Functional asymmetry of the F0 motor in bacterial ATP synthases

F₁F₀ ATP synthases use the electrochemical potential of H⁺ or Na⁺ across biological membranes to synthesize ATP by a rotary mechanism. In bacteria, the enzymes can act in reverse as ATP-driven ion pumps creating the indispensable membrane potential. Here, we demonstrate that the F₀ parts of a Na⁺- and H⁺-dependent enzyme display major asymmetries with respect to their mode of operation, reflected by the requirement of ∼100 times higher Na⁺ or H⁺ concentrations for the synthesis compared with the hydrolysis of ATP. A similar asymmetry is observed during ion transport through isolated F₀ parts, indicating different affinities for the binding sites in the a/c interface. Together with further data, we propose a model that provides a rationale for a differential usage of membrane potential and ion gradient during ATP synthesis as observed experimentally. The functional asymmetry might also reflect an important property of the ATP synthesis mechanism in vivo . In Escherichia coli , we observed respiratory chain-driven ATP production at pH 7–8, while P -site pH values < 6.5 were required for ATP synthesis in vitro . This discrepancy is discussed with respect to the hypothesis that during respiration lateral proton diffusion could lead to significant acidification at the membrane surface.     

Cell envelope changes in Bifidobacterium animalis ssp. lactis as a response to bile

Bifidobacterium animalis ssp. lactis is a probiotic frequently used as adjunct culture in fermented dairy products. In order to ensure its proper function at the intestinal level, this bacterium has to be tolerant to physiological concentrations of bile. This study examined the influence of bile on the fatty acid composition and the membrane characteristics of B. animalis IPLA 4549 and its mutant with acquired resistance to bile, B. animalis 4549dOx. Bile adaptation triggers in B. animalis 4549dOx a decrease in membrane fluidity and in the protein : phospholipid ratio, as well as a shift in the fatty acid composition of the cell. Remarkably, the presence of bile in the growth medium induced similar changes in both B. animalis cells. Furthermore, transmission electron microscopy analysis showed that bile promotes a severe distortion of the cell surface. This study provides new insights of the action of bile on the cell envelope of bifidobacteria.     

Phosphorylation of F1F0 ATPase δ-Subunit Is Regulated by Platelet-Derived Growth Factor in Mouse Cortical Neurons In Vitro

Abstract: The δ subunit of F₁F₀ ATPase (ATP synthase complex) is part of the stalk connecting the F₁ and F₀ moieties. Studies in Escherichia coli suggest that the analogous bacterial subunit, called ε, is essential for the ATPase assembly energy coupling. Platelet-derived growth factor (PDGF) is an important growth factor for various cell types, including neurons of the CNS. Using two-dimensional gel electrophoresis, microsequencing, western blot analysis, and immunoprecipitation techniques, we have found that PDGF induces phosphorylation of the δ subunit or a closely related peptide in cultured mouse cortical neurons.     

A bile salt-resistant derivative of Bifidobacterium animalis has an altered fermentation pattern when grown on glucose and maltose

The growth of Bifidobacterium animalis subsp. lactis IPLA 4549 and its derivative with acquired resistance to bile, B. animalis subsp. lactis 4549dOx, was evaluated in batch cultures with glucose or the glucose disaccharide maltose as the main carbon source. The acquisition of bile salt resistance caused a change in growth pattern for both sugars, which mainly resulted in a preferential use of maltose compared to glucose, whereas the mother strain used both carbohydrates in a similar way. High-performance liquid chromatography and gas chromatography-mass spectrometry analyses were performed to determine the amounts of glucose consumption and organic acid and ethanol formation from glucose by buffered resting cells taken at different points during growth. Resting cells of the bile-adapted strain generally consumed less glucose than those of the nonadapted one but showed an enhanced production of ethanol and higher acetic acid-to-lactic acid as well as formic acid-to-lactic acid ratios. These findings suggest a shift in the catabolism of carbohydrates promoted by the acquisition of bile resistance that may cause changes in the redox potential and improvements in the cellular ATP yield.     

