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Dietary intake of L-carnitine can promote cardiovascular diseases in humans through microbial production of trimethylamine (TMA) and its subsequent oxidation to trimethylamine N-oxide by hepatic flavin-containing monooxygenases. Although our microbiota are responsible for TMA formation from carnitine, the underpinning molecular and biochemical mechanisms remain unclear. In this study, using bioinformatics approaches, we first identified a two-component Rieske-type oxygenase/reductase (CntAB) and associated gene cluster proposed to be involved in carnitine metabolism in representative genomes of the human microbiota. CntA belongs to a group of previously uncharacterized Rieske-type proteins and has an unusual "bridging" glutamate but not the aspartate residue, which is believed to facilitate intersubunit electron transfer between the Rieske center and the catalytic mononuclear iron center. Using Acinetobacter baumannii as the model, we then demonstrate that cntAB is essential in carnitine degradation to TMA. Heterologous overexpression of cntAB enables Escherichia coli to produce TMA, confirming that these genes are sufficient in TMA formation. Site-directed mutagenesis experiments have confirmed that this unusual "bridging glutamate" residue in CntA is essential in catalysis and neither mutant (E205D, E205A) is able to produce TMA. Taken together, the data in our study reveal the molecular and biochemical mechanisms underpinning carnitine metabolism to TMA in human microbiota and assign the role of this novel group of Rieske-type proteins in microbial carnitine metabolism. methylated amine metabolism | comparative genomics | gut microbiota

3Methane oxidation can occur in both aerobic and anaerobic environments; however, these are completely different processes involving different groups of prokaryotes. Aerobic methane oxidation is carried out by aerobic methanotrophs, and anaerobic methane oxidizers, discovered recently, thrive under anaerobic conditions and use sulfate or nitrate as electron donors for methane oxidation (11,104). This review will focus on the aerobic oxidation of methane.Aerobic methanotrophs are a unique group of methylotrophic bacteria that utilize methane as a sole carbon and energy source (52). Based on their cell morphology, ultrastructure, phylogeny, and metabolic pathways, methanotrophs can be divided into two taxonomic groups: type I and type II. Type I methanotrophs include the genera Methylobacter, Methylomicrobium, Methylomonas, Methylocaldum, Methylosphaera, Methylothermus, Methylosarcina, Methylohalobius, Methylosoma, and Methylococcus, which belong to the gamma subdivision of the Proteobacteria (Fig. 1). The type II methanotrophs Methylocystis, Methylosinus, Methylocella, and Methylocapsa are in the alpha subdivision of the Proteobacteria (52) (Fig. 1). Recently, two filamentous methane oxidizers have been described, Crenothrix polyspora (113), which has a novel pmoA, and Clonothrix fusca (125), which has a conventional pmoA. Both are gammaproteobacteria and are closely related to the type I methanotrophs. Most extant methanotrophs are cultured at 20 to 45°C and neutral pH but have also recently been isolated from extreme environments (reviewed in reference 122).The first step in the oxidation of methane to CO 2 is the conversion of methane to methanol by the enzyme methane monooxygenase. There are two forms of this enzyme: a particulate membrane bound form (pMMO) and a soluble cytoplasmic form (sMMO). The pMMO has been reported in all methanotrophs except for the genus Methylocella (121), whereas the sMMO is present only in certain methanotroph strains (94). The pMMO is a membrane bound copper and iron containing enzyme (reviewed in reference 49). The structural genes for this enzyme have been cloned and sequenced from Methylococcus capsulatus Bath (107, 114), Methylocystis sp. strain M, and Methylosinus trichosporium OB3b (45). They lie in a three-gene operon, pmoCAB, which code for three integral membrane polypeptides of approximately 23, 27, and 45 kDa, respectively. These operons are present in duplicate copies in all three organisms. These duplicate copies of pmoCAB are virtually identical and are transcribed from 70 -type promoters found upstream of the pmoC gene (45, 110).The sMMO is a cytoplasmic enzyme containing a unique di-iron site at its catalytic center. It has a broad substrate range, including trichloroethylene, alkanes, alkenes, and aromatic compounds. The biochemistry of the sMMO has been studied in detail (reviewed in reference 75). It consists of three components: a hydroxylase, which is a dimer of three subunits, (␣␤␥) 2 ; a regulatory protein (protein B); and a reductase (protein C). It is e...

