1887

Abstract

Over the past few years, the genetic ‘toolkit’ available for use with 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 (HF) pathway in methylotrophy. Previously, it has not been possible to generate null mutants lacking either (encoding an NADP-dependent methylene-HF/methylene-tetrahydromethanopterin dehydrogenase) or (encoding methenyl-HF cyclohydrolase). An unmarked strain was generated that expressed the analogous gene (encoding a bifunctional NADP-dependent methylene-HF dehydrogenase/methenyl-HF cyclohydrolase) from CM4. In this strain, null mutants could be obtained that grew normally on multicarbon substrates but were defective for growth on C substrates. Additionally, null mutants of and/or could also be generated in the wild-type by supplementing the succinate medium with formate. These strains were unable to grow on C compounds but were not methanol-sensitive. These approaches have demonstrated that the apparent essentiality of and is due to the need for formyl-HF for biosynthesis of purines and other compounds, and have provided clear genetic evidence that the HF pathway is required for methylotrophy.

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2004-01-01
2019-11-18
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