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

Abstract

FocA translocates formate/formic acid bi-directionally across the cytoplasmic membrane when Escherichia coli grows by fermentation. It remains unclear, however, what physiological benefit is imparted by FocA, because formic acid (pK a=3.75) can diffuse passively across the membrane, especially at low pH. Here, we monitored changes in intra- and extracellular formate levels during batch-culture fermentation, comparing a parental E. coli K-12 strain with its isogenic focA mutant. Our results show that, regardless of the initial pH in the culture, FocA functions to maintain relatively constant intracellular formate levels during growth. Analysis of a strain synthesizing a FocAT91A variant with an exchange in a conserved threonine residue within the translocation pore revealed the strain accumulated formate intracellularly and imported formate poorly, but in a pH-dependent manner, which was different to uptake by native FocA. We conclude that FocA maintains formate homeostasis, using different mechanisms for efflux and uptake of the anion.

FocA and its central role in fine-tuning pH homeostasis of enterobacterial formate metabolism

“…When the pH in the periplasmic space of the E. coli cell becomes too acidic, FocA working together with FHL-1 alleviates pH stress [8, 23, 64, 78–80]. In this situation, the proton associated with formic acid might not be relayed back to H209, but instead would be translocated directly into the cytoplasm along with the anion (); this might result from the H209/T91 proton-relay system being overwhelmed by the high concentration of formic acid, with FocA adopting an inwardly directed channel-like function (see ).…”

“…A summary of the amino acid exchanges introduced and tested is shown in Table 1. Cells synthesizing a chromosomally encoded T91A variant of FocA accumulate formate intracellularly, indicating that the threonine residue is particularly important for anion efflux [23]. That serine can partially substitute for threonine suggests that the hydroxyl group of the side chain has a key role in formic acid translocation to the periplasm [66].…”

“…When the pH in the periplasmic space of the E. coli cell becomes too acidic, FocA working together with FHL-1 alleviates pH stress [8,23,64,[78][79][80]. In this situation, the proton associated with formic acid might not be relayed back to H209, but instead would be translocated directly into the cytoplasm along with the anion (Fig.…”

Distinguishing functional from structural roles of conserved pore residues during formate translocation by the FocA anion channel

“…An in vivo assay using a formate‐sensitive fdhF P :: lacZ ‐based reporter was established to study FocA from E. coli (Beyer et al, 2013; Suppmann & Sawers, 1994). The reporter assay allows monitoring of changes in intracellular formate concentration and has shown that H209 is crucial for pH‐dependent uptake of formate by FocA (Kammel, Trebbin, Pinske, et al, 2022), while T91 has an essential function in formate efflux out of the cell (Hunger et al, 2014; Kammel & Sawers, 2022a, 2022b). X‐ray crystal structure data have revealed that T91 can adopt two positions relative to the fixed location of H209; T91 either forms a hydrogen bond with H209, or it moves roughly 5 Å away from it, toward the cytoplasmic vestibule (Lü, Schwarzer, et al, 2012; Waight et al, 2010).…”

Microbial Musings – Spring 2022

“…As well as gaining insight into the role of the conserved histidine in bidirectional transporter, the His209Asn mutant grows more poorly than both the wild-type and a strain lacking focA completely, suggesting that allowing efflux only leads to a physiological imbalance that disrupts normal growth. In fact Michelle and Gary have just published another Short Communication in Microbiology to study this further [25], where they study a different focA allele containing a point mutant that makes uptake of formate pH-dependent. Using this strain and similar methods to the first paper they conclude that FocA likely uses different mechanisms for the efflux and uptake of formate that together control the intracellular level of formate.…”