Three putative cation/proton antiporters from the soda lake alkaliphine Alkalimonas amylolytica N10 complement an alkali-sensitive Escherichia coli mutant

Attempts to identify members of the antiporter complement of the alkali- and saline-adapted soda lake alkaliphile Alkalimonas amylolytica N10 have used screens of DNA libraries in antiporter-deficient Escherichia coli KNabc. Earlier screens used Na⁺ or Li⁺ for selection but only identified one NhaD-type antiporter whose properties were inconsistent with a robust role in pH homeostasis. Here, new screens using elevated pH for selection identified three other putative antiporter genes that conferred resistance to pH ≥8.5 as well as Na⁺ resistance. The three predicted gene products were in the calcium/cation antiporter (CaCA), cation/proton antiporter-2 (CPA2) and cation/proton antiporter-1 (CPA1) families of membrane transporters, and were designated Aa-CaxA, Aa-KefB and Aa-NhaP respectively, reflecting homology within those families. Aa-CaxA conferred the poorest Na⁺ resistance and also conferred modest Ca²⁺ resistance. Aa-KefB and Aa-NhaP inhibited growth of a K⁺ uptake-deficient E. coli mutant (TK2420), suggesting that they catalysed K⁺ efflux. For Aa-NhaP, the reversibility of the growth inhibition by high K⁺ concentrations depended upon an organic nitrogen source, e.g. glutamine, rather than ammonium. This suggests that NH₄ ⁺ as well as K⁺ efflux is catalysed by Aa-NhaP. Vesicles of E coli KNabc expressing Aa-NhaP, which conferred the strongest alkali resistance, exhibited K⁺/ H⁺ antiport activity in a pH range from 7.5 to 9.5, and with an apparent K_m for K⁺ of 0.5 mM at pH 8.0. The properties of this antiporter are consistent with the possibility that this soda lake alkaliphile uses K⁺(NH₄ ⁺)/H⁺ antiport as part of its alkaline pH homeostasis mechanism and part of its capacity to reduce potentially toxic accumulation of cytoplasmic K⁺ or NH₄ ⁺ respectively, under conditions of high osmolarity or active amino acid catabolism.