Inheritance of Anthracnose Resistance in the Common Bean Differential Cultivar G 2333 and Identification of a New Molecular Marker Linked to the Co-42 gene

The inheritance of anthracnose resistance of the common bean ( Phaseolus vulgaris L.) differential cultivar G 2333 to Colletotrichum lindemuthianum races 73 and 89 was studied in crosses with the susceptible cultivar Rudá. The segregation ratios of 15 : 1 in the F₂ and 3 : 1 in the backcrosses to Rudá indicate that for each of the races tested there are two independent resistance loci in G 2333. A random amplified polymorphic DNA (RAPD) molecular marker (OPH18_1200C) linked in resistance to race 73 was identified in a BC₃F_2:3 population derived from crosses between Rudá and G 2333. A RAPD molecular marker OPAS13_950C, previously identified as linked to gene Co-4² , was also amplified in this population. Co-segregation analyses showed that these two markers are located at 5.6 (OPH18_1200C) and 11.2 (OPAS13_950C) cM of the Co-4² gene. These markers were not present in BC₁F_2:3 plants resistant to race 89 indicating that this population carries a different resistance gene. DNA amplification of BC₁F_2:3 plants with RAPD molecular marker OPAB_450C, previously identified as linked to gene Co-5 , indicated that this gene is present in this population.     

The F1FO ATP synthase genes in Methanosarcina acetivorans are dispensable for growth and ATP synthesis

There is a long-standing discussion in the literature, based on biochemical and genomic data, whether some archaeal species may have two structurally and functionally distinct ATP synthases in one cell: the archaeal A₁A_O together with the bacterial F₁F_O ATP synthase. To address a potential role of the bacterial F₁F_O ATP synthase, we have exchanged the F₁F_O ATPase gene cluster in Methanosarcina acetivorans against a puromycin resistance cassette. Interestingly, the mutant was able to grow with no difference in growth kinetics to the wild type, and cellular ATP contents were identical in the wild type and the mutant. These data demonstrate that the F₁F_O ATP synthase is dispensable for the growth of M. acetivorans .     

Adaptation and response of Bifidobacterium animalis subsp. lactis to bile: a proteomic and physiological approach

Bile salts are natural detergents that facilitate the digestion and absorption of the hydrophobic components of the diet. However, their amphiphilic nature makes them very inhibitory for bacteria and strongly influences bacterial survival in the gastrointestinal tract. Adaptation to and tolerance of bile stress is therefore crucial for the persistence of bacteria in the human colonic niche. Bifidobacterium animalis subsp. lactis, a probiotic bacterium with documented health benefits, is applied largely in fermented dairy products. In this study, the effect of bile salts on proteomes of B. animalis subsp. lactis IPLA 4549 and its bile-resistant derivative B. animalis subsp. lactis 4549dOx was analyzed, leading to the identification of proteins which may represent the targets of bile salt response and adaptation in B. animalis subsp. lactis. The comparison of the wild-type and the bile-resistant strain responses allowed us to hypothesize about the resistance mechanisms acquired by the derivative resistant strain and about the bile salt response in B. animalis subsp. lactis. In addition, significant differences in the levels of metabolic end products of the bifid shunt and in the redox status of the cells were also detected, which correlate with some differences observed between the proteomes. These results indicate that adaptation and response to bile in B. animalis subsp. lactis involve several physiological mechanisms that are jointly dedicated to reduce the deleterious impact of bile on the cell's physiology.     

Bile salt deconjugation by Lactobacillus plantarum 80 and its implication for bacterial toxicity

The effects of bile salts on the survival of lactobacilli were investigated using glycocholic acid, cholic acid and deoxycholic acid as model compounds and the bile salt hydrolase active Lactobacillus plantarum 80 (BSH⁺) and its BSH negative mutant. The detrimental effects of cholic acid, i.e. growth inhibition and cytotoxicity at a concentration of 1 and 5 mmol l⁻¹, respectively, were considered to be due to the hydrophobic protonated form of the molecule, which brings about membrane damage. The conversion of glycocholic acid to cholic acid by the BSH active L. plantarum 80 caused a growth inhibition which was comparable with the inhibition observed in the broth supplemented with 1 mmol l⁻¹ cholic acid. Deoxycholic acid caused toxicity through membrane damage when the compound was in solution. Its toxicity disappeared in the culture broth as the molecule precipitated. In case of cholic acid, the toxicity could be removed by buffering the solution at pH 7·0. It was calculated that at this pH most of the cholic acid molecules were ionized. The results led to the formulation of an extended hypothesis about the ecological significance of bile salt transformations. Primary deconjugation is carried out to counteract intracellular acidification. Yet, the deconjugated molecule can be harmful at moderately acidic pH-values. In this case, the BSH⁺ strains could effectively profit from their activity in case they are associated with 7α-dehydroxylating bacteria which dehydroxylate the deconjugated bile salts. The dehydroxylated molecule has a low solubility and precipitates at moderately acidic pH.     

