A single amino acid exchange converts FocA into a unidirectional efflux channel for formate

Abstract

During mixed-acid fermentation, Escherichia coli initially translocates formate out of the cell, but re-imports it at lower pH. This is performed by FocA, the archetype of the formate-nitrite transporter (FNT) family of pentameric anion channels. Each protomer of FocA has a hydrophobic pore through which formate/formic acid is bidirectionally translocated. It is not understood how the direction of formate/formic acid passage through FocA is controlled by pH. A conserved histidine residue (H209) is located within the translocation pore, suggesting that protonation/deprotonation might be linked to the direction of formate translocation. Using a formate-responsive lacZ-based reporter system we monitored changes in formate levels in vivo when H209 in FocA was exchanged for either of the non-protonatable amino acids asparagine or glutamine, which occur naturally in some FNTs. These FocA variants (with N or Q) functioned as highly efficient formate efflux channels and the bacteria could neither accumulate formate nor produce hydrogen gas. Therefore, the data in this study suggest that this central histidine residue within the FocA pore is required for pH-dependent formate uptake into E. coli cells. We also address why H209 is evolutionarily conserved and provide a physiological rationale for the natural occurrence of N/Q variants of FNT channels.

Keywords: FNT family; anion channel; conserved histidine; formate uptake; hydrophobic pore.

Fig. 1.

Conservation of histidine residue 209 within the pore of FNT channels. (a) Illustration of the central position of histidine 209 (stick representation, blue) within FocA’s translocation pore. The protomer (PDB 3KCU [8]) is displayed in green and ribbon representation and the pore (grey) was modelled using the MOLE 2.5 online tool (for parameters, see Methods). (b) Conservation plot comparing the amino acid residues between positions 205 and 215 using 306 FNT channels to create a WebLogo 3 stacking plot (see Methods). The residues are aligned with native FocA from E. coli , whose sequence (residues 205–215) is presented below the plot. Amino acid residues displayed in black possess an apolar side chain (A, F, I, L, M, P, V, W) and polar side chains are indicated in green (C, G, N, Q, S, T, Y). Acidic amino acids are shown in red (D, E) and basic amino acid residues are shown in blue (H, K, R).

Fig. 2.

Histidine-209 is essential for bi-directional translocation of formate by FocA. Parameters determined to monitor changes in formate translocation were investigated in the focA mutant DH701 (focA) and in DH701 complemented with plasmids carrying a focA gene encoding the indicated H209 variants. Strains were grown anaerobically in glucose-M9-minimal medium. (a) Schematic representation of the output of the fdhF_P::lacZ reporter system used to monitor changes in intracellular formate (red circles) levels. (b) β-Galactosidase enzyme activity of the respective strains was analysed in cells grown in culture medium without (black histogram) and with exogenously added 20 mM formate (white histogram). (c) The concentration of formate in the culture medium was determined by HPLC analysis. (d) H₂ evolved by cells accumulated in the headspace of the cultures during growth in glucose-M9-minimal medium. H₂ was determined quantitatively using GC. All experiments were performed with minimally three biological replicates, with each assay performed in duplicate.

Fig. 3.

Cells synthesizing the FocA_H209N variant function as unidirectional formate efflux channels. Strains DH701 (focA), DH701/pfocA, DH701/pfocAH209N and DH701/pfocAH209N-∆K2-P31 were grown anaerobically in glucose-M9-minimal medium in crimp-sealed serum bottles. Samples were taken every hour to analyse optical density (OD₆₀₀), determine β-galactosidase enzyme activity, pH and formate levels of the culture medium. (a, h) Results obtained for strain DH701, (b, f) for strain DH701/pfocA, (c, g) for strain DH701/pfocAH209N and (d, h) for strain DH701/pfocAH209N-∆K2-P31. Parameters obtained for strains, whose focA gene expression was elevated by addition of 0.2 mg l⁻¹ AHT (anhydrotetracycline) after entering the exponential growth phase, are presented as filled squares. Data for strains grown without AHT treatment are displayed as circles. The colour-coding is as follows: light grey, OD₆₀₀; blue, β-galactosidase activity; dark grey, pH; and red, external formate concentration, which was calculated with respect to OD₆₀₀. All experiments were performed with three biological replicates.

Fig. 4.

The FocA_H209N variant is not able to take up formate. Formate translocation was investigated in the focA and pflA mutant DH601 (focA pflA) and in DH601 complemented with plasmids carrying a gene encoding either native FocA or FocA_H209N. β-Galactosidase enzyme activity of the respective strains was analysed in cells grown anaerobically in glucose-M9-minimal medium with exogenously added 20 mM formate. The experiment was carried out with minimally three biological replicates, with each assay performed in duplicate.

Fig. 5.

Histidine-209 is essential for uptake of hypophosphite by FocA. Sensitivity of strain DH701 (focA) synthesizing different FocA amino acid exchange variants toward hypophosphite was assessed by determining anaerobic growth rates in glucose-minimal medium in the absence (black histograms) or presence of 0.5 mM sodium hypophosphite (white histograms). The experiments were performed in triplicate.

Fig. 6.

DH4200 (FocA_H209N) is deficient in formate uptake and exhibits reduced anaerobic growth in minimal medium. Strain DH4200 (FocA_H209N) was characterized with respect to both intra- and extracellular formate levels and its growth phenotype. (a) β-Galactosidase enzyme activity of DH4100 and DH4200 was analysed for cells grown anaerobically in glucose-M9-minimal medium. (b) The concentration of formate in the culture medium was determined by HPLC analysis. (c) The respective strains, DH4100 (closed symbols) and DH4200 (open symbols), were grown anaerobically in glucose-M9-minimal medium (circles) and rich medium TGYEP (squares), both supplemented with 0.8 % (w/v) glucose as carbon source. All experiments were performed with minimally three biological replicates, with each assay performed in duplicate.

Fig. 7.

Native FocA shows dominance over the N209 exchange variant. Determination of β-galactosidase enzyme activity after anaerobic growth of the wild-type strain DH4100 (black histogram), the focA mutant DH701 (no FocA synthesized, grey histogram) and strain DH702 (10-fold elevated FocA levels; white histogram) transformed with a plasmid carrying a gene encoding either native FocA or FocA_H209N. The strains were grown anaerobically in glucose-M9-minimal medium. The experiment was performed with minimally three biological replicates.