RESEARCH

Background

The Na + /H + antiporters catalyze the exchange of Na + for H + across membranes and play a variety of functions, such as the regulation of internal pH, cell volume, and sodium level in the cytoplasm. In Escherichia coli and other bacteria three antiporters (NhaA, NhaB, and ChaA) are known, they are single gene products that function as monomers or homo-oligomers and their functional characteristics are well described. Instead, little information is available about Mrp systems. Multiple-resistant/pH regulation Na + /H + antiporters (Mrp antiporters) are encoded for six or seven genes ( mrp ABCDEFG operon) that are predicted to encode integral membrane proteins and has ben proposed to function as hetero-oligomeric complex. It has been proposed that Na + /H + MrpA subunit is involved in the pH homeostasis and stress induced by Na + as well. This antiporter complex operon has been reported in bacteria like Bacillus sp and Vibrio sp . The mrp ABCDEFG operon is also present in the methane-producing marine archeon Methanosarcina acetivorans that is expressed higher in acetate-grown cells vs methanol-grown cells. NaCl is an essential component in the medium of culture. The antiporter is energized by either an artificial D Y or D pH characteristic of a secondary antiporter (H + /Na + ¹ 1).

OBJECTIVE

The physiological function of Mrp complex in bacteria and Archaea is actually far to be completely elucidated. Because expression of the Mrp complex seems to be regulated by the growth substrate rather than toxic concentrations of Na + or alkaline pH, is probable that the Mrp in M. acetivorans exchanges the Na + gradient to an H + gradient necessary for ATP synthesis when cells are cultured on acetate. If so, a new and important role for Mrp antiporter will be proposed and, most important a new point of energy conservation in methanogenic pathway. Thus, my project consists in the characterization and elucidation of the physiological role of the Mrp complex in M. acetivorans . Genetic and biochemical approaches have been developed in our laboratory and in collaboration with Dr. Sowers lab (Biotechnology Institute, University of Maryland ). Currently, a knock-out ( mrpA - ) mutant was constructed in order to elucidate if the MrpA subunit plays a role as either intracellular pH regulator or as supplier of essential-Na + ions for the energy conserving reactions in the electron chain transporter in this anaerobic methane-producing archeon. The identification of some factors involved in the up-regulated expression of the Mrp complex is also under research in our laboratory.