186 Chapter 6 lysine 2,3 aminomutase (LAM) L-lysine β-L-lysine diaminopimelate decarboxylase (LysA) β-lysine N6-acetyltransferase (AT) lysA L-Aspartate Diaminopimelate (DAP) N(ε)-acetyl-β-L-lysine ablB kamA D E C kamA ablB ynaI unk. unk. unk. D 0 5 10 15 0 0.5 1 1.5 1.5 acl. 3 0 10 20 30 0 5 10 15 salinity % kamAexpression (ΔΔCt) salinity % kamA expression (∆∆Ct) Salinity (%) B A 0 20 40 ablBexpression (ΔΔCt) ablB expression (∆∆Ct) 0 0.5 1 1.5 1.5 acl. 3 0 0.5 1 1.5 1.5 acl 3 0 0.5 1 1.5 1.5 acl 3 A 80 100 120 140 160 180 ���� ��� 0 �� 100 MS2 189.1289 RT: 3.6 2e+8 relative intensity B C Intensity peak NMDS2 NMDS1 1e+8 0e+0 -1e+8 -2e+8 -2e+8 -1e+8 0 1e+8 2e+8 ������ 1e+8 ���� 0 0 ���� ��� ���� 1 ���� ��� salinity (%) 0 ���� ��� ���� 1 ���� ��� m/z Reference Unknown m/z 189.1289 salinity (%) A 80 100 120 140 160 180 ���� ��� 0 �� 100 MS2 RT: 3.6 MS2 189.1289 RT: 3.6 2e+8 relative intensity B C Intensity peak NMDS2 NMDS1 1e+8 0 -1e+8 3e+8 -2e+8 -2e+8 -1e+8 0 2e+8 1e+8 1.5e+8 1.e+8 5e+7 0e+0 0 0.25 0.5 0.75 1 1.25 1.5 Salinity (%) 0 ���� ��� ���� ���� ��� 1 m/z Reference Unknown 189.125 80 100 120 140 160 180 �100 0 MS2 RT: 3.6 189.125 RT: 3.6 Relative intensity B Intensity peak NMDS1 ������ 1e+8 ���� 0 0 ���� ��� ���� 1 ���� ��� salinity (%) m/z Reference Unknown m/z 189.1289 A B C F G 100 Salinity (%) lysine 2,3 aminomutase (LAM) L-lysine β-L-lysine diaminopimelate decarboxylase (LysA) β-lysine N6-acetyltransferase (AT) lysA L-Aspartate Diaminopimelate (DAP) N(ε)-acetyl-β-L-lysine ablB kamA D E C kamA ablB ynaI unk. unk. unk. D 0 5 10 15 0 0.5 1 1.5 1.5 acl. 3 0 10 20 30 kamA expression (∆∆Ct) Salinity (%) ablB expression (∆∆Ct) 0 0.5 1 1.5 1.5 acl. 3 A 80 100 120 140 160 180 ���� ��� 0 �� 100 MS2 189.1289 RT: 3.6 2e+8 relative intensity B C Intensity peak NMDS2 NMDS1 1e+8 0e+0 -1e+8 -2e+8 -2e+8 -1e+8 0 1e+8 2e+8 ������ 1e+8 ���� 0 0 ���� ��� ���� 1 ���� ��� salinity (%) 0 ���� ��� ���� 1 ���� ��� m/z Reference Unknown m/z 189.1289 Intensity peak 1.5e+8 1.e+8 5e+7 0e+0 0 0.25 0.5 0.75 1 1.25 1.5 Salinity (%) 189.125 80 100 120 140 160 180 �100 0 189.125 RT: 3.6 Relative intensity B m/z Reference Unknown A B C F G 100 Salinity (%) Figure 2. N(ε)-acetyl-β-L-lysine and its biosynthetic gene transcripts increase upon salt stress. A Non-Metric Multidimensional Scaling (NMDS) plot of metabolomics samples collected at 7 different salinities (in %): 0,0.25,0.5,0.75,1, 1.25 and 1.5 (n=4 per salinity). Each salinity is depicted using a different color (see legend). B Box and whisker plot of m/z 189.125 [M+H]+ (n=4 per salinity). C Mirror plot with MS2 spectra of m/z 189.125 (± 10ppm) from “Ca. Methanoperedens” (unknown) and Methanosarcina mazei (reference) demonstrating a spectral match. D Osmolyte N(ε)-acetyl-β-L-Lysine is a derivative of the L-Lysine amino acid family. Synthesis includes the central metabolism core gene encoding diaminopimelate decarboxylase (enzyme LysA encoded by lysA) (KEGG: K01586, EC: 4.1.1.20), followed by lysine 2,3-aminomutase (enzyme LAM encoded by kamA) (KEGG: K01843, EC: 5.4.3.2) and a β-lysine N6-acetyl transferase (AT encoded by ablB) (KEGG: K21935, EC: 2.3.1.264) exclusively found in the “Ca. Methanoperedens” MAG in the present enrichment culture studied. (E) Operon gene organization of kamA (1326bp)-ablB (870bp). kamA-ablB are preceded (with 1bp of difference) by the ynaI gene (1098bp) (KEGG: K16052), encoding an enzyme similar to a small conductance mechanosensitive channel (MscS) (PFAM: IPR045042) and downstream (41bp difference) by genes of unknown function. F-G Normalized gene expression of “Ca. Methanoperedens” specific-kamA and ablB genes in the form of Δ ΔCt at six different salinities (in %): 0,0.5,1,1.5, 1.5-acclimated and 3 at weeks 0,4,8,12,22 and 26, respectively, determined by quantitative PCR (in two to three biological replicates per condition, n=2-3).
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