Chistoserdova Ludmila, Lidstrom Mary E
Department of Chemical Engineering, Box 351750, University of Washington, Seattle, WA 98195-1750, USA.
Department of Microbiology, Box 35742, University of Washington, Seattle, WA 98195-1750, USA.
Microbiology (Reading). 1997 May;143 ( Pt 5):1729-1736. doi: 10.1099/00221287-143-5-1729.
A region of 14.2 kb has been analysed that is a part of a locus on the Methylobacterium extorquens AM1 chromosome containing a number of genes involved in one-carbon (C1) metabolism, including serine cycle genes, pqq genes, regulatory methanol oxidation genes and the gene for N5,N10-methylene tetrahydrofolate dehydrogenase (mtdA). Fifteen new ORFs have been identified within the new region, and their sequences suggest that they encode the following polypeptides: the C-terminal part of phosphoenolpyruvate carboxylase, malyl-CoA lyase, polypeptides of 9.4 and 31 kDa of unknown function, three putative subunits of an ABC-type transporter, two polypeptides similar to the products of mxaF and mxaJ from M. extorquens AM1 and other methylotrophs, a cytochrome c, three enzymes of folate metabolism, and polypeptides of 13 and 20.5 kDa with no homologues in the protein database. Ten insertion mutations have been generated in the region to determine if the newly identified genes are associated with C1 metabolism. A mutation in mclA, encoding malyl-CoA lyase, resulted in a C1-minus phenotype, while mutations in the other genes all showed a C1-plus phenotype. It was not possible to obtain null mutants in a putative folate metabolism gene, folC, implying the necessity of these folate synthesis genes for metabolism of C1 and multicarbon compounds. Mutations in the putative ABC transporter genes, the genes similar to mxaG and mxaJ, and other unidentified ORFs produced double-crossover recombinants with a C1-positive phenotype. Promoter regions have been investigated upstream of orf3 and orf4 using the promoter probe vector pHX200. Transcription from these promoters was weak in wild-type M. extorquens AM1 but increased in regulatory mox mutants.
对一个14.2 kb的区域进行了分析，该区域是甲基营养型生丝微菌AM1染色体上一个基因座的一部分，包含许多参与一碳（C1）代谢的基因，包括丝氨酸循环基因、pqq基因、调节甲醇氧化基因以及N5,N10-亚甲基四氢叶酸脱氢酶（mtdA）基因。在这个新区域内鉴定出了15个新的开放阅读框（ORF），它们的序列表明它们编码以下多肽：磷酸烯醇式丙酮酸羧化酶的C末端部分、苹果酰辅酶A裂解酶、功能未知的9.4 kDa和31 kDa多肽、一种ABC型转运蛋白的三个假定亚基、两种与甲基营养型生丝微菌AM1和其他甲基营养菌的mxaF和mxaJ产物相似的多肽、一种细胞色素c、三种叶酸代谢酶，以及在蛋白质数据库中没有同源物的13 kDa和20.5 kDa多肽。在该区域产生了10个插入突变，以确定新鉴定的基因是否与C1代谢相关。编码苹果酰辅酶A裂解酶的mclA中的一个突变导致了C1缺陷型表型，而其他基因中的突变均表现出C1阳性表型。在一个假定的叶酸代谢基因folC中无法获得缺失突变体，这意味着这些叶酸合成基因对于C1和多碳化合物的代谢是必需的。假定的ABC转运蛋白基因、与mxaG和mxaJ相似的基因以及其他未鉴定的ORF中的突变产生了具有C1阳性表型的双交换重组体。使用启动子探针载体pHX200对orf3和orf4上游的启动子区域进行了研究。这些启动子在野生型甲基营养型生丝微菌AM1中的转录较弱，但在调节性mox突变体中有所增加。
