Leinfelder W, Forchhammer K, Zinoni F, Sawers G, Mandrand-Berthelot M A, Böck A
Lehrstuhl für Mikrobiologie, Universität München, Federal Republic of Germany.
J Bacteriol. 1988 Feb;170(2):540-6. doi: 10.1128/jb.170.2.540-546.1988.
Mutants of Escherichia coli were isolated which were affected in the formation of both formate dehydrogenase N (phenazine methosulfate reducing) (FDHN) and formate dehydrogenase H (benzylviologen reducing) (FDHH). They were analyzed, together with previously characterized pleiotropic fdh mutants (fdhA, fdhB, and fdhC), for their ability to incorporate selenium into the selenopolypeptide subunits of FDHN and FDHH. Eight of the isolated strains, along with the fdhA and fdhC mutants, maintained the ability to selenylate tRNA, but were unable to insert selenocysteine into the two selenopolypeptides. The fdhB mutant tested had lost the ability to incorporate selenium into both protein and tRNA. fdhF, which is the gene coding for the 80-kilodalton selenopolypeptide of FDHH, was expressed from the T7 promoter-polymerase system in the pleiotropic fdh mutants. A truncated polypeptide of 15 kilodaltons was formed; but no full-length (80-kilodalton) gene product was detected, indicating that translation terminates at the UGA codon directing the insertion of selenocysteine. A mutant fdhF gene in which the UGA was changed to UCA expressed the 80-kilodalton gene product exclusively. This strongly supports the notion that the pleiotropic fdh mutants analyzed possess a lesion in the gene(s) encoding the biosynthesis or the incorporation of selenocysteine. The gene complementing the defect in one of the isolated mutants was cloned from a cosmid library. Subclones were tested for complementation of other pleiotropic fdh mutants. The results revealed that the mutations in the eight isolates fell into two complementation groups, one of them containing the fdhA mutation. fdhB, fdhC, and two of the new fdh isolates do not belong to these complementation groups. A new nomenclature (sel) is proposed for pleiotropic fdh mutations affecting selenium metabolism. Four genes have been identified so far: selA and selB (at the fdhA locus), selC (previously fdhC), and selD (previously fdhB).
分离出了大肠杆菌的突变体，这些突变体在甲酸盐脱氢酶N（吩嗪硫酸甲酯还原型）（FDHN）和甲酸盐脱氢酶H（苄基紫精还原型）（FDHH）的形成过程中均受到影响。将它们与先前已鉴定的多效性fdh突变体（fdhA、fdhB和fdhC）一起分析，以研究它们将硒掺入FDHN和FDHH的硒代多肽亚基中的能力。所分离出的8个菌株以及fdhA和fdhC突变体保持了硒化tRNA的能力，但无法将硒代半胱氨酸插入这两种硒代多肽中。所测试的fdhB突变体已丧失将硒掺入蛋白质和tRNA的能力。fdhF是编码FDHH的80千道尔顿硒代多肽的基因，它在多效性fdh突变体中由T7启动子 - 聚合酶系统表达。形成了一个15千道尔顿的截短多肽；但未检测到全长（80千道尔顿）基因产物，这表明翻译在指导硒代半胱氨酸插入的UGA密码子处终止。一个将UGA突变为UCA的突变fdhF基因仅表达80千道尔顿的基因产物。这有力地支持了这样一种观点，即所分析的多效性fdh突变体在编码硒代半胱氨酸生物合成或掺入的基因中存在损伤。从黏粒文库中克隆出了一个能够弥补其中一个分离突变体缺陷的基因。对亚克隆进行了测试，以检测其对其他多效性fdh突变体的互补作用。结果表明，8个分离株中的突变分为两个互补组，其中一组包含fdhA突变。fdhB、fdhC以及两个新的fdh分离株不属于这些互补组。对于影响硒代谢的多效性fdh突变，提出了一种新的命名法（sel）。到目前为止，已鉴定出四个基因：selA和selB（位于fdhA位点）、selC（先前的fdhC）和selD（先前的fdhB）。
