Preferential Translocation of Formate Over Other Mixed-Acid Fermentation

26 Enterobacteria like Escherichia coli generate formate, lactate, acetate and succinate as major 27 acidic fermentation products. Accumulation of these products in the cytoplasm would lead to 28 uncoupling of the membrane potential and therefore they must be either metabolized rapidly 29 or exported from the cell. E. coli has three membrane-localized formate dehydrogenases 30 (FDH) that oxidize formate. Two of these have their respective active site facing the 31 periplasm, the other in the cytoplasm. The bi-directional FocA channel translocates formate 32 across the membrane delivering substrate to these FDHs. FocA synthesis is tightly coupled to 33 synthesis of pyruvate formate-lyase (PflB), which generates formate. In this study we 34 analysed the consequences on the fermentation product spectrum of altering FocA levels, 35 uncoupling FocA from PflB synthesis or blocking formate metabolism. Changing the focA 36 translation initiation codon from GUG to AUG resulted in a 20-fold increase in FocA during 37 fermentation and an approximate 3-fold increase in PflB. Nevertheless, the fermentation 38 product spectrum thoughout the growth phase remained similar to that of the wild type. 39 Formate, acetate and succinate were exported but only formate was re-imported by these 40 cells. Lactate accumulated in the growth medium only in mutants lacking FocA, despite 41 retaining active PflB, or when formate could not be metabolized intracellularly. Together, 42 these results indicate that FocA has a strong preference for formate as a substrate in vivo and 43 not other acidic fermentation products. The tight coupling between FocA and PflB synthesis 44 ensures adequate substrate delivery to the appropriate FDH. 45 46 47 48 on Jauary 2, 2018 by gest http/jb.asm .rg/ D ow nladed fom