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.

Ultrastructural investigation of acidocalcisomes and ATPase activity in yeast Candida guilliermondii NP-4 as 'complementary' stress-targets.

As a result of electron microscopic studies of morphogenesis in yeast Candida guilliermondii NP-4, the formation of new structures of volutin acidocalcisomes has been established within the cell cytoplasm. Under influence of X-irradiation, the changes in morphometric and electron-dense properties of yeast cells were identified: in yeast cytoplasm, the electron-dense volutin granules were increased up to 400 nm in size. After 24-h post-irradiation incubation of yeasts, the large volutin pellets are fragmented into smaller number particles in size up to 25-150 nm. The ATPase activity in yeast mitochondria was changed under X-irradiation. In latent phase of growth, ATPase activity was decreased 1·35-fold in comparison with non-irradiated yeasts. In logarithmic phase of growth, ATPase activity was three times higher than in latent phase, and in stationary phase of growth it has a value similar to the latent phase. Probably, the cells receive the necessary energy from alternative energy sources, such as volutin. Electron microscopy of volutin granule changes might serve as convenient method for evaluation of damages and repair processes in cells under influence of different environmental stress-factors.

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.

Novel approach to combat antibiotic resistance: evaluation of some Armenian herb crude extracts for their antibiotic modulatory and antiviral properties.

AIMS: One of the strategies to combat antibiotic resistance can be the use of plant materials in combination with antibiotics, taking into account that phytochemicals can act as antibiotic resistance-modifying agents. This can give a second life to the traditional antibiotics. The aim was to evaluate antibiotic modulatory effect of crude extracts from Agrimonia eupatoria, Hypericum alpestre, Rumex obtusifolius and Sanguisorba officinalis herbs towards several commercial antibiotics using some Gram-positive and Gram-negative bacteria. METHODS: The antibiotic modulatory activity was tested by determining MICs of antibiotics in the absence and presence of plant crude extracts at subinhibitory concentrations. Antiviral potential of different extracts of tested plant materials was also explored by double overlay plaque assay. RESULTS: The tested plant crude extracts exhibited high modulatory activity towards used antibiotics. Particularly, high modulatory activity was observed with extracts of H. alpestre and R. obtusifolius. Many plant-antibiotic combinations induced the decrease in MICs of antibiotics up to ~fourfold indicating synergy. Moreover, the similar change was observed at both subinhibitory concentrations (MIC/2 and MIC/4) of the same plant crude extract. High anti-phage activity of plants with the exception of Lilium armenum against T4 phage of Escherichia coli C-T4 was also shown. CONCLUSIONS: Plant crude extract or commercial antibiotic combinations significantly increased the efficiency of antibiotics. Tested plant materials with exception of L. armenum have antiviral property. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, antibiotic modulatory activity of tested herb extracts was shown, which could have potential in practical applications. Tested plant materials with exception of L. armenum could have prospective, as a source of new antiviral compounds.

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.

Effects of iron oxide (Fe3 O4 ) nanoparticles on Escherichia coli antibiotic-resistant strains.

AIMS: Antibiotic resistance of different bacteria requires the development of alternative approaches for overcoming this phenomenon. The antibacterial effects of iron oxide (Fe3 O4 ) nanoparticles (NPs) (from 50 to 250 µg ml-1 ) on Escherichia coli antibiotic-resistant strains have been aimed. METHODS AND RESULTS: The study was performed with ampicillin-resistant E. coli DH5α-pUC18 and kanamycin-resistant E. coli pARG-25 stains. Specific growth rate of bacteria (µ), lag phase duration and colony-forming units (CFU) were determined to evaluate growth properties. Fe3 O4 NPs (average size of 10·64 ± 4·73 nm) coated with oleic acid and synthesized by modified co-precipitation method were used. The medium pH, H+ efflux, membrane H+ conductance, redox potential determinations and H2 yield assay were done using potentiometer methods. Growth properties were changed by NPs in concentration-dependent manner. NPs decreased (up to twofold) H+ -fluxes through bacterial membrane more in E. coli in the presence of the N,N'-dicyclohexylcarbodiimide, inhibitor of ATPase, indicating that antibacterial activity of these NPs was connected with ATP-associated metabolism. Membrane-associated H2 production was lowered up to twofold. Moreover, the synergetic interactions of NPs and antibiotics were found: combination of NPs and antibiotics provided the higher H+ conductance, lower H+ -fluxes and H2 yield. CONCLUSIONS: Fe3 O4 NPs can be suggested as alternative antibacterial agents, which can substitute antibiotics in different applications. SIGNIFICANCE AND IMPACT OF THE STUDY: The antibacterial effects of Fe3 O4 NPs on the growth properties and membrane activity of E. coli antibiotic-resistant strains have been demonstrated. These NPs have potential as antibacterial agents, which can substitute for antibiotics in bacterial disease treatment in biomedicine, pharmaceutical and environmental applications.

