Reconstruction of C(3) and C(4) metabolism in Methylobacterium extorquens AM1 using transposon mutagenesis.
The growth of Methylobacterium extorquens AM1 on C(1) compounds has been well-studied, but little is known about how this methylotroph grows on multicarbon compounds. A Tn5 transposon mutagenesis procedure was performed to identify genes involved in the growth of M. extorquens AM1 on succinate and pyruvate. Of the 15000 insertion colonies screened, 71 mutants were found that grew on methanol but either grew slowly or were unable to grow on one or both of the multicarbon substrates. For each of these mutants, the chromosomal region adjacent to the insertion site was sequenced, and 55 different genes were identified and assigned putative functions. These genes fell into a number of predicted categories, including central carbon metabolism, carbohydrate metabolism, regulation, transport and non-essential housekeeping functions. This study focused on genes predicted to encode enzymes of central heterotrophic metabolism: 2-oxoglutarate dehydrogenase, pyruvate dehydrogenase and NADH : ubiquinone oxidoreductase. In each case, the mutants showed normal growth on methanol and impaired growth on pyruvate and succinate, consistent with a role specific to heterotrophic metabolism. For the first two cases, no detectable activity of the corresponding enzyme was found in the mutant, verifying the predictions. The results of this study were used to reconstruct multicarbon metabolism of M. extorquens AM1 during growth on methanol, succinate and pyruvate.
Van Dien
Stephen J
SJ
Departments of Chemical Engineering, University of Washington, Seattle, WA 98195, USA.
Okubo
Yoko
Y
Departments of Chemical Engineering, University of Washington, Seattle, WA 98195, USA.
Hough
Melinda T
MT
Departments of Microbiology, University of Washington, Seattle, WA 98195, USA.
Korotkova
Natalia
N
Departments of Chemical Engineering, University of Washington, Seattle, WA 98195, USA.
Taitano
Tricia
T
Departments of Microbiology, University of Washington, Seattle, WA 98195, USA.
Lidstrom
Mary E
ME
Departments of Microbiology, University of Washington, Seattle, WA 98195, USA.|Departments of Chemical Engineering, University of Washington, Seattle, WA 98195, USA.
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism*; D003470:Culture Media; D004251:DNA Transposable Elements / Q000235:genetics*; D042967:Electron Transport Complex I; D007655:Ketoglutarate Dehydrogenase Complex / Q000235:genetics / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000432:Methanol / Q000378:metabolism; D020601:Methylobacterium extorquens / Q000201:enzymology / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism; D019802:Succinic Acid / Q000378:metabolism
D001426:Bacterial Proteins; D003470:Culture Media; D004251:DNA Transposable Elements; D002244:Carbon; D019289:Pyruvic Acid; D019802:Succinic Acid; D007658:Ketone Oxidoreductases; C053164:pyruvate dehydrogenase (NADP+); D007655:Ketoglutarate Dehydrogenase Complex; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I; D000432:Methanol