N-epsilon-acetyl-beta-lysine is a unique compatible solute found in methanogenic archaea grown at high salinities. Deletion of the genes that encode the lysine-2,3-aminomutase (ablA) and the beta-lysine acetyltransferase (ablB) abolished the production of N-epsilon-acetyl-beta-lysine in Methanosarcina mazei Go1. The mutant grew well at low and intermediate salinities. Interestingly, growth at high salt (800 mM NaCl) was only slowed down but not impaired demonstrating that in M. mazei Go1 N-epsilon-acetyl-beta-lysine is not essential for growth at high salinities. Nuclear magnetic resonance (NMR) analysis revealed an increased glutamate pool in the mutant. In addition to alpha-glutamate, a novel solute, alanine, was produced. The intracellular alanine concentration was as high as 0.36 +/- 0.05 mu mol (mg protein)(-1) representing up to 18% of the total solute pool at 800 mM NaCl. The cellular alanine concentration increased with the salinity of the medium and decreased in the presence of glycine betaine in the medium, indicating that alanine is used as compatible solute by M. mazei Go1.