Coordination of FocA and Pyruvate Formate-Lyase Synthesis in Escherichia coli Demonstrates Preferential Translocation of Formate over Other Mixed-Acid Fermentation Products

Abstract

Enterobacteria such as Escherichia coli generate formate, lactate, acetate, and succinate as major acidic fermentation products. Accumulation of these products in the cytoplasm would lead to uncoupling of the membrane potential, and therefore they must be either metabolized rapidly or exported from the cell. E. coli has three membrane-localized formate dehydrogenases (FDHs) that oxidize formate. Two of these have their respective active sites facing the periplasm, and the other is in the cytoplasm. The bidirectional FocA channel translocates formate across the membrane delivering substrate to these FDHs. FocA synthesis is tightly coupled to synthesis of pyruvate formate-lyase (PflB), which generates formate. In this study, we analyze the consequences on the fermentation product spectrum of altering FocA levels, uncoupling FocA from PflB synthesis or blocking formate metabolism. Changing the focA translation initiation codon from GUG to AUG resulted in a 20-fold increase in FocA during fermentation and an ϳ3-fold increase in PflB. Nevertheless, the fermentation product spectrum throughout the growth phase remained similar to that of the wild type. Formate, acetate, and succinate were exported, but only formate was reimported by these cells. Lactate accumulated in the growth medium only in mutants lacking FocA, despite retaining active PflB, or when formate could not be metabolized intracellularly. Together, these results indicate that FocA has a strong preference for formate as a substrate in vivo and not other acidic fermentation products. The tight coupling between FocA and PflB synthesis ensures adequate substrate delivery to the appropriate FDH.

Pyruvate Formate-Lyase Interacts Directly with the Formate Channel FocA to Regulate Formate Translocation

“…A similar result was observed with a crude extract derived from anaerobically grown REK702 (Fig. 2a, lane 2), which synthesizes higher levels of both FocA and PflB due to conversion of the focA GUG translation initiation codon to the more efficiently translated AUG codon [1,17]. In contrast, when the experiment was repeated with a crude extract of the pflB mutant RM220, no 85-kDa polypeptide corresponding to PflB was observed (Fig.…”

“…Recent studies have shown that FocA non-selectively translocates formate and other weak acids in an in vitro system [8], while E. coli cells have a mechanism to transport formate selectively [1,17]. Because PflB is responsible for formate generation in bacteria such as E. coli and its synthesis is tightly coupled with that of FocA, we decided to examine whether PflB in its active or inactive form might exert a regulatory influence on the ability of fermenting E. coli to take up formate.…”

“…If no acceptor is available, however, which occurs during fermentative growth, formate can be re-imported into the cell where it is metabolized by a formate-inducible formate hydrogenlyase complex to the gaseous products carbon dioxide and hydrogen [1,16]. Formate uptake by the cell also occurs through FocA; however, this is dependent upon an active formate hydrogenlyase complex, which dissipates intracellularly accumulated formate [17]. …”

“…Physiological studies on fermentative formate metabolism in E. coli have provided evidence that FocA translocates the anion bidirectionally [1,17]. The mechanism underlying control of bidirectional formate translocation by FocA is unknown.…”

“…Furthermore, a recent electrophysiological study demonstrated that isolated FocA is capable of translocating not only formate but also acetate and lactate in vitro , leading to the suggestion that FocA might have a comparatively broad substrate specificity [8]. However, the tight coupling between FocA and PflB synthesis in vivo, together with the fact that a strong correlation between formate translocation and FocA could be demonstrated for E. coli cells but not for other mixed acids [1,17], suggests that a mechanism exists in vivo , ensuring that only formate is translocated by the channel. Therefore, identifying how in vivo substrate specificity and “gating” of the FNT proteins is governed is an important question to address.…”