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.

Changes in ATPase activity, antioxidant enzymes and proline biosynthesis in yeast Candida guilliermondii NP-4 under X-irradiation.

This study investigates the effects of X-radiation on ATPase activity and antioxidant enzyme activity, particularly enzymes involved in proline biosynthesis, in yeast C. guilliermondii NP-4. Moreover, the study examined the post-irradiation repair processes in these cells. Results showed that X-irradiation at a dose of 300 Gy led to an increase in catalase (CAT) and superoxide dismutase (SOD) activity, as well as, an increase in the CAT/SOD ratio in C. guilliermondii NP-4. The repair of radiation-induced damage requires a substantial amount of energy, resulting in an increased demand for ATP in the irradiated and repaired yeasts. Consequently, the total and FoF1-ATPase activity in yeast homogenates and mitochondria increased after X-irradiation and post-irradiation repair. It was showed an increase in the activity of proline biosynthesis enzymes (ornithine transaminase and proline-5-carboxylate reductase) in X-irradiated C. guilliermondii NP-4, which remained elevated even after post-irradiation repair. As a result, the proline levels in X-irradiated and repaired yeasts were higher than those in non-irradiated cells. These findings suggest that proline may have a radioprotective effect on X-irradiated C. guilliermondii NP-4 yeasts. Taken together this study provides insights into the effects of X-radiation on ATPase activity, antioxidant enzyme activity, and proline biosynthesis in C. guilliermondii NP-4 yeast cells, highlighting the potential radioprotective properties of proline in X-irradiated yeasts.

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.

Gold nanoparticles activate hydrogenase synthesis and improve heterotrophic growth of Ralstonia eutropha H16.

Ralstonia eutropha is a facultative chemolithoautotrophic aerobic bacterium that grows using organic substrates or H2 and CO2. Hydrogenases (Hyds) are synthesized under lithoautotrophic, or energy-limited heterotrophic conditions and are used in enzyme fuel cells (EFC) as anodic catalysts. The effects of chemically synthesized gold nanoparticles (Au-NPs) on R. eutropha H16 growth, oxidation-reduction potential (ORP) kinetics, and H2-oxidizing Hyd activity were investigated in this study. Atomic force microscopy showed that thin, plate-shaped Au-NPs were in the nanoscale range with an average size of 5.68 nm. Compared with growth in medium without Au-NPs (control), the presence of Au-NPs stimulated growth, and resulted in a decrease in ORP to negative values. H2-oxidizing activity was not detected in the absence of Au-NPs, but activity was significantly induced (12 U/g CDW) after 24 h of growth with 18 ng/ml, increasing a further 4-fold after 72 h of growth. The results demonstrate that Au-NPs primarily influence the membrane-bound Hyd. In contrast to R. eutropha, Au-NPs had a negligible or negative effect on the growth, Hyd activity, and H2 production of Escherichia coli. The findings of this study offer new perspectives for the production of oxygen-tolerant Hyds and the development of EFCs.

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.

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.

Valorization of whey-based side streams for microbial biomass, molecular hydrogen, and hydrogenase production.

