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Journal of Bacteriology, December 2007, p. 9076-9081, Vol. 189, No. 24
0021-9193/07/$08.00+0     doi:10.1128/JB.01229-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of a Fourth Formate Dehydrogenase in Methylobacterium extorquens AM1 and Confirmation of the Essential Role of Formate Oxidation in Methylotrophy ^,

Departments of Chemical Engineering,¹ Microbiology, University of Washington, Seattle, Washington,² Institute of Microbiology, ETH Zurich 8093, Switzerland,³ INSA Toulouse, Complexe Scientifique de Rangueil, 31077 Toulouse, France⁴

Received 30 July 2007/ Accepted 26 September 2007

A mutant of Methylobacterium extorquens AM1 with lesions in genes for three formate dehydrogenase (FDH) enzymes was previously described by us (L. Chistoserdova, M. Laukel, J.-C. Portais, J. A. Vorholt, and M. E. Lidstrom, J. Bacteriol. 186:22-28, 2004). This mutant had lost its ability to grow on formate but still maintained the ability to grow on methanol. In this work, we further investigated the phenotype of this mutant. Nuclear magnetic resonance experiments with [¹³C]formate, as well as ¹⁴C-labeling experiments, demonstrated production of labeled CO₂ in the mutant, pointing to the presence of an additional enzyme or a pathway for formate oxidation. The tungsten-sensitive phenotype of the mutant suggested the involvement of a molybdenum-dependent enzyme. Whole-genome array experiments were conducted to test for genes overexpressed in the triple-FDH mutant compared to the wild type, and a gene (fdh4A) was identified whose translated product carried similarity to an uncharacterized putative molybdopterin-binding oxidoreductase-like protein sharing relatively low similarity with known formate dehydrogenase alpha subunits. Mutation of this gene in the triple-FDH mutant background resulted in a methanol-negative phenotype. When the gene was deleted in the wild-type background, the mutant revealed diminished growth on methanol with accumulation of high levels of formate in the medium, pointing to an important role of FDH4 in methanol metabolism. The identity of FDH4 as a novel FDH was also confirmed by labeling experiments that revealed strongly reduced CO₂ formation in growing cultures. Mutation of a small open reading frame (fdh4B) downstream of fdh4A resulted in mutant phenotypes similar to the phenotypes of fdh4A mutants, suggesting that fdh4B is also involved in formate oxidation.

Published ahead of print on 5 October 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

This article has been cited by other articles:

• Crowther, G. J., Kosaly, G., Lidstrom, M. E. (2008). Formate as the Main Branch Point for Methylotrophic Metabolism in Methylobacterium extorquens AM1. J. Bacteriol. 190: 5057-5062 [Abstract] [Full Text]