Downloaded from www.microbiologyresearch.org by IP: 54.162.190.106 On: Thu, 25 Feb 2016 22:54:00 MicrObiology (1997), 143, 1729-1 736 Printed in Great Britain Molecular and mutational analysis of a DNA region separating two methylotrophy gene clusters in Methylobacterium extorquens AM1 Ludmila Chistoserdova' and Mary E. Lidstrom1n2 Author for correspondence: Mary E. Lidstrom. Tel: + 1 206 616 5282. Fax: + 1 206 616 5721. e-mail : lidstrom@cheme.washington.edu 1.2 Department of Chemical A region of 14.2 kb has been analysed that is a part of a locus on the Methylobacteriumextorquens AM1 chromosome containing a number of Engineering', Box 351750, and Department of Microbiologyz, Box genes involved in one-carbon (C,) metabolism, including serine cycle genes, 35742, University of pqq genes, regulatory methanol oxidation genes and the gene for N5,NlO- Washingtoni wA methylene tetrahydrofolate dehydrogenase (rntdA). 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 94 and 31 kDa of unknown function, three putative subunits of an ABC-type transporter, two polypeptides similar to the products of m0F and mxal 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, metabolism. A mutation in rnc/A, encoding malyl-CoA lyase, resulted in a C,-minus phenotype, while mutations in the other genes all showed a C,-plus phenotype. It was not possible to obtain null mutants in a putative folate metabolism gene, WC, implying the necessity of these folate synthesis genes for metabolism of C, and multicarbon compounds. Mutations in the putative ABC transporter genes, the genes similar to maG and mxal, and other unidentified ORFs produced double-crossover recombinants with a C,-positive phenotype. Promoter regions have been investigated upstream of Orf3 and orf4 using the promoter probe vector pHX2OO. Transcription from these promoters was weak in wild-type M. extorquens AM1 but increased in regulatory MOX mutants. 98195-1750, USA 1 Keywords : Methylobacterium extorquens AM1, methylotrophy INTRODUCTION compounds are induced (Anthony, 1982). More than 20 genes have been shown to be specifically involved in Metbylobacterium extorquens AM1 is a pink-pigmented methanol oxidation to formaldehyde (Lidstrom et al., facultative methylotroph of the a-proteobacteria class 1994), and at least 11 genes have been identified to be (Peel & Quayle, 1961 ; Tsuji et al., 1990). During growth involved in methylamine oxidation (Chistoserdov et af., of M . extorquens AM1 on C, compounds such as 1994). The formaldehyde produced by methanol de- methanol or methylamine, specific enzyme systems hydrogenase (MDH) or methylamine dehydrogenase involved in the oxidation and assimilation of C, (MADH) is assimilated via the serine cycle, and the levels of the assimilatory enzymes are found to be increased on C, substrates as compared to multicarbon Abbreviations : MADH, methylamine dehydrogenase; MCL, malyl-CoA lyase; MDH, methanol dehydrogenase. substrates (Large & Quayle, 1962). Recently, a number The GenBank accession numbers for the 12312 nt (EcoRI-Pstl fragment) Of chromosomal regions Of extorquens have and 1849 nt (pstl-Bg/ll fragment) sequences reported in this paper are been characterized which are involved in C, metabolism, U72662 and U60993, respectively. showing that many C, genes are clustered. Genes 0002-1339 0 1997 SGM 1729 ... ..............................................................................................................................................