Proton conductance and regulation of proton/potassium fluxes in Escherichia coli FhlA-lacking cells during fermentation of mixed carbon sources.

Escherichia coli uptake potassium ions with the coupling of proton efflux and energy utilization via proton FOF1-ATPase. In this study contribution of formate hydrogen lyase (FHL) complexes in the proton/potassium fluxes and the formation of proton conductance (CMH+) were investigated using fhlA mutant strain. The proton flux rate (JH+) decreased in fhlA by âˆ¼ 25 % and ∼70 % during the utilization of glucose and glycerol, respectively, at 20 h suggesting H+ transport via or through FHL complexes. The decrease in JK+ in fhlA by ∼40 % proposed the interaction between FHL and Trk secondary transport system during mixed carbon fermentation. Moreover, the usage of N,N'-dicyclohexylcarbodiimide (DCCD) demonstrated the mediation of FOF1-ATPase in this interaction. CMH+ was 13.4 nmol min-1 mV-1 in WT at 20 h, which decreased by 20 % in fhlA. Taken together, FHL complexes have a significant contribution to the modulation of H+/K+ fluxes and the CMH + for efficient energy transduction and regulation of the proton motive force during mixed carbon sources fermentation.

Regulation of metabolism and proton motive force generation during mixed carbon fermentation by an Escherichia coli strain lacking the FOF1-ATPase.

Proton FOF1-ATPase is the key enzyme in E. coli under fermentative conditions. In this study the role of E. coli proton ATPase in the µ and formation of metabolic pathways during the fermentation of mixture of glucose, glycerol and formate using the DK8 (lacking FOF1) mutant strain was investigated. It was shown that the contribution of FOF1-ATPase in the specific growth rate was ∼45 %. Formate was not taken up in the DK8 strain during the initial hours of the growth. The utilization rates of glucose and glycerol were unchanged in DK8, however, the production of succinate, lactate and ethanol was decreased causing a reduction of the redox state up to -450 mV. Moreover, the contribution of FOF1-ATPase in the interplay between H+ and H2 cycles was described depending on the bacterial growth phase and main utilizing substrate. Besides, the H2 production rate in the DK8 strain was decreased by ∼60 % at 20 h and was absent at 72 h. Δp was decreased from -157 ± 4.8 mV to -140 ± 4.2 mV at 20 h and from -195 ± 5.9 mV to -148 ± 4.4 mV at 72 h, compared to WT. Taken together it can be concluded that during fermentation of mixed carbon sources metabolic cross talk between FOF1-ATPase-TrkA-Hyd-Fdh-H is taking place for maintaining the cell energy balance via regulation proton motive force.

Propionic and valproic acids have an impact on bacteria viability, proton flux and ATPase activity.

Short-chain fatty acids like propionic (PPA) and valproic acids (VP) can alter gut microbiota, which is suggested to play a role in development of autism spectrum disorders (ASD). In this study we investigated the role of various concentrations of PPA and VP in gut enteric gram-negative Escherichia coli K12 and gram-positive Enterococcus hirae ATCC 9790 bacteria growth properties, ATPase activity and proton flux. The specific growth rate (µ) was 0.24 h-1 and 0.82 h-1 in E. coli and E. hirae, respectively. Different concentrations of PPA reduced the value of µ similarly in both strains. PPA affects membrane permeability only in E. hirae. PPA decreased DCCD-sensitive ATPase activity in the presence of K+ ions by 20% in E. coli and 40% in E. hirae suggesting the importance of the FOF1-K+ transport system in the regulation of PPA-disrupted homeostasis. Moreover, the H+ flux during PPA consumption could be the protective mechanism for enteric bacteria. VP has a selective effect on the µ depending on bacteria. The overwhelming effect of VP was detected on the K+-promoted ATPase activity in E. hirae. Taken together it can be suggested that PPA and VP have a disruptive effect on E. coli and E. hirae growth, viability, bioenergetic and biochemical properties, which are connected with the alteration of FOF1-ATPase activity and H+ flux rate or direction.

Metabolic pathways and ΔpH regulation in Escherichia coli during the fermentation of glucose and glycerol in the presence of formate at pH 6.5: the role of FhlA transcriptional activator.

Escherichia coli is able to ferment mixed carbon sources and produce various fermentation end-products. In this study, the function of FhlA protein in the specific growth rate (µ), metabolism, regulation of ΔpH and proton ATPase activity was investigated. Reduced µ in fhlA mutant of ∼25% was shown, suggesting the role of FhlA in the growth process. The utilization rate of glycerol is decreased in fhlA ∼ 2 fold, depending on the oxidation-reduction potential values. Bacteria regulate the activity of hydrogenase enzymes during growth depending on the external pH, which manifests as a lack of hydrogen gas generation during glycerol utilization at pH values below 5.9. It is suggested that cells maintain ΔpH during the fermentative growth via formate-lactate-succinate exchange. The decrement of the value of pHin, but not of pHex in mutant cells, is regulating ΔpH and consequently proton motive force generation.

