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

¹ Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10029, USA
² Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China

Correspondence
Terry A Krulwich
terry.krulwich{at}mssm.edu

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 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⁺()/H⁺ antiport as part of its alkaline pH homeostasis mechanism and part of its capacity to reduce potentially toxic accumulation of cytoplasmic K⁺ or respectively, under conditions of high osmolarity or active amino acid catabolism.

The GenBank/EMBL/DDBJ accession numbers for the sequences of the A. amylolytica N10 gene products are CaxA (ABG37980), KefB (ABG37986) and NhaP (ABG37987). DNA sequences of the larger cloned fragments containing these genes were deposited as pNAK7 (DG649017), pNAK9/10 (DG649019) and pNAK11 (DG649020) respectively, and a fourth cloned fragment that was not further studied, pNAK8, was deposited as DQ649018.

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