Role of the Escherichia coli FocA and FocB formate channels in controlling proton/potassium fluxes and hydrogen production during osmotic stress in energy-limited, stationary phase fermenting cells.

Escherichia coli FocA and FocB formate channels export formate or import it for further disproportionation by the formate hydrogenlyase (FHL) complex to H2 and CO2. Here, we show that under pH and osmotic stress FocA and FocB play important roles in regulating proton and potassium fluxes and couple this with H2 production in stationary-phase cells. Using whole-cell assays with glucose as electron donor, a focB mutant showed a 50 % decrease in VH2, while N'N'-dicyclohexylcarbodiimide (DCCD) treatment of osmotically stressed cells underlined the role of FOF1 ATPase in H2 production. At pH 7.5 and under osmotic stress FocB contributed to the proton flux but not to the potassium flux. At pH 5.5 both formate channels contributed to the proton and potassium fluxes. Particulalry, a focA mutant had 40 % lower potassium flux whereas the proton flux increased approximately two-fold. Moreover, at pH 5.5H2 production was totally inhibited by DCCD in the focA mutant. Taken together, our results suggest that depending on external pH, the formate channels play an important role in osmoregulation by helping to balance proton/potassium fluxes and H2 production, and thus assist the proton FOF1-ATPase in maintenance of ion gradients in fermenting stationary-phase cells.

Escherichia coli Dcu C4-dicarboxylate transporters dependent proton and potassium fluxes and FOF1-ATPase activity during glucose fermentation at pH 7.5.

During fermentation in Escherichia coli succinate is transported via Dcu transporters, encoded dcuA, dcuB, dcuC and dcuD although the role of DcuD protein has not been elucidated yet. It has been shown contribution of Dcu transporters in the N,N'-dicyclohexylcarbodiimide (DCCD) sensitive proton and potassium transport through the cytoplasmic membrane and membrane-associated ATPase activity. Total H± efflux was decreased ~ 40% while K± uptake was absent in dcuD mutant. DCCD-sensitive H± flux was absent in dcuD nevertheless it was increased ~ 3 fold in dcuACB. K± uptake in dcuACB was stimulated ~ 30% compared to wild type but in DCCD assays K± ions were effluxed with the rate of 0.15 mmol/min per 109 cells/ml. In dcuACB mutant membrane potential (ΔΨ) was ~ 30 mV higher than in wild type. dcuD gene expression was increased in the dcuACB mutant respect to wild type at pH 7.5 (~120%), suggesting that an increment of DcuD activity compensates the lack of DcuA, DcuC and DcuB carriers. It can be concluded that active DcuD is important for H± efflux via the FOF1-ATPase and K± uptake at pH 7.5. In addition, DcuA, DcuB and DcuC transporters are crucial for regulating DCCD-sensitive K± transport and ΔΨ in E. coli.

External succinate and potassium ions influence Dcu dependent FOF1-ATPase activity and H+ flux of Escherichia coli at different pHs.

During fermentation Escherichia coli transport succinate mainly via Dcu family carriers. Current paper describes the role of externally added succinate on N'N'-dicyclohexylcarbodiimide (DCCD) sensitive ATPase activity and H+ flux depending on potassium ions. At pH 7.5 in wild type membrane vesicles DCCD-sensitive ATPase activity was the same as in dcuACBD quadruple mutant. In dcuACB it was increased ~ 3.3 fold while in dcuD DCCD-sensitive ATPase activity was absent. The DCCD-sensitive H+ efflux was fully dependent on FOF1 only in dcuACB mutant. This activity depended on potassium ions and only in dcuACBD mutant DCCD-sensitive ATPase activity was stimulated ~ 3 fold. At pH 5.5 DCCD-sensitive ATPase activities were determined in dcuACB or dcuD mutants but not in wild type. Interestingly, addition of potassium ions enhanced DCCD-sensitive ATPase activity in dcuD mutant ~ 3-fold compared to wild type. In dcuD mutant ~ 3-fold higher H+ uptake was registered, compared to wild type. Taken together it can be concluded that at pH 7.5 the FOF1-activity depends on DcuACB. Moreover, DcuACB but not DcuD are working towards H+ uptake direction. DcuD contributes to H+ efflux at pH 7.5 while at pH 5.5 it affects H+ influx when external succinate is present.

