Trimethylamine N -oxide metabolism by abundant marine heterotrophic bacteria

Abstract

Trimethylamine N-oxide (TMAO) is a common osmolyte found in a variety of marine biota and has been detected at nanomolar concentrations in oceanic surface waters. TMAO can serve as an important nutrient for ecologically important marine heterotrophic bacteria, particularly the SAR11 clade and marine Roseobacter clade (MRC). However, the enzymes responsible for TMAO catabolism and the membrane transporter required for TMAO uptake into microbial cells have yet to be identified. We show here that the enzyme TMAO demethylase (Tdm) catalyzes the first step in TMAO degradation. This enzyme represents a large group of proteins with an uncharacterized domain (DUF1989). The function of TMAO demethylase in a representative from the SAR11 clade (strain HIMB59) and in a representative of the MRC (Ruegeria pomeroyi DSS-3) was confirmed by heterologous expression of tdm (the gene encoding Tdm) in Escherichia coli. In R. pomeroyi, mutagenesis experiments confirmed that tdm is essential for growth on TMAO. We also identified a unique ATP-binding cassette transporter (TmoXWV) found in a variety of marine bacteria and experimentally confirmed its specificity for TMAO through marker exchange mutagenesis and lacZ reporter assays of the promoter for genes encoding this transporter. Both Tdm and TmoXWV are particularly abundant in natural seawater assemblages and actively expressed, as indicated by a number of recent metatranscriptomic and metaproteomic studies. These data suggest that TMAO represents a significant, yet overlooked, nutrient for marine bacteria. (1) and is predicted to have a number of important physiological roles (2). In marine elasmobranchs (sharks and rays), TMAO accumulates at high concentrations (up to 500 mM), helping to offset the destabilizing effects of urea on cellular proteins (1, 3, 4). TMAO can be metabolized to small methylated amines, for example, tri-, di-, and monomethylamine (TMA, DMA, and MMA, respectively). These volatile organic N compounds are precursors of marine aerosols and the potent greenhouse gas nitrous oxide in the marine atmosphere (5). In anoxic sediments or pockets of hypoxic conditions, such as in marine snow, they are precursors for the potent greenhouse gas methane (6). In marine surface waters, TMAO concentrations can reach up to 79 nM; however, owing to the technical difficulties associated with quantifying TMAO in seawater, reports of in situ concentrations of TMAO are limited (7,8). In a previously published study in which TMAO and TMA were quantified in the marine environment, TMAO had a higher average concentration throughout the water column and over a seasonal cycle (8).TMAO is a well-studied terminal electron acceptor for anaerobic microbial respiration (9, 10), but its catabolism in aerobic surface seawater is not well understood. Recent studies have shown that TMAO in the Sargasso Sea is predominantly oxidized by bacterioplankton as an energy source (11) and that the marine methylotrophic bacterium Methylophilales sp. HTCC2181 oxidizes TMAO to CO 2 to generate energy (1...

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

“…Gentamicin (10 mg ml À 1 ) was added to maintain mutant strains Dtmm::Gm and Dtdm::Gm (Lidbury et al, 2014). For all experiments R. pomeroyi (wild type and mutants) was grown in marine ammonium mineral salts (MAMS) medium (Schäfer, 2007) Figure 1 Proposed model for methylated amine catabolism in the marine bacterium Ruegeria pomeroyi DSS-3.…”

“…Text in brackets denotes the locus tag of the corresponding gene in R. pomeroyi. CH 2 ¼ H 4 F, 5,10-methylene tetrahydrofolate; CO 2 , carbon dioxide; DMA, dimethylamine; Dmm, dimethylamine monooxygenase; GMA, gamma-glutamylmethylamide; GmaS, gamma-glutamylmethylamide synthetase; MgdABCD, N-methylglutamate dehydrogenase; MgsABC, N-methylglutamate synthase; MMA, monomethylamine; NMG, N-methylglutamate; TMA, trimethylamine; TMAO, trimethylamine N-oxide; TmoXWV, ATP-dependent TMAO transporter (Lidbury et al, 2014). was modified from Schäfer (2007) (Schäfer, 2007). Vitamins were prepared as described previously (Chen, 2012).…”

“…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). The genome of R. pomeroyi was sequenced in 2004 (Moran et al, 2004), and this bacterium is now a model organism enabling a better understanding of how and why marine bacteria metabolise a wide range of substrates (Moran et al, 2004;Cunliffe, 2012;Todd et al, 2012;Lidbury et al, 2014).…”

“…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.…”

Quantification of glycine betaine, choline and trimethylamine N-oxide in seawater particulates: Minimisation of seawater associated ion suppression

“…Similarly, MRC have been shown to use TMAO as an energy source which also resulted in ammonia production [12], and the capacity for TMAO binding in MRC is thought to be widespread [13]. Members of the Pelagibacterales bacteria (SAR11 clade) also have the capacity to degrade TMAO [14]. Marine or estuarine methanogens can also grow on nitrogenous osmolytes [15e17] indicating a link between quaternary amines and biological methane production in marine environments.…”