MrpA Functions in Energy Conversion during Acetate-Dependent Growth of Methanosarcina acetivorans

Abstract

bThe role of the multisubunit sodium/proton antiporter (Mrp) of Methanosarcina acetivorans was investigated with a mutant deleted for the gene encoding the MrpA subunit. Antiporter activity was 5-fold greater in acetate-grown versus methanol-grown wild-type cells, consistent with the previously published relative levels of mrp transcript. The rate, final optical density, and dry weight/methane ratio decreased for the mutant versus wild type when cultured with a growth-limiting concentration of acetate. All growth parameters of the mutant or wild type were identical when grown with methanol in medium containing a growthlimiting Na ؉ concentration of 1.04 M. The lag phase, growth rate, and final optical density for growth of the mutant were suboptimal compared to the wild type when cultured with acetate in medium containing either 0.54 or 1.04 M Na ؉ . The addition of 25 mM NaCl to resting cell suspensions stimulated ATP synthesis driven by a potassium diffusion potential. ATP synthesis was greater in wild-type than mutant cells grown with acetate, a trend that held for methanol-grown cells, albeit less pronounced. Both sodium and proton ionophores reduced ATP synthesis in the wild type grown with either substrate. The results indicated that the Mrp complex is essential for efficient ATP synthesis and optimal growth at the low concentrations of acetate encountered in the environment.

Purification and Functional Reconstitution of a Seven-Subunit Mrp-Type Na+/H+ Antiporter

“…Studies of seven-subunit Bacillus Mrp antiporters show that they form hetero-oligomeric complexes containing all seven subunits, and the active form of Mrp appears to be a dimer of the hetero-oligomeric Mrp complex (15,16). Mrp-type antiporters have been found in bacteria and archaea from diverse ecological niches and genera and are among the antiporters that have critical roles in bacterial pathogens (11,12,(17)(18)(19)(20)(21)(22). However, the structural complexity of Mrp antiporters has made it difficult to unravel the antiport mechanism, which is needed to gain an understanding of the advantages conferred by its unusual complexity in settings in which it is important and often essential.…”

Functional Role of MrpA in the MrpABCDEFG Na + /H + Antiporter Complex from the Archaeon Methanosarcina acetivorans

“…Comparisons of the wild type versus a ΔmrpA mutant show that the Mrp complex is essential for optimal ATP synthesis and growth of cells converting acetate to methane and carbon dioxide (15). Growth with acetate generates both Na ϩ and H ϩ gradients that drive ATP synthesis by an ATP synthase dependent on both Na ϩ and H ϩ gradients (29).…”

“…Even though the sequenced genomes of species in the domain Archaea are annotated with genes encoding Mrp complexes, the only reported investigation is that for the seven-subunit MrpABCDEFG complex from the methane-producing (methanogen) Methanosarcina acetivorans (15). Comparisons of the wild type versus a ΔmrpA mutant show that the Mrp complex is essential for optimal ATP synthesis and growth of cells converting acetate to methane and carbon dioxide (15).…”

“…Growth with acetate generates both Na ϩ and H ϩ gradients that drive ATP synthesis by an ATP synthase dependent on both Na ϩ and H ϩ gradients (29). It is postulated that the Mrp complex from M. acetivorans functions to adjust the Na ϩ /H ϩ ratio optimally for ATP synthase (15). Here, we report properties of the MrpA subunit of M. acetivorans produced in E. coli strains with Na ϩ /H ϩ antiporter deficiencies that establishes a role for MrpA in the MrpABCDEFG complex important for acetotrophic growth.…”

“…Analyses of genomic sequences show Mrp complexes are broadly distributed among species with diverse physiologies from the domains Bacteria and Archaea, although investigations have focused on Bacteria to the exclusion of Archaea. Mrp complexes from Bacteria have been implicated in functions that include Na ϩ /H ϩ antiport (2-8), K ϩ /H ϩ antiport (9), pH homeostasis (7,10,11), resistance to bile salts (7,12), arsenite oxidation (8), pathogenesis (12,13), and energy conversion (14,15). Mrp complexes and their subunits are anticipated to have undiscovered functions (1).…”