Introduction: Formate is a central intermediate in enterobacterial mixed-acid fermentation of glucose. During exponential-phase growth, formic acid passes from the cytoplasm to the periplasm via the pentameric channel, FocA. Upon entry into the late exponential-phase of growth, or upon a decrease in external pH below 6.5, FocA re-imports formate or formic acid into the cytoplasm, where it is disproportionated into CO2 and H2 by the membrane-associated formate hydrogenlyase (FHL) complex. Uptake of formic acid by FocA is dependent on an active FHL complex, suggesting coupling between both systems. Each protomer of the FocA pentamer has a narrow hydrophobic pore through which neutral formic acid can pass. Conserved histidine (H209) and threonine (T91) residues are required to control uptake of formate, implying that the pore of FocA does not function simply as a "channel". Objectives: FocA is suggested to have two functions: 1, as a bidirectional channel for formic acid; 2, as a formate importer. It is proposed that FocA channel/import functions are coupled to the FHL complex to maintain pH homeostasis and optimize fermentative growth. We tested this hypothesis in the current study.

Methods: We use a formate-responsive reporter system, FocA amino acid-exchange variants, together with fhl mutants to assess various physiological parameters, including H2 production and formate translocation to test the dependence of FocA on the FHL complex.

Results: FocA variants, respectively, either rapidly translocate formate out of the cell (H209N variant) and produce low levels of H2, or accumulate formate intracellularly (T91A variant). Mutants that lack an FHL complex fail to produce H2 and also show poorer early exponential-phase growth than the parental strain.

Conclusion: Together, our findings indicate that FocA functions together with the FHL complex to maintain relatively constant intracellular formate levels, to improve growth by helping maintain pH homeostasis, and possibly also to contribute to energy conservation.