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

Christopher J. Marx^1,

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 (H₄F) pathway in methylotrophy. Previously, it has not been possible to generate null mutants lacking either mtdA (encoding an NADP-dependent methylene-H₄F/methylene-tetrahydromethanopterin dehydrogenase) or fch (encoding methenyl-H₄F cyclohydrolase). An unmarked strain was generated that expressed the analogous folD gene (encoding a bifunctional NADP-dependent methylene-H₄F dehydrogenase/methenyl-H₄F 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 C₁ 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 C₁ 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-H₄F for biosynthesis of purines and other compounds, and have provided clear genetic evidence that the H₄F pathway is required for methylotrophy.

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).

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