Carbonic anhydrase plays multiple roles in acetotrophic growth of a model marine methanogen from the domain Archaea

Carbonic anhydrase (CA) catalyzes the reversible hydration of CO2 to bicarbonate and a proton. The enzyme is universally distributed in all three domains of life and plays diverse physiological roles in the domains Eukarya and Bacteria. Remarkably, a physiological role has not been identified for any CA from the domain Archaea. Herein are described roles for a gamma class CA (Cam) from the methane-producing marine archaeon Methanosarcina acetivorans. Acetate-dependent growth of a {Delta}cam mutant showed an extended lag phase, lower final cell density, and metabolized acetate to a threshold of 20.0 mM compared to 1.0 mM for wild-type. Molar growth yields (Ymethane) were substantially greater for wild-type compared to the mutant. In contrast, growth parameters were identical for the methanol-grown wild-type and mutant. Rates of methane formation in resting cell suspensions containing 20.0 mM acetate were significantly less in the mutant versus wild-type and dependent on the presence of CO2. Rates for the wild-type decreased with increasing pH that was more pronounced for the mutant. CA activity was 100-fold greater in the membrane versus soluble fraction of acetate-grown cells. Addition of a surrogate CA stimulated acetate-dependent methanogenesis in resting cell suspensions of the mutant. The results support a role for Cam to supply protons for symport of acetate by the AceP symporter that also optimizes and facilitates growth at low acetate concentrations and high pH values encountered in the marine environment where M. acetivorans was isolated. Significance StatementAlthough CA plays major physiological roles in the domains Eukarya and Bacteria, a role has not been reported for the domain Archaea in which methanogens comprise the major group with abundant genomic annotations for CAs. Acetotrophic methanogens account for most of the methane produced in Earths biosphere where it is a major greenhouse gas. Although the biochemistry of the conversion of acetate to methane and carbon dioxide is well known, little is understood of acetate transport. The finding that CA has multiple roles facilitating thermodynamically constrained growth of a model marine acetotrophic methanogen has implications for advancing ecological understanding of the methane cycle that impacts global warming and climate change. Finally, the work is an introduction to anticipated physiological roles of CAs in the domain Archaea for which genomic annotations are abundant.