The contribution of electrostatic forces to the process of adherence of Candida albicans yeast cells to substrates

Abstract Thermoanaerobacter thermohydrosulfuricus Rt8.B1 catabolized xylose by the pentose phosphate pathway, and xylose isomerase and xylulokinase were inducible. The uptake of xylose was by two low-affinity, inducible systems. Both systems were resistant to the protonophore, tetrachlorosalicylanilide, the F₁F₀-ATPase inhibitor, N , N -dicyclohexylcarboiimide, and the sodium/proton antiporter, monensin. The high capacity system (100 nmol min⁻¹ (mg protein)⁻¹) was only expressed when the bacterium was grown with a high concentration of xylose (50 mM). It took more than 60 mM xylose to saturate the high capacity system. When T. thermohydrosulfuricus was grown with a low concentration of xylose (5 mM), xylose uptake was saturated by as little as 10 mM xylose (18 nmol min⁻¹ (mg protein)⁻¹). Cells grown with 50 mM xylose could not transport glucose, and high capacity xylose transport was not inhibited by glucose or non-metabolizable glucose analogues. Cells grown with 5 mM xylose transported glucose at a rapid rate (30 nmol min⁻¹ (mg protein)⁻¹), and low capacity xylose uptake was competitively inhibited by either glucose or 2-deoxy-glucose. Because the glucose uptake of cells grown on 5 mM xylose was competitively inhibited by xylose, it appeared that the low capacity xylose uptake system was a glucose/xylose carrier.     

The ATP-dependent synthesis of factor 390 by cell-free extracts of Methanobacterium thermoautotrophicum (strain ΔH)

Abstract Cell-free extracts of Methanobacterium thermoautotrophicum (strain ΔH) converted the 8-OH-5-deazaflavin coenzyme F₄₂₀ to factor 390, a 8-adenylyl derivative (F₄₂₀-AMP). Activity was only observed upon exposure of the crude cell-free extract to oxygen. The ability to synthesize F₃₉₀ was lost when crude cell-free extract was subsequently brought to an anaerobic reducing environment. The enzymatic reaction used ATP and oxidized coenzyme F₄₂₀ as substrates and inorganic pyrophosphate was formed next to F₃₉₀. GTP could be used instead of ATP resulting in a guanylylated derivative. The crude cell-free extract showed K _m values of 154 μM for coenzyme F₄₂₀ and 2.4 mM for ATP. A partially purified enzyme preparation exhibited a K _eq of 0.32. In accordance, coenzyme F₄₂₀ and ATP could be synthesized from F₃₉₀ and PPi by the reverse reaction.     

Calcium taste preferences: genetic analysis and genome screen of C57BL/6J x PWK/PhJ hybrid mice

To characterize the genetic basis of voluntary calcium consumption, we tested C57BL/6J mice (B6; with low avidity for calcium), PWK/PhJ mice (PWK; with high avidity for calcium) and their F₁ and F₂ hybrids. All mice received a series of 96-h two-bottle preference tests with a choice between water and the following: 50 m m CaCl₂, 50 m m calcium lactate, 50 m m MgCl₂, 100 m m KCl, 100 m m NH₄Cl, 100 m m NaCl, 5 m m citric acid, 30 μ m quinine hydrochloride and 2 m m saccharin. Most frequency distributions of the parental and F₁ but not F₂ groups were normally distributed, and there were few sex differences. Reciprocal cross analysis showed that B6 × PWK F₁ mice had a non-specific elevation of fluid intake relative to PWK × B6 F₁ mice. In the F₂ mice, trait correlations were clustered among the divalent salts and the monovalent chlorides. A genome screen involving 116 markers showed 30 quantitative trait loci (QTLs), of which six involved consumption of calcium chloride or lactate. The results show pleiotropic controls of calcium and magnesium consumption that are distinct from those controlling consumption of monovalent chlorides or exemplars of the primary taste qualities.     

Production of tylosin in solid-state fermentation by Streptomyces fradiae NRRL-2702 and its gamma-irradiated mutant (γ-1)

Aims:  To develop solid-state fermentation system (SSF) for hyper production of tylosin from a mutant γ-1 of Streptomyces fradiae NRRL-2702 and its parent strain.
Methods and Results:  Various agro-industrial wastes were screened to study their effect on tylosin production in SSF. Wheat bran as solid substrate gave the highest production of 2500 μg of tylosin g⁻¹ substrate by mutant γ-1 against parent strain (300 μg tylosin g⁻¹ substrate). The tylosin yield was further improved to 4500 μg g⁻¹ substrate [70% moisture, 10% inoculum (v/w), pH 9·2, 30°C, supplemental lactose and sodium glutamate on day 9]. Wild-type strain displayed less production of tylosin (655 μg of tylosin g⁻¹ substrate) in SSF even after optimization of process parameters.
Conclusion:  The study has shown that solid-state fermentation system significantly enhanced the tylosin yield by mutant γ-1.
Significance and Impact of the Study:  This study proved to be very useful and resulted in 6·87 ± 0·30-fold increase in tylosin yield by this mutant when compared to that of wild-type strain.     

