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Link between the Membrane-Bound Pyridine Nucleotide Transhydrogenase and Glutathione-Dependent Processes in Rhodobacter sphaeroides

Jason W. Hickman,¹ Robert D. Barber,^2, Eric P. Skaar,^1, and Timothy J. Donohue^1*

Department of Bacteriology,¹ Graduate Program in Cell and Molecular Biology, University of Wisconsin—Madison, Madison, Wisconsin 53706²

Received 30 August 2001/ Accepted 22 October 2001

The presence of a glutathione-dependent pathway for formaldehyde oxidation in the facultative phototroph Rhodobacter sphaeroides has allowed the identification of gene products that contribute to formaldehyde metabolism. Mutants lacking the glutathione-dependent formaldehyde dehydrogenase (GSH-FDH) are sensitive to metabolic sources of formaldehyde, like methanol. This growth phenotype is correlated with a defect in formaldehyde oxidation. Additional methanol-sensitive mutants were isolated that contained Tn5 insertions in pntA, which encodes the subunit of the membrane-bound pyridine nucleotide transhydrogenase. Mutants lacking transhydrogenase activity have phenotypic and physiological characteristics that are different from those that lack GSH-FDH activity. For example, cells lacking transhydrogenase activity can utilize methanol as a sole carbon source in the absence of oxygen and do not display a formaldehyde oxidation defect, as determined by whole-cell ¹³C-nuclear magnetic resonance. Since transhydrogenase can be a major source of NADPH, loss of this enzyme could result in a requirement for another source for this compound. Evidence supporting this hypothesis includes increased specific activities of other NADPH-producing enzymes and the finding that glucose utilization by the Entner-Doudoroff pathway restores aerobic methanol resistance to cells lacking transhydrogenase activity. Mutants lacking transhydrogenase activity also have higher levels of glutathione disulfide under aerobic conditions, so it is consistent that this strain has increased sensitivity to oxidative stress agents like diamide, which are known to alter the oxidation reduction state of the glutathione pool. A model will be presented to explain the role of transhydrogenase under aerobic conditions when cells need glutathione both for GSH-FDH activity and to repair oxidatively damaged proteins.

Present address: Department of Biological Sciences, University of Wisconsin—Parkside, Kenosha, WI 53141.

Present address: Department of Microbiology-Immunology, Northwestern University, Chicago, IL 60611.

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