The survival of irradiated lactobacilli in the simulated gastrointestinal conditions with antibiotic ceftazidime.

Lactobacillus acidophilus is one of the widespread probiotic bacteria that can overcome acid and bile barrier of stomach and intestine, respectively, and then have beneficial effects on the host improving its intestinal microbial balance. The cell membrane FO F1 -ATPase is an important factor in the response and tolerance to low pH through the action of controlling the H+ concentration between the cell cytoplasm and external medium. In this study, the effects of extremely high-frequency EMI at the frequencies of 51·8 GHz and 53 GHz and cetfazidime ( µmol l-1 ) on survival of L. acidophilus VKM B-1660 in the gastrointestinal model in vitro and on ATPase activity of their membrane vesicles were investigated. Irradiated L. acidophilus survived in media with acid pH; the irradiation stimulated N,N'-dicyclohexylcarbodiimide-sensitive FO F1 -ATPase activity under acidic conditions, but enhanced the inhibitory effects of ceftazidime. Probably irradiated L. acidophilus is overcoming the acid barrier even in the presence of ceftazidime due to the FO F1 -ATPase. The obtained results can allow the use of L. acidophilus in food industry, veterinary and medicine. SIGNIFICANCE AND IMPACT OF THE STUDY: The probiotic property of lactobacilli is defined with survival in different conditions of human digestive tract even in the presence of antibiotics and subjected to electromagnetic irradiation (EMI) at the extremely high frequency. Despite the fact that EMI and antibiotic ceftazidime affected Lactobacillus acidophilus; the viable number of bacterial cells was decreased in in vitro gastrointestinal model, but they could to grow in fresh growth medium. The changes in the FO F1 -ATPase activity were obtained at acidic pH. Thus, these bacteria can overcome acid barrier due to the FO F1 -ATPase: the irradiation stimulates the FO F1 -ATPase activity in the acidic conditions, but enhances the effects of ceftazidime. The results are important for identifying the mechanisms of lactobacilli survival for physical and chemical factors and valuable for use.

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.

Aggregation and Adhesion Activity of Lactobacilli Isolated from Fermented Products In Vitro and In Vivo: a Potential Probiotic Strain.

Approximately 25 strains of lactobacilli isolated from different dairy products and fermented vegetables were screened according to their possibility to show the high auto-aggregation and co-aggregation. The strains Lactobacillus helveticus INRA-2010-H11, Lactobacillus rhamnosus INA-5.1, and Lactobacillus acidophilus JM-2012 were determined to have the high auto-aggregation (approximately 73, 46, and 70.5% correspondingly). A high co-aggregation capacity (75.53%) for strains INRA-2010-H11 and JM-2012 was shown. The adhesion degree of INRA-2010-H11 on the surface of buccal epithelial cells was 88.23%. The study of INRA-2010-H11, JM-2012, and both strains' mixture (1:1) adhesion capacity on the surface of epithelial HeLa cells revealed the adhesion of 1.1 × 106, 6.3 × 104, and 2.3 × 105 CFU, respectively, from starter amount of CFU 107 and 108 for both strains. In vivo experiments of LAB adhesion in gastrointestinal tract of mouse revealed the presence of 2.5 × 109, 1.2 × 109, and 1.5 × 109 CFU of LAB in control and groups of mouse, fed by INRA-2010-H11 and mixture, respectively. Feeding by investigated lactobacilli was suggested to lead to microbiota biodiversity reduction in small intestine and colon and its augmentation in stomach. Thus, INRA-2010-H11 demonstrated a high aggregation and adhesion activity so it has the potential as a good probiotic strain.

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.

The distinguishing effects of low-intensity electromagnetic radiation of different extremely high frequencies on Enterococcus hirae: growth rate inhibition and scanning electron microscopy analysis.

