Ammonia assimilation and synthesis of alanine, aspartate, and glutamate in Methanosarcina barkeri and Methanobacterium thermoautotrophicum

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Abstract
The mechanism of NH3 assimilation in M. barkeri and M. thermoautotrophicum was documented by analysis of enzyme activities, 13NH3 incorporation studies and comparison of growth and enzyme activity levels in continuous culture. Glutamate accounted for 65 and 52% of the total amino acids in the soluble pools of M. barkeri and M. thermoautotrophicum. Both organisms contained significant activities of glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase. Hydrogen-reduced deazaflavin-factor 420 or FMN but not NAD, NADP or ferredoxin was used as the electron donor for glutamate synthase in M. barkeri. Glutamate dehydrogenase activity was not detected in either oragnism but alanine dehydrogenase activity was present in M. thermoautotrophicum. The in vivo activity of the glutamine synthetase was verified in M. thermoautotrophicum by analysis of 13NH3 incorporation into glutamine, glutamate and alanine. Alanine dehydrogenase and glutamine synthetase activity varied in response to [NH4+] when M. thermoautotrophicum was cultured in a chemostat with cysteine as the S source. Alanine dehydrogenase activity and growth yield (g cells/mol of methane) were highest when the organism was cultured with excess NH3; growth yield was lower and glutamine synthetase was maximal when NH3 was limiting.