Rousset M, Dermoun Z, Wall J D, Belaich J P
Laboratoire de Chimie Bactérienne, Centre National de la Recherche Scientifique, Marseille, France.
J Bacteriol. 1993 Jun;175(11):3388-93. doi: 10.1128/jb.175.11.3388-3393.1993.
Two genes, hynA and hynB, encode the two subunits of the periplasmic [NiFe] hydrogenase in Desulfovibrio fructosovorans. Sequencing downstream from hynB revealed a third open reading frame (hynC) that has the potential for encoding a polypeptide showing 21% identity with the HyaD, HoxM, and HupD proteins, belonging to putative operons encoding Escherichia coli hydrogenase 1, Alcaligenes eutrophus H16 membrane-bound hydrogenase, and Rhizobium leguminosarum uptake hydrogenase, respectively. Northern (RNA) blotting with a structural gene probe revealed the existence of a major transcript of 2.9 kb, which is the appropriate length to contain the two hydrogenase subunits only. In addition, two minor 4.4- and 5.8-kb transcripts that could contain hynABC and additional genes were found. The 5' end of the most abundant [NiFe] hydrogenase mRNA was found 170 bp upstream from the translational start site of hynA. The sequences at -10 and -35 relative to the transcriptional starting site showed 55% homology with the consensus sequences of the Escherichia coli sigma 70-type promoter. The cloning of that particular region as a promoter to control transcription of the lacZ gene in E. coli DH5 alpha or the hynA, hynB, and hynC genes in D. fructosovorans MR400 led to strong expression in both systems.
在果糖脱硫弧菌中，hynA和hynB这两个基因编码周质[NiFe]氢化酶的两个亚基。对hynB下游进行测序发现了第三个开放阅读框（hynC），它有可能编码一种与HyaD、HoxM和HupD蛋白具有21%同一性的多肽，这三种蛋白分别属于推定的操纵子，这些操纵子编码大肠杆菌氢化酶1、产碱杆菌H16膜结合氢化酶和豌豆根瘤菌摄取氢化酶。用结构基因探针进行Northern（RNA）印迹分析显示存在一个2.9 kb的主要转录本，其长度恰好只包含两个氢化酶亚基。此外，还发现了两个较小的4.4 kb和5.8 kb转录本，它们可能包含hynABC及其他基因。最丰富的[NiFe]氢化酶mRNA的5'端位于hynA翻译起始位点上游170 bp处。相对于转录起始位点的-10和-35序列与大肠杆菌σ7-0型启动子的共有序列具有55%的同源性。将该特定区域作为启动子进行克隆，以控制大肠杆菌DH5α中lacZ基因的转录或果糖脱硫弧菌MR400中hynA、hynB和hynC基因的转录，结果在这两个系统中均导致了强表达。