A low-intensity electromagnetic field of extremely high frequency has inhibitory and stimulatory effects on bacteria, including Enterococcus hirae. It was shown that the low-intensity (the incident power density of 0·06 mW cm-2 ) electromagnetic field at the frequencies of 51·8 GHz and 53 GHz inhibited E. hirae ATCC 9790 bacterial growth rate; a stronger effect was observed with 53 GHz, regardless of exposure duration (0·5 h, 1 h or 2 h). Scanning electron microscopy analysis of these effects has been done; the cells were of spherical shape. Electromagnetic field at 53 GHz, but not 51·8 GHz, changed the cell size-the diameter was enlarged 1·3 fold at 53 GHz. These results suggest the difference in mechanisms of action on bacteria for electromagnetic fields at 51·8 GHz and 53 GHz. SIGNIFICANCE AND IMPACT OF THE STUDY: A stronger inhibitory effect of low-intensity electromagnetic field on Enterococcus hirae ATCC 9790 bacterial growth rate was observed with 53 GHz vs 51·8 GHz, regardless of exposure duration. Scanning electron microscopy analysis showed that almost all irradiated cells in the population have spherical shapes similar to nonirradiated ones, but they have increased diameters in case of irradiated cells at 53 GHz, but not 51·8 GHz. The results are novel, showing distinguishing effects of low-intensity electromagnetic field of different frequencies. They could be applied in treatment of food and different products in medicine and veterinary, where E. hirae plays an important role.

Comparative Analysis of UV Irradiation Effects on Escherichia coli and Pseudomonas aeruginosa Bacterial Cells Utilizing Biological and Computational Approaches.

Microorganisms have a large number of tools to withstand different, and sometimes strong, environmental stresses, including irradiation, but this ability should be further evaluated for certain applications. Growth inhibition and morphological alterations of Escherichia coli M-17 and Pseudomonas aeruginosa GRP3 wild-type cells caused by UV-A irradiation have been detected in the present study. Comparative analysis was carried out using well-established microbiological methods (determination of specific growth rate, growth lag phase duration, and colony-forming unit number-CFU) and computational approaches, employing light microscopy and digital image analysis to evaluate bacterial cell morphology. Decreases in the specific growth rate, prolonged lag-phases, and lowered CFUs were observed after 5 and 10 min of UV irradiation (approx. 40 Gy) compared to the control (nonirradiated) cells. Accordingly, two computational parameters-the average bacterial cell surface area and the bacterial cell perimeter (i.e., of the 2D projection of bacterial cells in microscopy image)-were reduced. The ratio of bacterial cell surface area (S) to the square of the perimeter (p (2) ) was reduced after 5 min of irradiation, but after 10 min of irradiation the studied bacterial cells became flat cylinders. The revealed findings are concluded to be highly useful in developing new, rapid analysis methods to monitor environmental and UV irradiation effects on bacteria and to detect bacterial cell morphology alterations.

Adhesive Properties and Acid-Forming Activity of Lactobacilli and Streptococci Under Inhibitory Substances, Such as Nitrates.

One of the main requirements for probiotics is their ability to survive during passage through gastrointestinal tract and to maintain their activity at different adverse conditions. The aim of the study was to look for the strains of lactobacilli and streptococci with high adhesive properties even affected by inhibitory substances, such as nitrates (NO3 (-)). To study the adhesion properties hemagglutination reaction of bacterial cells with red blood cells of different animals and humans was used. The acid formation ability of bacteria was determined by the method of titration after 7 days of incubation in the sterile milk. These properties were investigated at different concentrations of NO3 (-). The high concentration (mostly ≥2.0 %) NO3 (-) inhibited the growth of both lactobacilli and streptococci, but compared with streptococcal cultures lactobacilli, especially Lactobacillus acidophilus Ep 317/402, have shown more stability and higher adhesive properties. In addition, the concentrations of NO3 (-) of 0.5-2.0 % decreased the acid-forming activity of the strains, but even under these conditions they coagulated milk and, in comparison to control, formed low acidity in milk. Thus, the L. acidophilus Ep 317/402 with high adhesive properties has demonstrated a higher activity of NO3 (-) transformation.

Protective effect of some amino acids synthesized derivatives and their chelates on Escherichia coli under X-ray irradiation.

