Enhanced glycosylation of an S-layer protein enables a psychrophilic methanogenic archaeon to adapt to elevated temperatures in abundant substrates.

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.