Identification of proteins involved in formaldehyde metabolism by Rhodobacter sphaeroides

Abstract

Formaldehyde is an intermediate formed during the metabolism of methanol or other methylated compounds. Many Gram-negative bacteria generate formaldehyde from methanol via a periplasmic pyrroloquinoline quinone (PQQ)-dependent dehydrogenase in which the alpha subunit of an alpha(2)beta(2) tetramer has catalytic activity. The genome of the facultative formaldehyde-oxidizing bacterium Rhodobacter sphaeroides encodes XoxF, a homologue of the catalytic subunit of a proposed PQQ-containing dehydrogenase of Paracoccus denitrificans. R. sphaeroides xoxF is part of a gene cluster that encodes periplasmic c-type cytochromes, including CycI, isocytochrome c(2) and CycB (a cyt c(553i) homologue), as well as adhI, a glutathione-dependent formaldehyde dehydrogenase (GSH-FDH), and gfa, a homologue of a glutathione-formaldehyde activating enzyme (Gfa). To test the roles of XoxF, CycB and Gfa in formaldehyde metabolism by R. sphaeroides, we monitored photosynthetic growth with methanol as a source of formaldehyde and whole-cell methanol-dependent oxygen uptake. Our data show that R. sphaeroides cells lacking XoxF or CycB do not exhibit methanol-dependent oxygen uptake and lack the capacity to utilize methanol as a sole photosynthetic carbon source. These results suggest that both proteins are required for formaldehyde metabolism. R. sphaeroides Gfa is not essential to activate formaldehyde, as cells lacking gfa are capable of both methanol-dependent oxygen uptake and growth with methanol as a photosynthetic carbon source.

Fig. 1

Succinate and methanol oxidation by R. sphaeroides. (a) Shown is the direction of gene transcription (arrowhead) and the intergenic distances within this region of the R. sphaeroides genome. Trimethoprim (Tp^R) cartridge insertion is indicated by ▲; the extent of wild-type DNA in cosmid pUI8747 is shown above the map. Known operons are shown by a dashed arrow (Barber et al., 1996; Rott et al., 1993), whereas dotted arrows show potential operons (Y. Dufour, personal communication). (b) Periplasmic oxidation of methanol to formaldehyde results in the concomitant transfer of electrons to CycB and to isocyt c₂, and the reduction of O₂ by a terminal cytochrome oxidase. Alternatively, under photosynthetic conditions, electrons can be transferred to RC complexes in the membrane. (c) During succinate-dependent respiration, succinate dehydrogenase (SDH) reduces ubiquinone (UQH₂) via a terminal quinol oxidase. Alternatively, electrons from quinone can pass through cyt bc₁, then to periplasmic cytochromes such as isocyt c₂, and finally onto membrane-bound cytochrome oxidase to reduce O₂.

Fig. 2

Whole-cell O₂ uptake by wild-type cells. Plotted is the percentage O₂ concentration as a function of time for each carbon source tested. (◆) Data used to calculate the endogenous rate of O₂ uptake (O₂ consumption without the addition of any carbon source); (■) samples containing methanol; (▲) samples containing succinate.

Fig. 3

Phylogenetic analysis of XoxF orthologues. Amino acid sequence identities among α-proteobacteria XoxF homologues are represented. The phylogenetic tree shows all three wild-type strains of R. sphaeroides grouping in one of two clades based on evolutionary distance. This analysis was generated with _MEGA (Kumar et al., 2004), and bootstrap values above 50 % from 500 replicates are shown. Scale bar, 0.02 substitutions per site.