Molecular and mutational analysis of a DNA region separating two methylotrophy gene clusters in Methylobacterium extorquens AM1
Molecular and mutational analysis of a DNA region separating two methylotrophy gene clusters in Methylobacterium extorquens AM1
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DOI：
10.1099/00221287-143-5-1729
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发表时间：
1997-05-01
期刊：
影响因子：
--
通讯作者：
Lidstrom, ME
中科院分区：
文献类型：
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作者：
Chistoserdova, L;Lidstrom, ME
已经分析了14.2的区域，其是扭脱甲基杆菌AM 1染色体上的基因座的一部分，所述基因座含有参与一碳（1）代谢的许多基因，包括丝氨酸循环基因、pqq基因、调节甲醇氧化基因和N5，N10-亚甲基四氢叶酸脱氢酶（mtdA）的基因。在新的区域内鉴定了15个新的开放阅读框，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端部分，苹果酰辅酶A裂解酶，未知功能的9.4和31 kDa多肽，ABC型转运蛋白的3个推定亚基，两个类似于M.扭脱醌AM 1和其他甲基营养菌，细胞色素c，叶酸代谢的三种酶，以及在蛋白质数据库中没有同源物的13和20.5 kDa的多肽。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C-1代谢相关。编码苹果酰辅酶A裂解酶的mclA突变导致C-1+表型。不可能在假定的叶酸代谢基因folC中获得无效突变体，这意味着这些叶酸合成基因对于C-1和多碳化合物的代谢是必要的。在推定的ABC转运蛋白基因，类似于mxaG和mxaJ的基因，和其他身份不明的ORF的突变产生的双交叉重组与C-1阳性表型。已使用启动子探针载体pHX 200研究了orf 3和orf 4上游的启动子区。这些启动子的转录在野生型M.但在调节性mox突变体中增加。
A region of 14.2 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 (1) 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 C-1 metabolism. A mutation in mclA, encoding malyl-CoA lyase, resulted in a C-1-plus phenotype. It is not possible to obtain null mutants in a putative folate metabolism gene, folC, implying the necessity of these folate synthesis genes for metabolism of C-1, 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 C-1-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.