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.

Fig. 1

Effects of potassium ion on Na⁺- and alkali-tolerance in E. coli KNabc. E. coli KNabc was grown in LBK (upper panel) or LBC (lower panel) with the indicated concentrations of added NaCl at a series of pH values from 7.0 to 9.0. A₆₀₀ was recorded after 9 hours of growth at 37°C with shaking. Results are the averages from duplicate determinations in two independent experiments and the error bars show the standard deviations from the mean.

Fig. 2

Diagrams of the inserts from plasmids bearing A. amylolytica N10 genes that complemented the alkali-sensitive phenotype of E. coli KNabc. The indicated plasmids are shown with a size scale at the bottom. The predicted ORFs are shown with arrows that reflect the direction of transcription. The closest homologue in a BLAST search is shown on top of most of the arrows.

Fig. 3

Complementation capacities of A. amylolytica N10 genes in alkali-, Na⁺- and Ca²⁺-sensitive E. coli KNabc. Plate assays were conducted as described under Methods. The test plasmids were: pYW136 (Aa-CaxA), pYW138 (Aa-KefB) and pYW139 (Aa-NhaP), pGerN (positive control except for panel D) and pGEM-3Zf(+) (negative control). E. coli DH5α transformed with an empty vector (pMW118 carrying kanamycin-resistant cassette) was the control for the experiments in panel D. The conditions were as indicated above the four panels. The experiments in panels A-C was terminated after 40 hours of incubation at 37°C. The experiment in panel D, conducted only with pYW136 and controls, was terminated after 7 days.

Fig. 4

Complementation assays of the high [K⁺] requirement of the K⁺ uptake-deficient E. coli TK2420 mutant strain with the cloned antiporter candidate genes from A. amylolytica N10. A. E. coli TK2420 transformants with pYW136, pYW138, pYW139 or pGEM-3Zf(+) were grown in standard minimal medium (Epstein et al., 1993), containing the indicated concentrations of added KCl, at 37°C. The A₆₀₀ values were recorded after 16 hours. Results are the average of duplicate determinations from at least 3 independent growth experiments and the error bars show the standard deviation. B. E. coli TK2420 transformants with either pGEM-3Zf(+) (control) or pYW139 (Aa-NhaP) were grown in minimal medium in which glutamine replaced (NH₄)₂SO₄ and KCl was added at the indicated concentrations. Two independent experiments were conducted as described for panel A.

Fig. 5

Assay of K⁺/H⁺, Na⁺/H⁺ and Li⁺/H⁺ antiport activity of Aa-NhaP. Assays were conducted at pH 7.5 on everted membrane vesicles from E. coli KNabc transformants with either pYW139 plasmid (upper panel) or pGEM-3Zf(+) (lower panel). At the time points indicated by the downward arrows at the top, Tris-D-lactate was added to a final concentration of 2.5 mM to initiate respiration. The downward arrows, after the consequent AO quenching had leveled off, indicate the addition 10 mM NaCl, LiCl or KCl. NH₄Cl was added 60 seconds later (the point of the upward arrow) to dissipate the pH gradient. A.U.= arbitrary units of fluorescence quenching.

Fig. 6

The pH and kinetic profile of the K⁺/H⁺ antiporter activity of Aa-NhaP. A. K⁺/H⁺ antiport activity was assayed as described in the legend to Fig. 5, except that assays were conducted at the different pH values shown. The pH of the BTP buffer was adjusted by addition of sulfuric acid. The values are the averages of duplicate determinations from at least two independent experiments and the error bars show the standard deviations. B. K⁺/H⁺ antiporter assays were conducted in BTP buffer at pH 8.0 where the background activity of vector control was zero. The Michaelis-Menten plot is shown with a reciprocal plot as an insert. The standard deviations derived from duplicate assays in two experiments were less than 10% of the mean values and are not shown.