LAUSR

Adaptation to higher temperatures would increase the environmental competitiveness of psychrophiles, organisms that thrive in low‐temperature environments. Methanolobus psychrophilus, a cold wetland methanogen, ‘evolved’ as a mesophile, growing optimally at 30 °C after subculturings, and cells grown with ample substrates exhibited higher integrity. Here, we investigated N‐glycosylation of S‐layer proteins, the major archaeal envelope component, with respect to mesophilic adaptation. Lectin affinity enriched a glycoprotein in cells grown at 30 °C under ample substrate availability, which was identified as the S‐layer protein Mpsy_1486. Four N‐glycosylation sites were identified on Mpsy_1486, which exhibited different glycosylation profiles, with N94 only found in cells cultured at 30 °C. An N‐linked glycosylation inhibitor, tunicamycin, reduced glycosylation levels of Mpsy_1486 and growth at 30 °C, thus establishing a link between S‐layer protein glycosylation and higher temperature adaptation of the psychrophilic archaeon M. psychrophilus.