Oxidation of trimethylamine to trimethylamine N-oxide facilitates high hydrostatic pressure tolerance in a generalist bacterial lineage

Abstract

High hydrostatic pressure (HHP) is a characteristic environmental factor of the deep ocean. However, it remains unclear how piezotolerant bacteria adapt to HHP. Here, we identify a two-step metabolic pathway to cope with HHP stress in a piezotolerant bacterium. Myroides profundi D25^T, obtained from a deep-sea sediment, can take up trimethylamine (TMA) through a previously unidentified TMA transporter, TmaT, and oxidize intracellular TMA into trimethylamine N-oxide (TMAO) by a TMA monooxygenase, MpTmm. The produced TMAO is accumulated in the cell, functioning as a piezolyte, improving both growth and survival at HHP. The function of the TmaT-MpTmm pathway was further confirmed by introducing it into Escherichia coli and Bacillus subtilis Encoded TmaT-like and MpTmm-like sequences extensively exist in marine metagenomes, and other marine Bacteroidetes bacteria containing genes encoding TmaT-like and MpTmm-like proteins also have improved HHP tolerance in the presence of TMA, implying the universality of this HHP tolerance strategy in marine Bacteroidetes.

Fig. 1. Effect of methylamine on the growth and survival of strain D25 under HHP.

(A) Growth curves of strains D25 and DSS-3 cultured with TMA or TMAO as the sole nitrogen source. (B) Changes of the concentrations of medium TMA and intracellular TMA and TMAO of strain D25 cultured in 2216E medium with an initial TMA concentration of 85 μM. (C) Growth curves of strain D25 at 4°C at 0.1, 20, and 40 MPa with or without 10 μM TMA. (D) Effect of 10 μM TMAO or TMA on the growth and survival of strain D25 at different pressures. The cell number at the start of the incubation (start) was ca. 1 × 10⁶ CFU/ml. The cultures were incubated at 4°C for 10 days, and then CFU was counted and compared with that of the starting culture (end/start). The red line refers to a complete death of cells in the culture. Control: Strain D25 cultured without TMAO or TMA. (E) Morphological observation of strain D25 cultivated at different pressures with or without 10 μM TMA. The error bars represent SDs from triplicate experiments. The asterisks show statistical difference from control (**P < 0.01, two-tailed t test). ETD, Everhart-Thornley detector; WD, working distance. SKLMT, State Key Laboratory of Microbial Technology.

Fig. 2. Characterization of MpTmm.

(A) Nonlinear fit curves for the oxidation of TMA by recombinant MpTmm at 4° and 25°C. (B) Effect of different concentrations of TMA on the expression of gene Mptmm in strain D25. Strain D25 was cultured in 2216E medium with or without TMA for 1 hour. (C) Effect of hydrostatic pressure on the expression of gene Mptmm in strain D25. Strain D25 was cultured in 2216E medium at atmosphere pressure, 20 or 40 MPa for 1 hour. (D) Comparison of the structures of MpTmm and RnTmm. MpTmm is colored in yellow, and RnTmm in purple. (E) Comparison of the locations of NADP⁺ (left) and FAD (right) in the crystal structures of MpTmm and RnTmm. The FAD and NADP⁺ molecules are shown in sticks colored in yellow for MpTmm and in purple for RnTmm. (F) Effect of temperature on the activity of MpTmm. (G) Measurement of the T_m value of MpTmm by DSC. (H) Effect of pressure on the activity of MpTmm. The enzymatic activities at different pressures were measured at 25° and 4°C, respectively. The error bars represent SDs from triplicate experiments.

Fig. 3. Characterization of TmaT.

