Multiple formate dehydrogenase enzymes in the facultative methylotroph Methylobacterium extorquens AM1 are dispensable for growth on methanol

Abstract

Formate dehydrogenase has traditionally been assumed to play an essential role in energy generation during growth on C(1) compounds. However, this assumption has not yet been experimentally tested in methylotrophic bacteria. In this study, a whole-genome analysis approach was used to identify three different formate dehydrogenase systems in the facultative methylotroph Methylobacterium extorquens AM1 whose expression is affected by either molybdenum or tungsten. A complete set of single, double, and triple mutants was generated, and their phenotypes were analyzed. The growth phenotypes of the mutants suggest that any one of the three formate dehydrogenases is sufficient to sustain growth of M. extorquens AM1 on formate, while surprisingly, none is required for growth on methanol or methylamine. Nuclear magnetic resonance analysis of the fate of [(13)C]methanol revealed that while cells of wild-type M. extorquens AM1 as well as cells of all the single and the double mutants continuously produced [(13)C]bicarbonate and (13)CO(2), cells of the triple mutant accumulated [(13)C]formate instead. Further studies of the triple mutant showed that formate was not produced quantitatively and was consumed later in growth. These results demonstrated that all three formate dehydrogenase systems must be inactivated in order to disrupt the formate-oxidizing capacity of the organism but that an alternative formate-consuming capacity exists in the triple mutant.

FIG. 1.

C₁ metabolism of M. extorquens AM1. H₄MPT, tetrahydromethanopterin; H₄F, tetrahydrofolate; Fae, H₄MPT-dependent formaldehyde activating enzyme (39); MtdA, NADP-dependent methylene-H₄MPT dehydrogenase (8, 38); MtdB, NAD(P)-dependent methylene-H₄MPT dehydrogenase (11); Mch, methenyl-H₄MPT cyclohydrolase (30); Fhc, formyltransferase/hydrolase complex (29); Fch, methenyl-H₄F cyclohydrolase (30); FtfL, formate-H₄F ligase (23); FDH, formate dehydrogenase (; this study).

FIG. 2.

Clustering of genes encoding the three formate dehydrogenases.

FIG. 3.

Time course showing product formation by suspensions of M. extorquens AM1 incubated with 120 mM [¹³C]methanol. Before the experiment, cell suspensions were washed and resuspended in mineral medium (12) supplemented with 1 μM each of Mo and W; 125-MHz ¹³C NMR spectra were recorded in blocks of 5 min after the addition of methanol and are shown in the region from 60 to 180 ppm. The density of the cell suspensions (6 ml) was 15 mg (dry weight)/ml, the aeration rate was 38 ml/min, and the temperature was 30°C. For each spectrum, 125 scans were collected. The chemical shift for CO₂ is at 125.7 ppm, that for HCO₃⁻ is at 161.3 ppm, and that for formate is at 172.1; the signal corresponding to methanol is at 50.1 ppm (not shown).

FIG. 4.

Transient formate accumulation upon growth of M. extorquens AM1 and an FDH triple mutant in the presence of methanol. Cells (600 ml) were grown in a standard minimal medium in 2-liter Erlenmeyer flasks at 180 rpm and 30°C. Formate concentrations (indicated by bars) were determined by HPLC as described (27).