The protective effects of novel synthesized derivatives of some amino acids--nicotinyl-L-tyrosinate and nicotinyl-L-tryptophanate schiff bases and their Cu(II) and Mn(II) chelates on growth, survival and membrane-associated ATPase activity of E. coli under X-ray irradiation were investigated. The specific growth rate and survival of E. coli were decreased at 10, 20 and 30 Gy doses. However, as 30 Gy was found to be the most effective irradiation dose, it was chosen for studying the radio-protective properties of different compounds. These compounds could increase the bacterial cell protection against X-ray irradiation in concentration-dependent manner. They had a role in stimulation of synthesis or regulation of activity of metal-dependent enzymes, required for reversing the X-ray irradiation damage. The study may prove useful for further estimation of the effectiveness of different compounds as radio-protectors on bacteria and other cells, especially mammalian cells under X-ray irradiation.

Structural and functional features of formate hydrogen lyase, an enzyme of mixed-acid fermentation from Escherichia coli.

Formate hydrogen lyase from Escherichia coli is a membrane-bound complex that oxidizes formic acid to carbon dioxide and molecular hydrogen. Under anaerobic growth conditions and fermentation of sugars (glucose), it exists in two forms. One form is constituted by formate dehydrogenase H and hydrogenase 3, and the other one is the same formate dehydrogenase and hydrogenase 4; the presence of small protein subunits, carriers of electrons, is also probable. Other proteins may also be involved in formation of the enzyme complex, which requires the presence of metal (nickel-cobalt). Its formation also depends on the external pH and the presence of formate. Activity of both forms requires F(0)F(1)-ATPase; this explains dependence of the complex functioning on proton-motive force. It is also possible that the formate hydrogen lyase complex will exhibit its own proton-translocating function.

Effect of diethylsulphoxide on growth, survival and ion exchange of Escherichia coli.

AIMS: To study the effect of diethylsulphoxide (DESO) on Escherichia coli growth, survival and ionic exchange in comparison with dimethylsulphoxide (DMSO). METHODS AND RESULTS: Bacterial survival was estimated by counting colony-forming units and by the most probable number (five-tube) technique; the K+ and H+ transport and H(2) formation were determined electrochemically. Diethylsulphoxide at concentrations between 0.01 and 0.5% (w/v) stimulated and above 5% decreased the anaerobic growth rate and survival. 2H+ : K+ exchange and H(2) formation were lost at 5% DESO. At 0.05% DESO the kinetic characteristics of H+ : K+ exchange and H(2) formation were typical for Delta micro (H(+)) -dependent TrkA uncoupled with F(0)F(1) under respiration. CONCLUSIONS: Diethylsulphoxide at low concentrations serves as an electron acceptor for an anaerobic respiratory chain stimulating bacterial growth and survival through the modulation of H+ : K+ exchange and H(2) formation activity. The effects of DESO were more pronounced than those of DMSO. SIGNIFICANCE AND IMPACT OF THE STUDY: Diethylsulphoxide determines essential biological and therapeutic properties that make its application preferable.

K+ influx by Kup in Escherichia coli is accompanied by a decrease in H+ efflux.

Escherichia coli accumulates K+ by means of multiple uptake systems of which Kup is the major transport system at acidic pH. In cells grown under fermentative conditions at pH 5.5, K+ influx by a wild-type strain upon hyper-osmotic stress at pH 5.5 was accompanied by a marked decrease in H+ efflux, with a 1:1 ratio of K+ to H+ fluxes. This was observed with cells treated with N,N'-dicyclohexylcarbodiimide. Similar results with a mutant defective in Kdp and TrkA but with a functional Kup system but not in a mutant defective in Kdp and Kup but having an active TrkA system suggest that Kup operates as a H+ -K+ -symporter.

Participation of hyf-encoded hydrogenase 4 in molecular hydrogen release coupled with proton-potassium exchange in Escherichia coli.

In a previous work (Trchounian et al., Biol. Membrany 16:416-428 (1999) (in Russian)) we reported the interrelations between production of H2 and H+-K+ exchange in fermenting Escherichia coli grown under anaerobic conditions at pH 7.5. The ion fluxes had stable stoichiometry 2H+/K+ and were N,N'-dicyclohexylcarbodiimide (DCC)-inhibitable at different external pH and K+ activity. In the present study, the H2 production was further studied in fermenting bacteria grown at pH 7.5 or 6.5. The H2 production was inhibited by DCC and did not occur if bacteria were grown at pH 7.5 in a medium containing formate or upon hypoosmotic stress. The H2 production was not sensitive to osmotic stress when bacteria were grown at pH 6.5. Formation of H2 and 2H+/K+ exchange were not observed in mutants with deletions of the hyfoperon genes, encoding membrane-associated hydrogenase 4. K+ influx in these mutants was not sensitive to valinomycin, in contrast to the K+ influx in the parental strain. If grown at pH 6.5, the mutants produced H2 and carried out 2H+/K+ exchange, when subjected to the hyperosmotic stress. The results suggest a participation of hydrogenase 4 in the production of H2 and proton-potassium exchange in fermenting E. coli grown at pH 7.5. In bacteria grown at pH 6.5 or in a medium containing formate, another membrane-bound hydrogenase, namely hydrogenase 3, may be responsible for the H2 production.

