Interactions between Nitrogen Fixation and Osmoregulation in the Methanogenic Archaeon Methanosarcina barkeri 227

The nitrogenase enzyme complex of Methanosarcina barkeri 227 was found to be more sensitive to NaCl than previously studied molybdenum nitrogenases are, with total inhibition of activity occurring at 190 mM NaCl, compared with >600 mM NaCl for Azotobacter vinelandii and Clostridium pasteurianum nitrogenases. Na(+) and K(+) had equivalent effects, whereas Mg(2+) was more inhibitory than either monovalent cation, even on a per-charge basis. The anion Cl(−) was more inhibitory than acetate was. Because M. barkeri 227 is a facultative halophile, we examined the effects of external salt on growth and diazotrophy and found that inhibition of growth was not greater with N(2) than with NH(4)(+). Cells grown with N(2) and cells grown with NH(4)(+) produced equal concentrations of α-glutamate at low salt concentrations and equal concentrations of N(ɛ)-acetyl-β-lysine at NaCl concentrations greater than 500 mM. Despite the high energetic cost of fixing nitrogen for these osmolytes, we obtained no evidence that there is a shift towards nonnitrogenous osmolytes during diazotrophic growth. In vitro nitrogenase enzyme assays showed that at a low concentration (approximately 100 mM) potassium glutamate enhanced activity but at higher concentrations this compound inhibited activity; 50% inhibition occurred at a potassium glutamate concentration of approximately 400 mM.