School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.
Proc Natl Acad Sci U S A. 2014 Feb 18;111(7):2710-5. doi: 10.1073/pnas.1317834111. Epub 2014 Feb 3.
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.
三甲胺 N-氧化物（TMAO）是一种常见的渗透物，存在于多种海洋生物群中，在海洋表面水中以纳摩尔浓度检测到。TMAO 可以作为生态重要的海洋异养细菌的重要营养物质，特别是 SAR11 类群和海洋玫瑰杆菌类群（MRC）。然而，负责 TMAO 分解代谢的酶和 TMAO 进入微生物细胞所需的膜转运蛋白尚未被鉴定。我们在这里表明，TMAO 脱甲基酶（Tdm）催化 TMAO 降解的第一步。这种酶代表一大类具有未表征结构域（DUF1989）的蛋白质。通过在大肠杆菌中异源表达 tdm（编码 Tdm 的基因），在 SAR11 类群的代表菌株 HIMB59 和 MRC 的代表 Ruegeria pomeroyi DSS-3 中证实了 TMAO 脱甲基酶的功能。在 Ruegeria pomeroyi 中，突变实验证实 tdm 对于 TMAO 生长是必需的。我们还鉴定了一种在多种海洋细菌中发现的独特的 ATP 结合盒转运蛋白（TmoXWV），并通过标记交换突变和编码该转运蛋白的基因启动子的 lacZ 报告基因测定实验，实验证实了其对 TMAO 的特异性。Tdm 和 TmoXWV 在天然海水中的混合物中特别丰富，并如许多最近的宏转录组学和宏蛋白质组学研究所示，积极表达。这些数据表明，TMAO 是海洋细菌的一种重要但被忽视的营养物质。