K+ uptake by fermenting Escherichia coli cells: pH dependent mode of the TrkA system operating.

Escherichia coli accumulates K+ by means of multiple transport systems, of which TrkA is the most prominent at neutral and alkaline pH while Kup is major at acidic pH. In the present study, K+ uptake was observed with cells grown under fermentative conditions at an initial pH of 9.0 and 7.3 (the medium pH decreased to 8.4 and 6.8, respectively, during the mid-logarithmic growth phase), washed with distilled water and resuspended in a K+ containing medium at pH 7.5 in the presence of glucose. The kinetics for this K+ uptake and the amount of K+ accumulated by the wild type and mutants having a functional TrkA or Kup could confirm that K+ uptake by E. coli grown either at pH 9.0 or pH 7.3 occurs mainly through TrkA. The following results distinguish pH dependent mode of TrkA operating: (1) K+ uptake was inhibited by DCCD in cells grown either at pH 9.0 or pH 7.3, although the stoichiometry of K+ influx to DCCD-inhibited H+ efflux for bacteria grown at pH 9.0 varied with external K+ concentration, but remained constant for cells grown at pH 7.3; (2) K+ uptake was observed with an atpD mutant grown at pH 9.0 but not at pH 7.3; (3) The DCCD-inhibited H+ efflux was increased 8-fold less by 5 mM K+ added into a K+ free medium for bacteria grown at pH 9.0 than that for cells grown at pH 7.3; (4) the DCCD-inhibited ATPase activity of membrane vesicles from bacteria grown at pH 9.0 was reduced a little in the presence of 100 mM K+, but stimulated more than 2.4-fold at pH 7.3.

Relationship between formate hydrogen lyase and proton-potassium pump under heterolactic fermentation in Escherichia coli: functional multienzyme associations in the cell membrane.

Anaerobically grown glucose-fermenting E. coli cells produce molecular hydrogen, acidify the medium and uptake potassium ions. It was shown that the H2 release and the proton-potassium exchange with the fixed (2H+/K+) stoichiometry of the initial DCC-sensitive fluxes were lost in mutants with the deleted fdhF gene or the hycA-H operon responsible for the biosynthesis of formate dehydrogenase H (FDH,H) or hydrogenase 3 (H3), respectively, which are the main components of the formate hydrogen lyase FHL(H). However, both processes occurred in mutants with the deleted hycE, hycF or hycG genes encoding the major and minor components of H3, respectively. The K+ uptake was sensitive to the osmotic shock resulting from glucose addition to the medium and decreased significantly in the presence of valinomycin. The H2 release and the 2H+/K+ exchange were absent in the mutant with the deleted hycB gene encoding the corresponding minor component of H3. This mutant acidified the medium and uptook K+ with Km typical for TrkA, but the stoichiometry of the DCC-inhibited fluxes was variable, and the K+ gradient between the cytoplasm and the medium in this mutant was lower than in the mutants lacking other minor components of H3. The results obtained suggest that the hycB gene product, FdhF and HycE, form probably the FHL(H) complex that directly interacts with the H+-ATPase complex F0F1 and the TrkA(H) system of K+ uptake. Such a multienzyme association is responsible for the H2 production and 2H+/K+ exchange. The major and other minor components of H3 have probably no direct role in the H2 production and 2H+/K+ exchange. H2 production by precursor's or hycE mutant's protoplasts treated with toluene was shown to occur upon addition of the thiol reagent dithiothreitol to the medium containing ATP, potassium ions, NAD+, and NADH. H2 production was inhibited by DCC. The quantity of available thiol groups in membrane vesicles of the precursor or the hycE, hycF or hycG mutants, in which the H2 production and 2H+/K+ exchange were observed, was larger than in other mutants. The number of SH groups decreased in the presence of DCC. These results indicate a significance of the thiol groups for the function of the proposed association.