The Activity Profile of the NhaD-Type Na + (Li + )/H + Antiporter from the Soda Lake Haloalkaliphile Alkalimonas amylolytica Is Adaptive for the Extreme Environment

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Abstract
In extreme alkaliphiles, Na+/H+ antiporters play a central role in the Na+ cycle that supports pH homeostasis, Na+ resistance, solute uptake, and motility. Properties of individual antiporters have only been examined in extremely alkaliphilic soil Bacillus spp., whereas the most alkaline natural habitats usually couple high pH with high salinity. Here, studies were conducted on a Na+(Li+)/H+ antiporter, NhaD, from the soda lake haloalkaliphile Alkalimonas amylolytica. The activity profile of A. amylolytica NhaD at different pH values and Na+ concentrations reflects its unique natural habitat. In membrane vesicles from antiporter-deficient Escherichia coli EP432 (ΔnhaA ΔnhaB), the pH optimum for NhaD-dependent Na+(Li+)/H+ antiport was at least 9.5, the highest pH that could be tested; no activity was observed at pH ≤8.5. NhaD supported low Na+/H+ antiport activity at pH 9.5 that was detectable over a range of Na+ concentrations from 10 mM to at least 800 mM, with a 600 mM optimum. Although A. amylolytica nhaD was isolated by complementing the Li+ sensitivity of the triple mutant E. coli strain KNabc (ΔnhaA ΔnhaB ΔchaA), sustained propagation of nhaD-bearing plasmids in this strain resulted in a glycine (Gly327)→serine mutation in a putative cytoplasmic loop of the mutant transporter. The altered activity profile of NhaD-G327S appears to be adaptive to the E. coli setting: a much higher activity than wild-type NhaD at Na+ concentrations up to 200 mM but lower activity at 400 to 600 mM Na+, with a pH optimum and minimal pH for activity lower than those of wild-type NhaD.