Side streams of the dairy industry are a suitable nutrient source for cultivating microorganisms, producing enzymes, and high-value chemical compounds. The heterotrophic Escherichia coli and chemolithoautotroph Ralstonia eutropha are of major biotechnological interest. R. eutropha is a model organism for producing O2-tolerant [NiFe]-hydrogenases (Hyds) (biocatalysts), and E. coli has found widespread use as an expression platform for producing recombinant proteins, molecular hydrogen (H2), and other valuable products. Aiming at developing suitable cultivation media from side streams of the dairy industry, the pre-treatment (filtration, dilution, and pH adjustment) of cheese (sweet) whey (SW) and curd (acid) whey (AW), with and without the use of ß-glucosidase, has been performed. Growth parameters (oxidation-reduction potential (ORP), pH changes, specific growth rate, biomass formation) of E. coli BW25113 and R. eutropha H16 type strains were monitored during cultivation on filtered and non-filtered SW and AW at 37 °C, pH 7.5 and 30 °C, pH 7.0, respectively. Along with microbial growth, measurements of pH and ORP indicated good fermentative growth. Compared to growth on fructose-nitrogen minimal salt medium (control), a maximum cell yield (OD600 4.0) and H2-oxidizing Hyd activity were achieved in the stationary growth phase for R. eutropha. Hyd-3-dependent H2 production by E. coli utilizing whey as a growth substrate was demonstrated. Moreover, good biomass production and prolonged H2 yields of ~ 5 mmol/L and cumulative H2 ~ 94 mL g/L dry whey (DW) (ß-glucosidase-treated) were observed during the cultivation of the engineered E. coli strain. These results open new avenues for effective whey treatment using thermostable ß-glucosidase and confirm whey as an economically viable commodity for biomass and biocatalyst production. KEY POINTS: • Archaeal thermostable ß-glucosidase isolated from the metagenome of a hydrothermal spring was used for lactose hydrolysis in whey. • Hydrogenase enzyme activity was induced during the growth of Ralstonia eutropha H16 on whey. • Enhanced biomass and H2 production was shown in a genetically modified strain of Escherichia coli.

L-amino acids affect the hydrogenase activity and growth of Ralstonia eutropha H16.

Ralstonia eutropha H16 is a chemolithoautotrophic bacterium with O2-tolerant hydrogenase (Hyds) enzymes. Hyds are expressed in the presence of gas mixtures (H2, O2, CO2) or under energy limitation and stress conditions. O2-tolerant Hyds are promising candidates as anode biocatalysts in enzymatic fuel cells (EFCs). Supplementation of 0.5% (w/v) yeast extract to the fructose-nitrogen (FN) growth medium enhanced H2-oxidizing Hyd activity ~ sixfold. Our study aimed to identify key metabolites (L-amino acids (L-AAs) and vitamins) in yeast extract that are necessary for the increased synthesis and activity of Hyds. A decrease in pH and a reduction in ORP (from + 240 ± 5 mV to - 180 mV ± 10 mV values) after 24 h of growth in the presence of AAs were observed. Compared to the FN-medium control, supplementation of 7.0 µmol/ml of the L-AA mixture stimulated the growth of bacteria ~ 1.9 to 2.9 fold, after 72 h. The whole cells' H2-oxidizing Hyd activity was not observed in control samples, whereas the addition of L-AAs, mainly glycine resulted in a maximum of ~ 22 ± 0.5 and 15 ± 0.3 U, g CDW-1 activity after 24 h and 72 h, respectively. Our results suggest a correlation between ORP, pH, and function of Hyds in R. eutropha H16 in the presence of key L-AAs. L-AAs used in small amounts can be proposed as signaling molecules or key components of Hyd maturation. These results are important for the optimization of O2-tolerant Hyds production as anode biocatalysts.

Osmotic stress as a factor for regulating E. coli hydrogenase activity and enhancing H2 production during mixed carbon sources fermentation.

Escherichia coli performs mixed-acid fermentation and produces molecular hydrogen (H2) via reversible hydrogenases (Hyd). H2 producing activity was investigated during hyper- and hypo-osmotic stress conditions when a mixture of carbon sources (glucose and glycerol) was fermented at different pHs. Hyper-osmotic stress decreased H2 production rate (VH2) ~30 % in wild type at pH 7.5 when glucose was supplemented, while addition of formate stimulated VH2 ~45% compared to hypo-stress conditions. Only in hyfG in formate assays was VH2 inhibited ~25% compared to hypo-stress conditions. In hypo-stress conditions addition of glycerol increased VH2 ~2 and 3 fold in hybC and hyfG mutants, respectively, compared to wild type. At pH 6.5 hyper-osmotic stress stimulated VH2 ~2 fold in all strains except hyaB mutant when glucose was supplemented, while in formate assays significant stimulation (~3 fold) was determined in hybC mutant. At pH 5.5 hyper-osmotic stress inhibited VH2 ~30% in wild type when glucose was supplemented, but in formate assays it was stimulated in all strains except hyfG. Taken together, it can be concluded that, depending on external pH and absence of Hyd enzymes in stationary-phase-grown osmotically stressed E. coli cells, H2 production can be stimulated significantly which can be applied in developing H2 production biotechnology.

