Evidence for bidirectional formic acid translocation in vivo via the Escherichia coli formate channel FocA.

Pentameric FocA permeates either formate or formic acid bidirectionally across the cytoplasmic membrane of anaerobically growing Escherichia coli. Each protomer of FocA has its own hydrophobic pore, but it is unclear whether formate or neutral formic acid is translocated in vivo. Here, we measured total and dicyclohexylcarbodiimide (DCCD)-inhibited proton flux out of resting, fermentatively grown, stationary-phase E. coli cells in dependence on FocA. Using a wild-type strain synthesizing native FocA, it was shown that using glucose as a source of formate, DCCD-independent proton efflux was ∼2.5 mmol min-1, while a mutant lacking FocA showed only DCCD-inhibited, FOF1-ATPase-dependent proton-efflux. A strain synthesizing a chromosomally-encoded FocAH209N variant that functions exclusively to translocate formic acid out of the cell, showed a further 20 % increase in FocA-dependent proton efflux relative to the parental strain. Cells synthesizing a FocAT91A variant, which is unable to translocate formic acid out of the cell, showed only DCCD-inhibited proton efflux. When exogenous formate was added, formic acid uptake was shown to be both FocA- and proton motive force-dependent. By measuring rates of H2 production, potassium ion flux and ATPase activity, these data support a role for coupling between formate, proton and K+ ion translocation in maintaining pH and ion gradient homeostasis during fermentation. FocA thus plays a key role in maintaining this homeostatic balance in fermenting cells by bidirectionally translocating formic acid.

HyfF subunit of hydrogenase 4 is crucial for regulating FOF1 dependent proton/potassium fluxes during fermentation of various concentrations of glucose.

Escherichia coli anaerobically ferment glucose and perform proton/potassium exchange at pH 7.5. The role of hyf (hydrogenase 4) subunits (HyfBDF) in sensing different concentrations of glucose (2 g L-1 or 8 g L-1) via regulating H+/K+ exchange was studied. HyfB, HyfD and HyfF part of a protein family of NADH-ubiquinone oxidoreductase ND2, ND4 and ND5 subunits is predicted to operate as proton pump. Specific growth rate was optimal in wild type and mutants grown on 2 g L-1 glucose reaching ~ 0.8 h-1. It was shown that in wild type cells proton but not potassium fluxes were stimulated ~ 1.7 fold reaching up to 1.95 mmol/min when cells were grown in the presence of 8 g L-1 glucose. Interestingly, cells grown on peptone only had similar proton/potassium fluxes as grown on 2 g L-1glucose. H+/K+ fluxes of the cells grown on 2 g L-1 but not 8 g L-1 glucose depend on externally added glucose concentration in the assays. DCCD-sensitive H+ fluxes were tripled and K+ fluxes doubled in wild type cells grown on 8 g L-1 glucose compared to 2 g L-1 when in the assays 2 g L-1glucose was added. Interestingly, in hyfF mutant when cells were grown on 2 g L-1glucose and in 2 g L-1 assays DCCD-sensitive fluxes were not determined compared to wild type while in hyfD mutant it was doubled reaching up to 0.657 mmol/min. In hyf mutants DCCD-sensitive K+ fluxes were stimulated in hyfD and hyfF mutants compared to wild type but depend on external glucose concentration. DCCD-sensitive H+/K+ ratio was equal to ~ 2 except hyfF mutant grown and assayed on 2 g L-1glucose while in 8 g L-1 conditions role of hyfB and hyfD is considered. Taken together it can be concluded that Hyd-4 subunits (HyfBDF) play key role in sensing glucose concentration for regulation of DCCD-sensitive H+/K+ fluxes for maintaining proton motive force generation.

Glucose concentration is determinant for the functioning of hydrogenase 1 and hydrogenase 2 in regulating the proton and potassium fluxes in Escherichia coli at pH 7.5.

This study examines how FOF1-ATPase, hydrogenases (Hyd-1 and Hyd-2), and potassium transport systems (TrkA) interact to maintain the proton motive force (pmf) in E. coli during fermentation of different glucose concentrations (2 g L-1 and 8 g L-1). Our findings indicate that mutants lacking the hyaA-hyaC genes exhibited a 30 % increase in total proton flux compared to the wild type when grown with 2 g L-1 glucose. This has been observed during assays where similar glucose levels were supplemented. Disruptions in proton pumping, particularly in hyaB and hyaC single mutants, led to increased potassium uptake. The hyaB mutant showed a threefold increase in the contribution of FOF1-ATPase to proton flux, suggesting a significant role for Hyd-1 in proton translocation. In the hybC mutant grown in 2 g L-1 glucose conditions, DCCD-sensitive fluxes decreased by 70 %, indicating critical role of Hyd-2 in proton transport and FOF1 function. When cells were grown with 8 g L-1 glucose, the 2H+/1K+ ratio was significantly disturbed in both wild type and mutants. Despite these perturbances, mutants with disruptions in Hyd-1 and Hyd-2 maintained constant FOF1 function, suggesting that this enzyme remains stable in glucose-rich environments. These results provide valuable insights into how Hyd-1 and Hyd-2 contribute to the regulation of ion transport, particularly proton translocation, in response to glucose concentration. Our study uncovered potential complementary mechanisms between Hyd-1 and Hyd-2 subunits, suggesting a complex interplay between these enzymes via metabolic cross talk with FOF1 in response to glucose concentrations to maintain pmf.

Roasted coffee wastes as a substrate for Escherichia coli to grow and produce hydrogen.

After brewing roasted coffee, spent coffee grounds (SCGs) are generated being one of the daily wastes emerging in dominant countries with high rate and big quantity. Escherichia coli BW25113 wild-type strain, mutants with defects in hydrogen (H2)-producing/oxidizing four hydrogenases (Hyd) (ΔhyaB ΔhybC, ΔhycE, ΔhyfG) and septuple mutant (ΔhyaB ΔhybC ΔhycA ΔfdoG ΔldhA ΔfrdC ΔaceE) were investigated by measuring change of external pH, bacterial growth and H2 production during the utilization of SCG hydrolysate. In wild type, H2 was produced with rate of 1.28 mL H2 (g sugar)-1 h-1 yielding 30.7 mL H2 (g sugar)-1 or 2.75 L (kg SCG)-1 during 24 h. In septuple mutant, H2 production yield was 72 mL H2 (g sugar)-1 with rate of 3 mL H2 (g sugar)-1 h-1. H2 generation was absent in hycE single mutant showing the main role of Hyd-3 in H2 production. During utilization of SCG wild type, specific growth rate was 0.72 ± 0.01 h-1 with biomass yield of 0.3 g L-1. Genetic modifications and control of external parameters during growth could lead to prolonged and enhanced microbiological H2 production by organic wastes, which will aid more efficiently global sustainable energy needs resulting in diversification of mobile and fixed energy sources.