179 Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress down and incubated for 10 min in 1.5 mL of the same extraction solvent in petridishes that were standing on dry-ice. To enhance cell disruption, we pipetted the extraction solvent about 20-25 times before transferring it to 1.5 mL tubes. Cell debris and proteins were removed by centrifugation for 10 min at 4ºC and 20,000xg. Samples were collected and stored at -70ºC until analysis on a Agilent 6546 Liquid Chromatography - Quadrupole Time of Flight (LC/Q-TOF) system, as specified previously (Jansen et al., 2020). In brief, samples were separated using aqueous normal phase chromatography, followed by MS detection in the positive ionization mode (m/z 50-1200). Vendor-specific datafiles were converted to mzXML format using MSconvert (Chambers et al., 2012) and analyzed using XCMS online (Smith et al., 2006). To filter for osmolytes, we selected for features with peak areas above half a million counts that increased with higher salinities. The unknown metabolite with m/z 189.125 was selected for MS2 fragmentation (unit isolation) and fragmented at collision energies of 10, 20 and 40 V. With the collected MS2 spectra, in silico structure prediction was performed using SIRIUS (Dührkop et al., 2019). Reference N(ε)-acetyl-β-L-lysine production with Methanosarcina mazei and negative control Reference osmolyte-producing Methanosarcina mazei DSMZ 7222 was grown in 23.8 g/L NaCl, as described before(Pflüger et al., 2003), which previously resulted in the production of N(ε)-acetyl-β-L-lysine. 50 ml of exponential-phase biomass was spun down at max speed for 10 min and the supernatant was discarded. The pellet was then resuspended in 1.5 ml of ice-cold acetonitrile:methanol:deionized water (40:40:20) and incubated on ice 30 min. Samples were later centrifuged for 10 min at 4ºC and 20,000xg, so as to recover the supernatant and remove cell debris. Isomer N(ε)-acetyl-L-lysine (Sigma-Aldrich, St Luis, USA) was dissolved in acetonitrile:methanol:deionized water (40:40:20) at a concentration of 40 µM to serve as negative control. All samples that were employed for the MS2 spectra analysis were diluted 100-fold, including the accumulating metabolite in “Ca. Methanoperedens”. 6
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