Genetic and Biochemical Characterization of the Na+/H+ Antiporters of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa has four Na⁺/H⁺ antiporters that interconvert and balance Na⁺ and H⁺ gradients across the membrane. These gradients are important for bioenergetics and ionic homeostasis. To understand these transporters, we constructed four strains, each of which has only one antiporter, i.e., NhaB, NhaP, NhaP2, and Mrp. We also constructed a quadruple deletion mutant that has no Na⁺/H⁺ antiporters. Although the antiporters of P. aeruginosa have been studied previously, the strains constructed here present the opportunity to characterize their kinetic properties in their native membranes and their roles in the physiology of P. aeruginosa. The strains expressing only NhaB or Mrp, the two electrogenic antiporters, were able to grow essentially like the wild-type strain across a range of Na⁺ concentrations and pH values. Strains with only NhaP or NhaP2, which are electroneutral, grew more poorly at increasing Na⁺ concentrations, especially at high pH values, with the strain expressing NhaP being more sensitive. The strain with no Na⁺/H⁺ antiporters was extremely sensitive to the Na⁺ concentration and showed essentially no Na⁺(Li⁺)/H⁺ antiporter activity, but it retained most K⁺/H⁺ antiporter activity of the wild-type strain at pH 7.5 and approximately one-half at pH 8.5. We also used the four strains that each express one of the four antiporters to characterize the kinetic properties of each transporter. Transcriptome sequencing analysis of the quadruple deletion strain showed widespread changes, including changes in pyocyanin synthesis, biofilm formation, and nitrate and glycerol metabolism. Thus, the strains constructed for this study will open a new door to understanding the physiological roles of these proteins and their activities in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa has four Na⁺/H⁺ antiporters that connect and interconvert its Na⁺ and H⁺ gradients. We have constructed four deletion mutants, each of which has only one of the four Na⁺/H⁺ antiporters. These strains made it possible to study the properties and physiological roles of each antiporter independently in its native membrane. Mrp and NhaB are each able to sustain growth over a wide range of pH values and Na⁺ concentrations, whereas the two electroneutral antiporters, NhaP and NhaP2, are most effective at low pH values. We also constructed a quadruple mutant lacking all four antiporters, in which the H⁺ and Na⁺ gradients are disconnected. This will make it possible to study the role of the two gradients independently.

Keywords: Na+/H+ antiporters; Pseudomonas aeruginosa; energy metabolism; ion transporters.

FIG 1

Cation/H⁺ antiporter activity measured by the acridine orange fluorescence quenching method for the wild-type (WT) P. aeruginosa strain and the antiporter deletion mutant in everted vesicles. Representative fluorescence versus time traces for the Na⁺/H⁺ antiporter activity in the wild-type strain (WT) and the quadruple antiporter deletion mutant (ΔnhaB ΔnhaP ΔnhaP2 ΔmrpA) are shown. The black arrows indicate the addition of 20 mM NaCl to stimulate Na⁺/H⁺ antiporter activity, and the blue arrows indicate the addition of 2 mM NH₄Cl to collapse the proton gradient. Fluorescence quenching is shown in arbitrary units.

FIG 2

Michaelis-Menten kinetics of the Na⁺/H⁺ antiporters of P. aeruginosa with varying NaCl concentrations at pH 7.5 (A) and pH 8.5 (B). Data for the wild-type (WT), NhaB-only, NhaP-only, NhaP2-only, and Mrp strains are shown. Activities were averaged from two biological replicates with three technical replicates each, and error bars represent the standard deviations.

FIG 3

(A) Comparison of wild-type (WT) cation/H⁺ antiporter activities with those of the triple deletion mutants (NhaB-only, NhaP-only, NhaP2-only, and Mrp-only strains) and the quadruple mutant (ΔnhaB ΔnhaP ΔnhaP2 ΔmrpA) at pH 7.5. (B) Comparison of wild-type cation/H⁺ antiporter activities with those of the triple deletion mutants (NhaB-only, NhaP-only, NhaP2-only, and Mrp-only strains) and the quadruple mutant (ΔnhaB ΔnhaP ΔnhaP2 ΔmrpA) at pH 8.5. Activities were averaged from two biological replicates with three technical replicates each, and error bars represent the standard deviations.