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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...

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.

Microbial diversity in Movile Cave (Romania) was studied using bacterial and archaeal 16S rRNA gene sequence and functional gene analyses, including ribulose-1,5-bisphosphate carboxylase/ oxygenase (RuBisCO), soxB (sulfate thioesterase/thiohydrolase) and amoA (ammonia monooxygenase). Sulfur oxidizers from both Gammaproteobacteria and Betaproteobacteria were detected in 16S rRNA, soxB and RuBisCO gene libraries. DNA-based stable-isotope probing analyses using 13 C-bicarbonate showed that Thiobacillus spp. were most active in assimilating CO 2 and also implied that ammonia and nitrite oxidizers were active during incubations. Nitrosomonas spp. were detected in both 16S rRNA and amoA gene libraries from the 'heavy' DNA and sequences related to nitriteoxidizing bacteria Nitrospira and Candidatus 'Nitrotoga' were also detected in the 'heavy' DNA, which suggests that ammonia/nitrite oxidation may be another major primary production process in this unique ecosystem. A significant number of sequences associated with known methylotrophs from the Betaproteobacteria were obtained, including Methylotenera, Methylophilus and Methylovorus, supporting the view that cycling of one-carbon compounds may be an important process within Movile Cave. Other sequences detected in the bacterial 16S rRNA clone library included Verrucomicrobia, Firmicutes, Bacteroidetes, alphaproteobacterial Rhodobacterales and gammaproteobacterial Xanthomonadales. Archaeal 16S rRNA sequences retrieved were restricted within two groups, namely the Deep-sea Hydrothermal Vent Euryarchaeota group and the Miscellaneous Crenarchaeotic group. No sequences related to known sulfur-oxidizing archaea, ammonia-oxidizing archaea, methanogens or anaerobic methane-oxidizing archaea were detected in this clone library. The results provided molecular biological evidence to support the hypothesis that Movile Cave is driven by chemolithoautotrophy, mainly through sulfur oxidation by sulfur-oxidizing bacteria and reveal that ammonia-and nitrite-oxidizing bacteria may also be major primary producers in Movile Cave.

Upon phosphorus (P) deficiency, marine phytoplankton reduce their requirements for P by replacing membrane phospholipids with alternative non-phosphorus lipids. It was very recently demonstrated that a SAR11 isolate also shares this capability when phosphate starved in culture. Yet, the extent to which this process occurs in other marine heterotrophic bacteria and in the natural environment is unknown. Here, we demonstrate that the substitution of membrane phospholipids for a variety of non-phosphorus lipids is a conserved response to P deficiency among phylogenetically diverse marine heterotrophic bacteria, including members of the Alphaproteobacteria and Flavobacteria. By deletion mutagenesis and complementation in the model marine bacterium Phaeobacter sp. MED193 and heterologous expression in recombinant Escherichia coli, we confirm the roles of a phospholipase C (PlcP) and a glycosyltransferase in lipid remodelling. Analyses of the Global Ocean Sampling and Tara Oceans metagenome data sets demonstrate that PlcP is particularly abundant in areas characterized by low phosphate concentrations. Furthermore, we show that lipid remodelling occurs seasonally and responds to changing nutrient conditions in natural microbial communities from the Mediterranean Sea. Together, our results point to the key role of lipid substitution as an adaptive strategy enabling heterotrophic bacteria to thrive in the vast P-depleted areas of the ocean.