Characterization of Two Na+(K+, Li+)/H+ Antiporters from Natronorubrum daqingense

Abstract

The Na⁺/H⁺ antiporter NhaC family protein is a kind of Na⁺/H⁺ exchanger from the ion transporter (IT) superfamily, which has mainly been identified in the halophilic bacteria of Bacillus. However, little is known about the Na⁺/H⁺ antiporter NhaC family of proteins in the extremely halophilic archaea. In this study, two Na⁺/H⁺ antiporter genes, nhaC1 and nhaC2, were screened from the genome of Natronorubrum daqingense based on the gene library and complementation of salt-sensitive Escherichia coli KNabc. A clone vector pUC18 containing nhaC1 or nhaC2 could make KNabc tolerate 0.6 M/0.7 M NaCl or 30 mM/40 mM LiCl and a pH of up to 8.5/9.5, respectively. Functional analysis shows that the Na⁺(K⁺, Li⁺)/H⁺ antiport activities of NhaC1 and NhaC2 are both pH-dependent in the range of pH 7.0-10.0, and the optimal pH is 9.5. Phylogenetic analysis shows that both NhaC1 and NhaC2 belong to the Na⁺/H⁺ antiporter NhaC family of proteins and are significantly distant from the identified NhaC proteins from Bacillus. In summary, we have identified two Na⁺(K⁺, Li⁺)/H⁺ antiporters from N. daqingense.

Keywords: Na+/H+ antiporter; Natronorubrum daqingense; NhaC family protein; extremely halophilic archaea.

Figure 1

Screening of ORFs that exert Na⁺/H⁺ antiport function. (A) The agarose gel electrophoresis of positive clone plasmids that could restore the growth of E. coli KNabc on LBK medium containing 0.2 M NaCl; (B) The agarose gel electrophoresis after double enzyme digestion of positive clone plasmids by EcoRI and HindIII (the corresponding lanes in A&B are Marker and pUC-1028-1 to pUC1028-10 from left to right); (C) Construction schematic of the expression vector pET28AK in this study; (D,E) E. coli KNabc transformants were grown on the LBK medium and LBK medium containing 0.2 M NaCl, (1) KNabc/pUC-1028-1-ORF3, (2) KNabc/pET-1028-1-ORF4, (3) KNabc/pUC-1028-7-ORF2, (4) KNabc/pUC-1028-9-ORF2, (5) KNabc/pET1028-9-ORF3, (6) KNabc/pET1028-9-ORF4, (7) KNabc/pUC18, (8) KNabc/pET28AK, (9) blank.

Figure 2

Hydrophobicity analysis and transmembrane segment (TMS) prediction of NhaC1 and NhaC2. (A,B) Hydrophobicity analysis of NhaC1 and NhaC2; (C,D) 11 and 12 predicted TMSs of NhaC1 and NhaC2.

Figure 3

Phylogenetic trees of NhaC1 (A) and NhaC2 (B) with their closest homologs and an identified protein with Na⁺/H⁺ antiport activity based on the neighbour-joining algorithm. To construct phylogenetic trees, the 24 and 33 closest homologs with 72.84–96.47% and 63.62–98.28% identities, respectively, were selected from 100 sequences. An identical protein from the NhaC family was selected, as shown in blue in Figure 3. Bootstrap values > 50% (based on 1000 replications) are shown at branch points. Both NhaC1 and NhaC2 and their closest homologs clustered with the bootstrap values of 100%; both are shown in bold red in Figure 3.

Figure 4

(A) Alignment between NhaC1 and homologs of NhaC family proteins. The 11 homologs with percent identities from 80.43% to 96.47% were selected from Halopiger, Haloterrigena, Natronorubrum, Halostagnicola, Natronococcus, and Natronorubrum. (B) Alignment between NhaC2 and 14 homologs of NhaC family proteins. A total of 14 homologs from Natronococcus, Halobiforma, Natronorubrum, and Halostagnicola with the percent identity ranging from 85.11% to 93.28% were selected. Shading homology corresponds to 100% (black), ≥75% (grey), ≥50% (light grey), and <50% (white) amino acid identity.

Figure 5

Salt tolerance and alkaline pH resistance of nhaC1 and nhaC2. To test the salt tolerance of KNabc/pUC-nhaC1 and KNabc/pUC-nhaC2, 1% overnight cultures (OD₆₀₀ was adjusted to 0.8 before inoculation) were inoculated to LBK medium containing 0–0.8 M NaCl (A) or 0–50 mM LiCl (B) and 50 mg∙mL⁻¹ of ampicillin, respectively, at pH 7.0, and cultured at 37 °C for 12 h, with KNabc/pUC18 as the negative control, and the OD600 nm was measured; (C) 1% overnight cultures (OD₆₀₀ was adjusted to 0.8 before inoculation) were inoculated to LBK medium containing 50 mM NaCl and 50 mg∙mL⁻¹ of ampicillin (pH 7.0–10.0) and cultured at 37 °C for 12 h; KNabc/pUC18 was used as negative control, and the OD₆₀₀ was measured. Data in the figure represent the average of three independent trials.

Figure 6

Detection of molecular weights and cell localizations of NhaC1 and NhaC2. (A,B) SDS-PAGE of NhaC1 fused with 6×His tag and NhaC2 fused with 6×His tag, purified by Ni-NTA affinity chromatography, and the staining method was Coomassie brilliant blue staining; (C,D) Cell localizations of NhaC1 and NhaC2 detected by Western blots. Total protein, cytoplasmic protein, and membrane protein of NhaC1 fused with 6×His tag and NhaC2 fused with 6×His tag are shown in Lanes 1, 3, and 5, respectively; Total protein, cytoplasmic protein, and membrane protein from KNabc/pET28AK are shown as a negative control in Lanes 2, 4, and 6, respectively.

Figure 7

Detection of Na⁺(K⁺, Li⁺)/H⁺ antiport activity. (A) The measurements for Na⁺(K⁺, Li⁺)/H⁺ antiport activities were performed in everted membrane vesicles prepared by E. coli KNabc containing pET28AK (as negative controls), pET28AK-nhaC1, and pET28AK-nhaC2 at pH 7.0 with monovalent cations (final concentration 5 mM), respectively; (B,C) Detection of pH-dependent activity profile of NhaC1 and NhaC2. Data in the figure represent the average of three independent trials.

Figure 8

Calculation of K_0.5 values of Na⁺, K⁺, and Li⁺ by NhaC1 and NhaC2. With the final concentration of monovalent cations as the abscissa and the fluorescence dequenching rate as the ordinate, origin2017 was used for nonlinear regression analysis to fit the curve and calculate the final concentration on monovalent cations added, which could reach half of the maximum fluorescence dequenching. (A–C) The curves and K_0.5 values of Na⁺, K⁺, and Li⁺ NhaC1, respectively; (D–F) The curves and K_0.5 values of Na⁺, K⁺, and Li⁺ NhaC2 respectively. Data in the figure represents the average of three independent trials.