Coordinated Expression of the Genes Encoding FocA and Pyruvate Formate-Lyase Is Important for Maintenance of Formate Homeostasis during Fermentative Growth of Escherichia coli

Abstract

FocA is a pentameric membrane channel that translocates formic acid bidirectionally across the cytoplasmic membrane of Escherichia coli during fermentation. The focA gene is co-transcribed with pflB, which encodes pyruvate formate-lyase, the enzyme that generates formate. Recent evidence has suggested that FocA serves to regulate intracellular formate levels and thus helps to maintain pH balance in fermenting cells. In this study, we aimed to provide support for this hypothesis by either altering FocA levels, mutating the chromosomal focA gene, or introducing additional copies of focA, either alone or with pflB, on a plasmid and monitoring the effect on intracellular and extracellular formate levels. Our results revealed that the expression of the native focA-pflB operon ensures that intracellular formate levels remain relatively constant during exponential phase growth, even when additional, mutated copies of focA that encode FocA variants are introduced in trans. Enhancing focA expression was balanced by higher formate excretion from the cell. Using chromosomal focA gene variants confirmed that FocA, and not PflB, sets intracellular formate homeostatic levels. Moreover, any chromosomal focA mutation that altered the formate concentration inside the cell caused a negative fermentative growth phenotype. Thus, FocA governs intracellular formate levels to ensure optimal growth during glucose fermentation.