Evidence for Escherichia coli DcuD carrier dependent FOF1-ATPase activity during fermentation of glycerol.

During fermentation Escherichia coli excrete succinate mainly via Dcu family carriers. Current work reveals the total and N,N'-dicyclohexylcarbodiimide (DCCD) inhibited ATPase activity at pH 7.5 and 5.5 in E. coli wild type and dcu mutants upon glycerol fermentation. The overall ATPase activity was highest at pH 7.5 in dcuABCD mutant. In wild type cells 50% of the activity came from the FOF1-ATPase but in dcuD mutant it reached ~80%. K+ (100 mM) stimulate total but not DCCD inhibited ATPase activity 40% and 20% in wild type and dcuD mutant, respectively. 90% of overall ATPase activity was inhibited by DCCD at pH 5.5 only in dcuABC mutant. At pH 7.5 the H+ fluxes in E. coli wild type, dcuD and dcuABCD mutants was similar but in dcuABC triple mutant the H+ flux decreased 1.4 fold reaching 1.15 mM/min when glycerol was supplemented. In succinate assays the H+ flux was higher in the strains where DcuD is absent. No significant differences were determined in wild type and mutants specific growth rate except dcuD strain. Taken together it is suggested that during glycerol fermentation DcuD has impact on H+ fluxes, FOF1-ATPase activity and depends on potassium ions.

pH and a mixed carbon-substrate spectrum influence FocA- and FocB-dependent, formate-driven H2 production in Escherichia coli.

Escherichia coli encodes two formate channels, FocA and FocB, that either export formate or import it for further disproportionation by the formate hydrogenlyase (FHL) complex to H2 and CO2. We show that FocA/B appear to change their substrate-translocation direction depending on pH and electron donor. When cells were grown on glucose and glycerol at pH 7.5, formate accumulated in focB or focA-focB mutants when glucose or formate was used as electron donor because H2 production increased ∼2- and ∼1.5-fold, respectively. Moreover, addition of external formate to the growth medium increased H2 production in a focA-focB mutant. This indicates that in the wild type, formate is preferentially exported at pH 7.5 and that another FocA/B-independent uptake system exists. At pH 6.5 and 5.5, the formate channel mutants showed reduced H2 production, suggesting that formate is usually imported by them to produce H2 at acidic pH. Addition of formate to the growth medium increased H2 production at these pHs. Notably, glycerol failed to act as an effective electron donor for formate production. Taken together, our results suggest that regulation of formate translocation direction by FocA/FocB channels is important for maintaining internal pH and proton motive force by modulating H2 production.

The Effects of Low Doses of Gamma-Radiation on Growth and Membrane Activity of Pseudomonas aeruginosa GRP3 and Escherichia coli M17.

Microorganisms are part of the natural environments and reflect the effects of different physical factors of surrounding environment, such as gamma (γ) radiation. This work was devoted to the study of the influence of low doses of γ radiation with the intensity of 2.56 µW (m2 s)-1 (absorbed doses were 3.8 mGy for the radiation of 15 min and 7.2 mGy-for 30 min) on Escherichia coli M-17 and Pseudomonas aeruginosa GRP3 wild type cells. The changes of bacterial, growth, survival, morphology, and membrane activity had been studied after γ irradiation. Verified microbiological (specific growth rate, lag phase duration, colony-forming units (CFU) number, and light microscopy digital image analysis), biochemical (ATPase activity of bacterial membrane vesicles), and biophysical (H+ fluxes throughout cytoplasmic membrane of bacteria) methods were used for assessment of radiation implications on bacteria. It was shown that growth specific rate, lag phase duration and CFU number of these bacteria were lowered after irradiation, and average cell surface area was decreased too. Moreover ion fluxes of bacteria were changed: for P. aeruginosa they were decreased and for E. coli-increased. The N,N'-dicyclohexylcarbodiimide (DCCD) sensitive fluxes were also changed which were indicative for the membrane-associated F0F1-ATPase enzyme. ATPase activity of irradiated membrane vesicles was decreased for P. aeruginosa and stimulated for E. coli. Furthermore, DCCD sensitive ATPase activity was also changed. The results obtained suggest that these bacteria especially, P. aeruginosa are sensitive to γ radiation and might be used for developing new monitoring methods for estimating environmental changes after γ irradiation.