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.

The FocA channel functions to maintain intracellular formate homeostasis during Escherichia coli fermentation

“…Previous structural analyses revealed a water molecule coordinated to T91 when it is hydrogen-bonded with H209 [7]. The requirement for T91 to allow effective formate translocation by FocA in vivo supports a role for the hydroxyl group of the threonine side-chain, the bound water and the imidazole/imidazolium side-chain of H209 [16] functioning together to control formate movement through the pore. The separation of T91 and H209 shown in the original structure of E.…”

“…One of these is the cytoplasmically oriented N -terminal domain of FocA, which gates formate permeation through an interaction with PflB [13, 14]. The other structural feature involves control of anion permeation through the hydrophobic protomer pore by two highly conserved amino acid residues: threonine at position 91 (T91) and histidine at position 209 (H209) () [15, 16]. Within the pore itself, exchange of the H209 residue for either of the amide amino acids (Asn or Gln) converts FocA into a channel that functions exclusively to efflux formate from the cell [16].…”

“…Next, we measured β-galactosidase enzyme activity for DH4100 and DH4300 in stationary-phase cultures, after growth for 22 h in M9-glucose minimal medium, pH 7 [14,16], and noted that activity for strain DH4300 was ~60 % higher than that measured for DH4100 (Fig. 3).…”

“…Three strains were used in this study and included the parental strain DH4100 [15], a focA mutant DH701 that cannot synthesize FocA [15], and DH4300, which synthesizes a chromosomally encoded FocA T91A variant. Strain DH4300 was constructed by first mutating codon 91 of the focA gene on a subcloning pJET-plasmid containing a focA-pflB fragment [16] using oligonucleotide primers CGGA GCCG ATCT CTTT GCTT CCAC CGTGT (forward primer) and AACA CGGT GGAA GCAA AGAG ATCG GCTCCGC (reverse primer), adopting a standard PCR-based mutagenesis approach [16]. After subsequent cloning of the DNA fragment including the mutated focA gene into pMAK705, the mutated gene was introduced onto the chromosome of MC4100 [21] to generate strain MC4300, using the method described [22].…”

Hydrogen production in the presence of oxygen by Escherichia coli K-12

“…Indeed, site-directed mutagenesis of focA has identified amino acid residues that are important for formic acid influx, but not efflux. For example, substitution of E. coli FocA His-209 by either Asn or Gln locked the channel in efflux mode only [37]. This hypothesis would also stand if the monoculture of E.…”