The role of Escherichia coli FhlA transcriptional activator in generation of proton motive force and FO F1 -ATPase activity at pH 7.5.

Escherichia coli is able to utilize the mixture of carbon sources and produce molecular hydrogen (H2 ) via formate hydrogen lyase (FHL) complexes. In current work role of transcriptional activator of formate regulon FhlA in generation of fermentation end products and proton motive force, N'N'-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase activity at 20 and 72 hr growth during utilization of mixture of glucose, glycerol, and formate were investigated. It was shown that in fhlA mutant specific growth rate was ~1.5 fold lower compared to wt, while addition of DCCD abolished the growth in fhlA but not in wt. Formate was not utilized in fhlA mutant but wt cells simultaneously utilized formate with glucose. Glycerol utilization started earlier (from 2 hr) in fhlA than in wt. The DCCD-sensitive ATPase activity in wt cells membrane vesicles increased ~2 fold at 72 hr and was decreased 70% in fhlA. Addition of formate in the assays increased proton ATPase activity in wt and mutant strain. FhlA absence mainly affected the ΔpH but not ΔΨ component of Δp in the cells grown at 72 hr but not in 24 hr. The Δp in wt cells decreased from 24 to 72 hr of growth ~40 mV while in fhlA mutant it was stable. Taken together, it is suggested that FhlA regulates the concentration of fermentation end products and via influencing FO F1 -ATPase activity contributes to the proton motive force generation.

Formate and potassium ions affect Escherichia coli proton ATPase activity at low pH during mixed carbon fermentation.

Escherichia coli is able to ferment not only single but also mixtures of carbon sources. The formate metabolism and effect of formate on various enzymes have been extensively studied during sole glucose but not mixed carbon sources utilization. It was revealed that in membrane vesicles (MV) of wild type cells grown at pH 7.5 during fermentation of the mixture of glucose (2 g/L), glycerol (10 g/L), and formate (0.68 g/L), in the assays, the addition of formate (10 mM) increased the N,N'-dicyclohexylcarbodiimide (DCCD)-inhibited ATPase activity on ~30% but no effect of potassium ions (100 mM) had been detected. In selC (coding formate dehydrogenases) and fdhF (coding formate dehydrogenase H) single mutants, formate increased DCCD-inhibited ATPase activity on ~40 and ~70%, respectively. At pH 5.5, in wild type cells MV, formate decreased the DCCD-inhibited ATPase activity ~60% but unexpectedly in the presence of potassium ions, it was stimulated ~5.8 fold. The accessible SH or thiol groups number in fdhF mutant was less by 28% compared with wild type. In formate assays, the available SH groups number was less ~10% in wild type but not in fdhF mutant. Taken together, the data suggest that proton ATPase activity depends on externally added formate in the presence of potassium ions at low pH. This effect might be regulated by the changes in the number of redox-active thiol groups via formate dehydrogenase H, which might be directly related to proton ATPase FO subunit.

Understanding the Role of Escherichia coli Hydrogenases and Formate Dehydrogenases in the FO F1 -ATPase Activity during the Mixed Acid Fermentation of Mixture of Carbon Sources.

During fermentation Escherichia coli produces di-hydrogen (H2 ) via reversible membrane-bound [Ni-Fe]-hydrogenases (Hyd). This study describes the total and N,N'-dicyclohexylcarbodiimide (DCCD) inhibited ATPase activity and H2 production at various pHs in E. coli wild type and mutants encoding Hyd enzymes and formate dehydrogenases (FDH) on fermentation of glucose, glycerol, and formate. The highest total ATPase activity was detected at pH 7.5 in hyaB hybC selC (lacking large subunits of Hyd-1 and Hyd-2 and FDH, respectively) triple mutant. This ATPase activity was mainly due to the proton-translocating ATPase but in FDH mutant the DCCD inhibition was less compared to wild type. Potassium ions stimulated total ATPase activity at pH 5.5 ~50% and ~35% in wild type and hypF (lacking all Hyd enzymes) mutant, respectively. Moreover, K+ also stimulated DCCD inhibited ATPase activity ~1.7-fold-2-fold in strains where FDH was absent only at pH 5.5. DCCD inhibited H2 production only at pH 5.5 in all assays. Taken together it is suggested that at low pH, FDH, and Hyd enzymes are linked with the FO F1 -ATPase for regulating and maintaining the cytoplasmatic pH and thus proton motive force generation. FDH and Hyd enzymes have impact on the FO F1 -ATPase activity depending on external pH and potassium ions. © 2018 IUBMB Life, 70(10):1040-1047, 2018.