:fumarate oxidoreductase activity was not restored in complemented hydL mutants. These latter strains may therefore lack, in addition to the labile hydrogenase, a second component (provisionally designated component R), possibly an electron carrier coupling H2 oxidation to the anerobic respiratory chain. Sequence analysis showed an open reading frame of 1,314 base pairs for hydG. It was preceded by a ribosome-binding site but apparently lacked a promoter. Minicell experiments revealed a single polypeptide of approximately 50 kilodaltons. Comparison of the predicted amino acid sequence with a protein sequence data base revealed strong homology to NtrC from Klebsiella pneumoniae, a DNA-binding transcriptional activator. The 411 base pairs upstream from pHG40 contained a second open reading frame overlapping hydG by four bases. The deduced amino acid sequence showed considerable homology with the C-terminal part of NtrB. This sequence was therefore assumed to be part of a second gene, encoding the NtrB-like component, and was designated hydH. The labile hydrogenase activity in E. coli is apparently regulated by a multicomponent system analogous to the NtrB-NtrC system. This conclusion is in agreement with the results of Birkmann et al. (A. Birkmann, R. G. Sawers, and A. Böck, Mol. Gen. Genet. 210:535-542, 1987), who demonstrated ntrA dependence for the labile hydrogenase activity.
为了从大肠杆菌中分离出调控不稳定氢化酶活性的基因，使用质粒文库转化缺乏不稳定氢化酶的hydL突变体。分离出了一种单一类型的基因，命名为hydG。该基因也部分恢复了hydF突变体（所有氢化酶同工酶均有缺陷）中的氢化酶活性，尽管氢化酶1和2的低水平未被诱导。因此，hydG显然特异性地调控不稳定氢化酶活性。hydF突变体中这种后一种活性的恢复伴随着H2摄取活性的成比例增加，表明存在功能关系。在互补的hydL突变体中，H2：延胡索酸氧化还原酶活性未恢复。因此，这些后一种菌株除了缺乏不稳定氢化酶外，可能还缺少第二种成分（暂定为成分R），可能是一种将H2氧化与厌氧呼吸链偶联的电子载体。序列分析显示hydG有一个1314个碱基对的开放阅读框。它前面有一个核糖体结合位点，但显然缺少启动子。微小细胞实验揭示了一条约50千道尔顿的单一多肽。将预测的氨基酸序列与蛋白质序列数据库进行比较，发现与肺炎克雷伯菌的NtrC有很强的同源性，NtrC是一种DNA结合转录激活因子。pHG40上游的411个碱基对包含第二个与hydG重叠四个碱基的开放阅读框。推导的氨基酸序列与NtrB的C末端部分有相当的同源性。因此，该序列被认为是第二个基因的一部分，编码NtrB样成分，并命名为hydH。大肠杆菌中不稳定氢化酶活性显然受一个类似于NtrB-NtrC系统的多组分系统调控。这一结论与Birkmann等人（A. Birkmann、R. G. Sawers和A. Böck，《分子遗传学与普通遗传学》210：535-542，1987年）的结果一致，他们证明了不稳定氢化酶活性对ntrA的依赖性。