(A) Effect of hydrostatic pressure on the expression of gene tmaT in strain D25 detected by quantitative real-time PCR. Strain D25 was cultured in 2216E medium at atmosphere pressure, 20 or 40 MPa for 1 hour. The error bars represent SDs from triplicate experiments. (B to E) ITC curves for titrations of TMA or TMAO into TmaT. ITC traces (top) and integrated binding isotherms (bottom) are shown. The titration substrates and temperatures are shown in the pictures. DP, differential power. (F) Molecular phylogenetic analysis of TmaT and its homologs with other BCCT transporters. Sequences are from the IMG/JGI database. BetP, BetT, and CaiT are BCCT transporters for glycine betaine, choline, and carnitine, respectively.

Fig. 4. The in vivo function of TmaT and MpTmm in recombinant E. coli DH5α, E. coli mutant ΔtorCAD, and B. subtilis 168.

(A, C, and D) Effect of expression of TmaT and MpTmm on the growth and survival of E. coli DH5α (A), ΔtorCAD (C), and B. subtilis 168 (D) cultured at different pressures for 48 hours. Control, strain containing the empty plasmid cultured with TMA; tmm, strain expressing MpTmm cultured with TMA; tmaT, strain expressing TmaT cultured with TMA; &, strain coexpressing MpTmm and TmaT cultured with TMA; and &-, strain coexpressing MpTmm and TmaT cultured without TMA. The cell number at the start of the incubation (start) was ca. 1 × 10⁵ CFU/ml. The strains were cultivated at 25°C for 48 hours with or without 20 μM TMA, and then CFU were counted and compared with those of the starting culture (end/start). The asterisks show statistical difference from control (**P < 0.01, two-tailed t test). The error bars represent SDs from triplicate experiments. (B) Morphological observation of cells of ΔtorCAD coexpressing TmaT and MpTmm cultured at 25°C for 48 hours under different pressures with or without 20 μM TMA. ETD, Everhart-Thornley detector; WD, working distance. SKLMT, State Key Laboratory of Microbial Technology.

Fig. 5. The HHP tolerance strategy of strain D25 and its universality in Bacteroidetes.

(A) The proposed model for HHP tolerance of strain D25 via the TMA metabolism pathway mediated by TmaT and MpTmm. Under HHP stress in deep sea, strain D25 expresses genes tmaT and Mptmm and produces the TMA transporter TmaT and the TMA monooxygenase MpTmm. Strain D25 takes up environmental TMA into its cell by TmaT and oxidizes the intracellular TMA into TMAO by MpTmm. TMAO is accumulated in the cell without further metabolism and is used as a piezolyte by strain D25 to cope with HHP stress. (B) Effect of the presence of TMA in medium on the growth of other Bacteroidetes strains at different pressures. Strains were cultured at 15°C for 10 days in the medium containing 3% artificial sea salt, 0.05% peptone, 0.01% yeast powder, and 20 μM TMA. The error bars represent SDs from triplicate experiments. Strains E. algicola MEBiC 12267, A. litoralis CNURIC011, and Cytophaga sp. I-545-C were all isolated from surface seawater in our lab. The asterisks show statistical difference from control (**P < 0.01, two-tailed t test). The error bars represent SDs from triplicate experiments.

Fig. 6. Three TMA/TMAO metabolism pathways in marine bacteria.

(A) A neighbor-joining phylogenetic tree showing the bacterial strains, which are capable of metabolizing TMA/TMAO. The tree was constructed on the basis of the 16S ribosomal RNA gene sequences of the strains. The red dot(s) indicates the phylogenetic position of the representative stains illustrated in (B). The detailed information of each strain is listed in table S4. (B) Schemes of the TMA/TMAO metabolism pathways and related genomic regions in R. pomeroyi DSS-3 (α-proteobacteria), Shewanella violacea DSS12 (γ-proteobacteria), and M. profundi D25 (Bacteroidetes). MpTmm, TMA monooxygenase gene; tdm, TMAO demethylase gene; tmaT, TMA transporter gene; dmm DABC, dimethylamine monooxygenase gene; tmo XVW, adenosine 5′-triphosphate–dependent TMAO transporter gene; and torA, TMAO reductase gene.