title: Three putative cation/proton antiporters from the soda lake alkaliphile Alkalimonas amylolytica N10 complement an alkali-sensitive Escherichia coli mutant.
abstract: 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 >or=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 Ca(2+) 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.
authors.affiliation: Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10029, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wei
authors.affiliation: Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10029, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.affiliation: Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China.
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10029, USA.
authors.forename: Terry A
authors.initials: TA
authors.lastname: Krulwich
mesh_terms: D000468:Alkalies / Q000494:pharmacology*; D017920:Antiporters; D001426:Bacterial Proteins; D002412:Cations; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D020563:Gammaproteobacteria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011522:Protons; D012965:Sodium Chloride; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
chemical_list: D000468:Alkalies; D017920:Antiporters; D001426:Bacterial Proteins; D002412:Cations; D011522:Protons; D017923:Sodium-Hydrogen Exchangers; D012965:Sodium Chloride