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...
SummaryBacteria that inhabit the rhizosphere of agricultural crops can have a beneficial effect on crop growth. One such mechanism is the microbial‐driven solubilization and remineralization of complex forms of phosphorus (P). It is known that bacteria secrete various phosphatases in response to low P conditions. However, our understanding of their global proteomic response to P stress is limited. Here, exoproteomic analysis of Pseudomonas putida BIRD‐1 (BIRD‐1), Pseudomonas fluorescens SBW25 and Pseudomonas stutzeri DSM4166 was performed in unison with whole‐cell proteomic analysis of BIRD‐1 grown under phosphate (Pi) replete and Pi deplete conditions. Comparative exoproteomics revealed marked heterogeneity in the exoproteomes of each Pseudomonas strain in response to Pi depletion. In addition to well‐characterized members of the PHO regulon such as alkaline phosphatases, several proteins, previously not associated with the response to Pi depletion, were also identified. These included putative nucleases, phosphotriesterases, putative phosphonate transporters and outer membrane proteins. Moreover, in BIRD‐1, mutagenesis of the master regulator, phoBR, led us to confirm the addition of several novel PHO‐dependent proteins. Our data expands knowledge of the Pseudomonas PHO regulon, including species that are frequently used as bioinoculants, opening up the potential for more efficient and complete use of soil complexed P.
Bacteria of the marine Roseobacter clade are characterised by their ability to utilise a wide range of organic and inorganic compounds to support growth. Trimethylamine (TMA) and trimethylamine N-oxide (TMAO) are methylated amines (MA) and form part of the dissolved organic nitrogen pool, the second largest source of nitrogen after N 2 gas, in the oceans. We investigated if the marine heterotrophic bacterium, Ruegeria pomeroyi DSS-3, could utilise TMA and TMAO as a supplementary energy source and whether this trait had any beneficial effect on growth. In R. pomeroyi, catabolism of TMA and TMAO resulted in the production of intracellular ATP which in turn helped to enhance growth rate and growth yield as well as enhancing cell survival during prolonged energy starvation. Furthermore, the simultaneous use of two different exogenous energy sources led to a greater enhancement of chemoorganoheterotrophic growth. The use of TMA and TMAO primarily as an energy source resulted in the remineralisation of nitrogen in the form of ammonium, which could cross feed into another bacterium. This study provides greater insight into the microbial metabolism of MAs in the marine environment and how it may affect both nutrient flow within marine surface waters and the flux of these climatically important compounds into the atmosphere.
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