FIG 4

(A) Effects of pH and NaCl on the growth of the wild-type P. aeruginosa strain and the triple antiporter deletion mutants. Wild-type (WT), ΔnhaP ΔnhaP2 ΔmrpA, ΔnhaB ΔnhaP2 ΔmrpA, ΔnhaB ΔnhaP ΔmrpA, and ΔnhaB ΔnhaP ΔnhaP2 strains were grown in LB medium with 50 mM, 170 mM, 300 mM, or 500 mM NaCl at pH 6.5, 7.5, or 8.5. (B) Growth comparison of the quadruple deletion mutant (ΔnhaB ΔnhaP ΔnhaP2 ΔmrpA) and the wild-type strain at pH 7.5 with 50 mM NaCl. (C) Growth of the quadruple mutant (ΔnhaB ΔnhaP ΔnhaP 2ΔmrpA) at pH 6.5 with 50 mM NaCl, 170 mM NaCl, 300 mM NaCl, or 500 mM NaCl. (D) Growth of the quadruple mutant (ΔnhaB ΔnhaP ΔnhaP2 ΔmrpA) at pH 7.5 with 0 mM NaCl, 50 mM NaCl, or 170 mM NaCl. (E) Growth of the quadruple mutant at pH 8.5 with 0 mM NaCl, 50 mM NaCl, or 170 mM NaCl. Changes in OD₅₀₀ were measured using a Tecan Infinite Magellan M1000 Pro plate reader during 20 h of growth at 37°C with continuous orbital shaking at 217 rpm. The growth curves are the average from two biological replicates with three technical replicates each, and the error bars represent the standard deviations. (F to H) Effects of NaCl at pH 6.5 (F), pH 7.5 (G), and pH 8.5 (H), plotted as the changes in doubling time per hour.

FIG 5

(A) Differential expression of genes encoding the pyocyanin biosynthetic and PQS pathways in the ΔnhaB ΔnhaP ΔnhaP2 ΔmrpA mutant, compared with the wild-type strain, during exponential and stationary phases of growth in LB medium (pH 7.5) with 50 mM NaCl. A negative value indicates a decrease in expression in the ΔnhaB ΔnhaP ΔnhaP2 ΔmrpA mutant, compared to the wild-type strain. Stars indicate significant changes in expression with P values of <0.05. (B) Pyocyanin production in the wild-type (WT) strain and quadruple mutant at 4 h (exponential phase) and 15 h (stationary phase) in LB medium (pH 7.5) with 50 mM NaCl. Pyocyanin concentrations were measured at 690 nm (E = 4,130 M⁻¹cm⁻¹). The error bars represent the standard deviations. Stars indicate P values from Student's t test of <0.01.

FIG 6

(A) Biofilm quantification by crystal violet staining measured at 600 nm, normalized by OD₅₀₀. Values are the average from two biological replicates with 12 technical replicates each, and the error bars represent the standard errors. (B) Differential expression of the pel operon in the ΔnhaB ΔnhaP ΔnhaP2 ΔmrpA mutant, compared with the wild-type (WT) strain, during exponential and stationary phases of growth in LB medium (pH 7.5) with 50 mM NaCl. A positive value indicates an increase in expression in the ΔnhaB ΔnhaP ΔnhaP2 ΔmrpA mutant, compared to the wild-type strain. All expression changes are significant (log₂FC of > 2, with P values of <0.05).

FIG 7

(A) Differential expression of the genes involved in denitrification in P. aeruginosa in the ΔnhaB ΔnhaP ΔnhaP2 ΔmrpA mutant, compared with the wild-type strain, during exponential and stationary phases of growth in LB medium (pH 7.5) with 50 mM NaCl. A negative value indicates a decrease in expression in the ΔnhaB ΔnhaP ΔnhaP2 ΔmrpA mutant, compared to the wild-type strain. All expression changes are significant (log₂FC of > 2, with P values of <0.05). (B) Growth of the wild-type (WT) strain and the quadruple mutant in LB medium (pH 7.5) under anaerobic conditions in the presence of 50 mM NaNO₃. The growth curves are the average from two experiments repeated three times, and the error bars represent the standard deviations.

FIG 8

(A) Differential expression of genes involved in glycerol uptake and metabolism in P. aeruginosa in the ΔnhaB ΔnhaP ΔnhaP2 ΔmrpA mutant, compared with the wild-type strain, during exponential and stationary phases of growth in LB medium (pH 7.5) with 50 mM NaCl. A negative value indicates a decrease in expression in the ΔnhaB ΔnhaP ΔnhaP2 ΔmrpA mutant, compared to the wild-type strain. All expression changes are significant (log₂FC of > 2, with P values of <0.05). (B) Growth of the wild-type (WT) strain and the quadruple deletion mutant (ΔnhaB ΔnhaP ΔnhaP2 ΔmrpA) in minimal medium (M9) (pH 7.5) with 20 mM glycerol as the sole carbon source. The growth curves are the average from two experiments repeated three times, and the error bars represent the standard deviations.