Bacterial flavin-containing monooxygenase is trimethylamine monooxygenase

Abstract

Flavin-containing monooxygenases (FMOs) are one of the most important monooxygenase systems in Eukaryotes and have many important physiological functions. FMOs have also been found in bacteria; however, their physiological function is not known. Here, we report the identification and characterization of trimethylamine (TMA) monooxygenase, termed Tmm, from Methylocella silvestris, using a combination of proteomic, biochemical, and genetic approaches. This bacterial FMO contains the FMO sequence motif (FXGXXXHXXXF/Y) and typical flavin adenine dinucleotide and nicotinamide adenine dinucleotide phosphate-binding domains. The enzyme was highly expressed in TMA-grown M. silvestris and absent during growth on methanol. The gene, tmm, was expressed in Escherichia coli, and the purified recombinant protein had high Tmm activity. Mutagenesis of this gene abolished the ability of M. silvestris to grow on TMA as a sole carbon and energy source. Close homologs of tmm occur in many Alphaproteobacteria, in particular Rhodobacteraceae (marine Roseobacter clade, MRC) and the marine SAR11 clade (Pelagibacter ubique). We show that the ability of MRC to use TMA as a sole carbon and/or nitrogen source is directly linked to the presence of tmm in the genomes, and purified Tmm of MRC and SAR11 from recombinant E. coli showed Tmm activities. The tmm gene is highly abundant in the metagenomes of the Global Ocean Sampling expedition, and we estimate that 20% of the bacteria in the surface ocean contain tmm. Taken together, our results suggest that Tmm, a bacterial FMO, plays an important yet overlooked role in the global carbon and nitrogen cycles.

Trimethylamine and trimethylamine N-oxide are supplementary energy sources for a marine heterotrophic bacterium: implications for marine carbon and nitrogen cycling

“…Methylated one-carbon compounds were originally thought to be substrates primarily for a specialised guild of bacteria, the methylotrophs (Chistoserdova et al, 2009;Chistoserdova, 2011); however, recent evidence has implicated marine heterotrophic bacteria in the catabolism of these compounds (Chen et al, 2011;Sun et al, 2011;Lidbury et al, 2014). Although a small percentage of isolates of the MRC can grow on TMA and TMAO as a sole C source, the majority appear to be able to only utilise these compounds as a sole N source, while maintaining the genes predicted to be involved in oxidation of the methyl groups (Chen, 2012).…”

“…HTCC2181), respectively, can couple TMAO oxidation to ATP production, which results in stimulation of growth (Sun et al, 2011;Halsey et al, 2012); however, these organisms fundamentally differ from members of the MRC. R. pomeroyi has the genes required for TMA catabolism (Figure 1) and can grow on TMA as a N source, but not on a sole C source, due to a lack of genes required for C assimilation via the serine cycle (Chen et al, 2011;Chen, 2012). Here we test the hypothesis that the oxidation of MAs is coupled to ATP production, providing an ecophysiological advantage to heterotrophic bacteria.…”

“…The ecological success of this clade may be in part due to their ability to utilise a variety of metabolic strategies to generate cellular energy, which allows for the more efficient utilisation of carbon (assimilation versus dissimilation; Sorokin et al, 2005;Moran and Miller, 2007;Boden et al, 2011b). For these reasons, the MRC bacteria have essential roles in both carbon and sulphur cycling, and more recently, nitrogen cycling (Buchan et al, 2005;Chen et al, 2011) within the marine environment. Ruegeria pomeroyi DSS-3 (basonym, Silicibacter pomeroyi DSS-3) is a member of the MRC, which was isolated off the coast of Georgia through enrichment with dimethylsulphoniopropionate (González et al, 2003).…”

“…While those MRC bacteria harbouring the genes necessary for TMA oxidation could all utilise TMA as a sole N source to support heterotrophic growth, only representatives from the genus Roseovarius of the MRC could grow on TMA as a sole carbon (C) source (methylotrophy). All marine bacteria that possess a functional TMA monooxygenase (Tmm; Chen et al, 2011) and a TMAO demethylase (Tdm; Lidbury et al, 2014) also have the genes necessary for the complete oxidation of methyl groups, cleaved off during catabolism of TMA (Sun et al, 2011;Chen, 2012;Halsey et al, 2012). Two different oligotrophic bacteria from the Alphaproteobacteria (Candidatus Pelagibacter ubique HTCC1062) and Betaproteobacteria (Methylophilales sp.…”

Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme

“…Choline is an essential nutrient for higher organisms, including humans, contributing to cell membrane function, methyl transfer events, and neurotransmission (1). The volatile odorant TMA is used as a carbon source by bacteria, is a precursor to the marine osmolyte trimethylamine-N-oxide (TMAO), and is converted to the powerful greenhouse gas methane by methanogenic archaea (2)(3)(4). The sole biochemical reaction directly connecting these two small molecules is the metabolism of choline to TMA by anaerobic microorganisms (Fig.…”