Darwin A, Tormay P, Page L, Griffiths L, Cole J
School of Biochemistry, University of Birmingham, UK.
J Gen Microbiol. 1993 Aug;139(8):1829-40. doi: 10.1099/00221287-139-8-1829.
The formate dehydrogenases of Escherichia coli involved in electron transfer from formate to nitrite (Nrf activity: nitrite reduction by formate) have been identified. No previously undescribed selenoprotein was detected in bacteria grown under conditions optimal for the expression of Nrf activity. The Nrf activities of single mutants defective in either FdhN or FdhH were between 50 and 60% that of the parental strain. A double mutant defective in both FdhN and FdhH retained less than 10% of the activity of the FdhN+ FdhH+ strain. No Nrf activity was detected in a triple mutant defective in FdhN, FdhH and FdhO or in the selC strain. It is concluded that all three of the known formate dehydrogenases of E. coli can contribute to the transfer of electrons from formate to the Nrf pathway. Mutants defective in Nrf activity and cytochrome c552 synthesis were isolated by insertion mutagenesis or identified amongst strains received from the E. coli Genetic Stock Center. The mutations were located in at least three regions of the chromosome, including the 92 to 94 minute region which includes fdhF, the gene encoding FdhH required for formate hydrogenlyase activity. Fine structure mapping by P1 transduction established that the nrf mutations in the fdhF region were due to defects in three separable loci, all of which were independent of but close to fdhF. Clones were isolated from a cosmid library that complemented a deletion extending from fdhF into a region essential for Nrf activity. From these clones, plasmids were isolated that complemented only some of the Nrf- mutations in the 92 to 94 minute region, confirming the presence of different operons essential for Nrf activity and cytochrome c552 synthesis in this region. Suggested reasons for this genetic complexity include the need for proteins involved in electron transfer from the various formate dehydrogenases to cytochrome c552, for the attachment of the haem group to the apocytochrome and for cytochrome c552 export into the periplasm.
已鉴定出大肠杆菌中参与从甲酸向亚硝酸盐进行电子转移的甲酸脱氢酶（Nrf活性：甲酸还原亚硝酸盐）。在最有利于Nrf活性表达的条件下培养的细菌中，未检测到以前未描述的硒蛋白。FdhN或FdhH有缺陷的单突变体的Nrf活性为亲本菌株的50%至60%。FdhN和FdhH均有缺陷的双突变体保留的活性不到FdhN+FdhH+菌株活性的10%。在FdhN、FdhH和FdhO有缺陷的三突变体或selC菌株中未检测到Nrf活性。得出的结论是，大肠杆菌的所有三种已知甲酸脱氢酶都可促进电子从甲酸向Nrf途径的转移。通过插入诱变分离出Nrf活性和细胞色素c552合成有缺陷的突变体，或在从大肠杆菌遗传菌种保藏中心获得的菌株中鉴定出此类突变体。这些突变位于染色体的至少三个区域，包括92至94分钟区域，该区域包含fdhF，即甲酸氢解酶活性所需的FdhH编码基因。通过P1转导进行的精细结构作图确定，fdhF区域中的nrf突变是由于三个可分离位点的缺陷所致，所有这些位点均独立于fdhF但与之接近。从黏粒文库中分离出的克隆可互补从fdhF延伸至Nrf活性必需区域的缺失。从这些克隆中分离出的质粒仅可互补92至94分钟区域中的部分Nrf-突变，证实该区域存在对Nrf活性和细胞色素c552合成必不可少的不同操纵子。造成这种遗传复杂性的可能原因包括需要参与从各种甲酸脱氢酶向细胞色素c552进行电子转移的蛋白质、需要将血红素基团附着到脱辅基细胞色素上以及需要将细胞色素c552输出到周质中。
