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

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 CM4T. 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.