Development of an insertional expression vector system for Methylobacterium extorquens AM1 and generation of null mutants lacking mtdA and/or fch

• Published in: Microbiology (Society for General Microbiology) Vol. 150; no. 1; pp. 9 - 19
• Main Authors: Marx, Christopher J, Lidstrom, Mary E
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.01.2004; Society for General Microbiology
• Subjects: Biological and medical sciences; Formates - metabolism; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Genetic Vectors; Methenyltetrahydrofolate Cyclohydrolase - genetics; Methylobacterium - enzymology; Methylobacterium - genetics; Methylobacterium chloromethanicum; Methylobacterium extorquens; Methylobacterium extorquens - genetics; Methylobacterium extorquens - growth & development; Methylobacterium extorquens - metabolism; Microbiology; Molecular Sequence Data; Mutagenesis, Insertional; Mutation; Oxidoreductases Acting on CH-NH Group Donors - genetics; Phenotype; Plasmids - genetics; Gene expression; Vector; Mutation
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.26587-0

1 Department of Microbiology, University of Washington, Seattle, WA 98195, USA 2 Department of Chemical Engineering, University of Washington, Seattle, WA 98195, USA Correspondence Mary E. Lidstrom lidstrom{at}u.washington.edu Over the past few years, the genetic ‘toolkit’ available for use with Methylobacterium extorquens AM1 has expanded significantly. Here a further advance is presented and demonstrated, an insertional expression system that allows expression of genes from a stable, unmarked chromosomal locus. This system has been used to better understand the role of the tetrahydrofolate (H 4 F) pathway in methylotrophy. Previously, it has not been possible to generate null mutants lacking either mtdA (encoding an NADP-dependent methylene-H 4 F/methylene-tetrahydromethanopterin dehydrogenase) or fch (encoding methenyl-H 4 F cyclohydrolase). An unmarked strain was generated that expressed the analogous folD gene (encoding a bifunctional NADP-dependent methylene-H 4 F dehydrogenase/methenyl-H 4 F cyclohydrolase) from Methylobacterium chloromethanicum CM4 T . In this strain, null mutants could be obtained that grew normally on multicarbon substrates but were defective for growth on C 1 substrates. Additionally, null mutants of mtdA and/or fch could also be generated in the wild-type by supplementing the succinate medium with formate. These strains were unable to grow on C 1 compounds but were not methanol-sensitive. These approaches have demonstrated that the apparent essentiality of mtdA and fch is due to the need for formyl-H 4 F for biosynthesis of purines and other compounds, and have provided clear genetic evidence that the H 4 F pathway is required for methylotrophy. Abbreviations: gfp /GFP, green fluorescent protein; H 4 F, tetrahydrofolate; H 4 MPT, tetrahydromethanopterin; MCS, multiple-cloning site; P prefix, promoter; t prefix, terminator of transcription Present address: 2215 Biomedical Physical Sciences, Michigan State University, East Lansing, MI 98824-4320, USA. The GenBank accession numbers for the vector sequences reported in this article are AY307999 (pCM168) and AY308000 (pCM172).