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.

Ribes nigrum L. Extract-Mediated Green Synthesis and Antibacterial Action Mechanisms of Silver Nanoparticles.

Silver nanoparticles (Ag NPs) represent one of the most widely employed metal-based engineered nanomaterials with a broad range of applications in different areas of science. Plant extracts (PEs) serve as green reducing and coating agents and can be exploited for the generation of Ag NPs. In this study, the phytochemical composition of ethanolic extract of black currant (Ribes nigrum) leaves was determined. The main components of extract include quercetin rutinoside, quercetin hexoside, quercetin glucuronide, quercetin malonylglucoside and quercitrin. The extract was subsequently employed for the green synthesis of Ag NPs. Consequently, R. nigrum leaf extract and Ag NPs were evaluated for potential antibacterial activities against Gram-negative bacteria (Escherichia coli ATCC 25922 and kanamycin-resistant E. coli pARG-25 strains). Intriguingly, the plant extract did not show any antibacterial effect, whilst Ag NPs demonstrated significant activity against tested bacteria. Biogenic Ag NPs affect the ATPase activity and energy-dependent H+-fluxes in both strains of E. coli, even in the presence of N,N'-dicyclohexylcarbodiimide (DCCD). Thus, the antibacterial activity of the investigated Ag NPs can be explained by their impact on the membrane-associated properties of bacteria.

Structural characterization and antibacterial activity of silver nanoparticles synthesized using a low-molecular-weight Royal Jelly extract.

In recent years silver nanoparticles (Ag NPs) gained increased and widespread applications in various fields of industry, technology, and medicine. This study describes the green synthesis of silver nanoparticles (Ag NPs) applying a low-molecular-weight fraction (LMF) of Royal Jelly, the nanoparticle characterization, and particularly their antibacterial activity. The optical properties of NPs, characterized by UV-Vis absorption spectroscopy, showed a peak at ~ 430 nm. The hydrodynamic radius and concentration were determined by complementary dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). The particle morphology was investigated using transmission electron microscopy (TEM), and the crystallinity of the silver was confirmed by X-ray diffraction (XRD). The antibacterial activities were evaluated utilizing Gram-negative and Gram-positive bacteria and colony counting assays. The growth inhibition curve method was applied to obtain information about the corresponding minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC) required. Obtained results showed that (i) the sizes of Ag NPs are increasing within the increase of silver ion precursor concentration, (ii) DLS, in agreement with NTA, showed that most particles have dimensions in the range of 50-100 nm; (iii) E. coli was more susceptible to all Ag NP samples compared to B. subtilis.

Biosynthesis of silver nanoparticles using extracts of Stevia rebaudiana and evaluation of antibacterial activity.

The present study reveals a simple, non-toxic and eco-friendly method for the "green" synthesis of Ag-NPs using hydroponic and soil medicinal plant Stevia rebaudiana extracts, the characterization of biosynthesized nanoparticles, as well as the evaluation of their antibacterial activity. Transmission electronic microscopy (TEM) and Dynamic Light Scattering (DLS) analysis confirmed that biosynthesized Ag-NPs are in the nano-size range (50-100 nm) and have irregular morphology. Biogenic NPs demonstrate antibacterial activity against Escherichia coli BW 25,113, Enterococcus hirae ATCC 9790, and Staphylococcus aureus MDC 5233. The results showed a more pronounced antibacterial effect on E. coli growth rate, in comparison with Gram-positive bacteria, which is linked to the differences in the structure of bacterial cell wall. Moreover, the Ag-NPs not only suppressed the growth of bacteria but also changed the energy-dependent H+-fluxes across the bacterial membrane. The change of H+-fluxes in presence of H+-translocating systems inhibitor, N,N'-dicyclohexylcarbodiimide (DCCD), proves the effect of Ag-NPs on the structure and permeability of the bacterial membrane. Overall, our findings indicate that the Ag-NPs synthesized by medicinal plant Stevia extracts may be an excellent candidate as an alternative to antibiotics against the tested bacteria.

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.

Antibacterial activity of royal jelly-mediated green synthesized silver nanoparticles.

The application of green synthesis in nanotechnology is growing day by day. It's a safe and eco-friendly alternative to conventional methods. The current research aimed to study raw royal jelly's potential in the green synthesis of silver nanoparticles and their antibacterial activity. Royal jelly served as a reducing and oxidizing agent in the green synthesis technology of colloidal silver nanoparticles. The UV-Vis maximum absorption at ~ 430 nm and fluorescence emission peaks at ~ 487 nm confirmed the presence of Ag NPs. Morphology and structural properties of Ag NPs and the effect of ultrasound studies revealed: (i) the formation of polydispersed and spherical particles with different sizes; (ii) size reduction and homogeneity increase by ultrasound treatment. Antibacterial activity of different concentrations of green synthesized Ag NPs has been assessed on Gram-negative S. typhimurium and Gram-positive S. aureus, revealing higher sensitivity on Gram-negative bacteria.

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.

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.

Influence of C4 -Dcu transporters on hydrogenase and formate dehydrogenase activities in stationary phase-grown fermenting Escherichia coli.

During mixed-acid fermentation, Escherichia coli transports succinate mainly via transporters of the Dcu family. Here, we analyze the influence of Dcu transporters on hydrogenase (Hyd) and fermentative formate dehydrogenase (FDH-H) activities and how this is affected by external pH and carbon source. Using selected dcu mutations, it was shown that Dcu carriers mainly affect Hyd and FDH-H activities during glycerol but not glucose fermentation at acidic pH. During glycerol fermentation at pH 5.5, inactivation of either one or all Dcu carriers increased total Hyd activity by 60% compared with wild type. Under the same growth conditions, a dcuACBD mutant had a twofold higher FDH-H activity. When glucose was fermented in dcuD single mutant at pH 5.5, the FDH-H activity was also increased twofold compared with wild type. Interestingly, in dcuD or dcuACBD mutants at pH 7.5, Hyd activity was lowered by 20%. Taken together, it can be concluded that during glucose fermentation at pH 7.5, lack of DcuD affects Hyd enzyme activity, but at pH 5.5, it has a stronger effect on FDH-H activity. During glycerol fermentation, lack of Dcu carriers increased Hyd and FDH-H activities as revealed at pH 5.5. The results suggest that impairing Dcu transport function increases intracellular formate levels and thus affects H2 cycling and 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.

Fermentation Revisited: How Do Microorganisms Survive Under Energy-Limited Conditions?

During fermentation FOF1 hydrolyzes ATP, coupling proton transport to proton-motive force (pmf) generation. Despite that, pmf generated by ATP hydrolysis does not satisfy the energy budget of a fermenting cell. However, pmf can also be generated by extrusion of weak organic acids such as lactate and by hydrogen cycling catalyzed by hydrogenases (Hyds). Here we highlight recent advances in our understanding of how the transport of weak organic acids and enzymes contributes to pmf generation during fermentation. The potential impact of these processes on metabolism and energy conservation during microbial fermentation have been overlooked and they not only expand on Mitchell's chemiosmotic theory but also are of relevance to the fields of microbial biochemistry and human and animal health.