Release of adenosine triphosphatase from Streptococcus lactis subsp. cremoris membrane: evaluation of carbohydrate in membrane and F1-ATPase preparations by polyacryacrylamide gel electrophoresis

The presence of lipoteichoic acid (LTA) in plasma membrane and released adenosine triphosphatase (ATPase) preparations of Streptococcus lactis subsp. cremoris HA was studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The membrane preparation gave two spots with periodic acid Schiff's (PAS) staining. The reference LTA revealed one CBB stainable band with MW 21 500, and was detected as a characteristic yellow spot in the molecular weight area 14 000–20 900 with silver staining and was also stainable with PAS. A slight colour was found with PAS in the F₁-ATPase (EC 3.6.1.3) preparation. The results suggest that the carbohydrate component in the membrane preparation and in the solubilized F₁-ATPase is LTA.     

Tonoplast energization: Two H+ pumps, one membrane

The vacuolar membrane (tonoplast) of plant cells contains two functionally and physically distinct phosphohydrolases, which catalyse electrogenic H⁺ -translocation: An ATPase (tp-ATPase; EC 3.6.1.3) and an inorganic pyrophosphatase (tp-PPase; 3.6.1.1). Neither enzyme belongs to the F₀F₁– or E₁E₂-categories of primary cation pumps, but instead belong to a third and fourth category of enzyme, respectively. Research priorities for the tp-ATPase are studies directed at understanding the roles of the 70 and 60 kDa subunits in catalysis and regulation; the involvement of the 16 kDa subunit in transmembrane H⁺ conduction; and investigations of F₀F₁- like structure/function partitioning. In the longer term, comparisons of sequence homology between the N,N'- dicyclohexylcarbodiimide -binding (16 kDa) proteins from different sources may enable elucidation of the evolutionary relationship of the tp-ATPase with other putative third-category H⁺– translocases. The tp-PPase, on the other hand, represents an exciting but largely unexplored biochemical entity, which necessitates a reconsideration of accepted views concerning the involvement of inorganic pyrophosphate (PPi) in transmembrane energy conservation. Just why the tonoplast should be endowed with two H⁺-translocases is a problem that can only be approached once consideration is given to the paramount question of H⁺/PPi stoichiometry. Once the stoichiometry is known, it should be possible to establish the physiological poise of the tp-PPase, and hence to speculate on its role in the metabolism of plant cells.     

Tributyltin-resistant Methanothermobacter thermautotrophicus mutant with mutational substitutions in the A1A0-ATP synthase operon

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant to tributyltin chloride (TBT) was isolated. TBT, the inhibitor of the A₀ domain of A₁A₀-ATP synthase, inhibits methanogenesis in the wild-type cells; however, the TBT-resistant mutant exhibited methanogenesis even in the presence of 800 μM TBT. ATP synthesis driven by methanogenic electron transport was markedly diminished in the mutant strain. While TBT profoundly inhibited ATP synthesis driven by methanogenic electron transport in the wild type, only a slight inhibition was observed in the mutant strain. These results suggested a modification in the ATP-synthesizing system of the mutant strain. The sequence of the complete A₁A₀-ATP synthase operon ( Mth952 – Mth961 ) in the wild-type and mutant strains was determined and compared. Three mutations leading to amino acid substitutions in two A₁A₀-ATP synthase subunits were identified – Val₃₃₈Ala in subunit A and Leu₂₅₂Ile and Ser₂₉₃Ala in subunit B. Moreover, this study revealed the differential expression of several proteins that may contribute to TBT resistance. The results imply that change of TBT sensitivities of TBT-resistant mutant is due to mutational substitutions in the A₁A₀-ATP synthase operon.     

Susceptibility to chlorine of Aeromonas hydrophila strains

The susceptibility of five Aeromonas hydrophila strains and one Escherichia coli strain to chlorine was studied under carefully controlled laboratory conditions. Of the Aer. hydrophila strains, two were from untreated water, two from tap water (immediately downstream of a water treatment plant) and one from the DSM collection. The study included disinfectant concentration (0·1, 0·2 and 0·5 mg l⁻¹), pH (6, 7 and 8) and temperature (4, 21 and 32 °C) as controlled variables. The results indicated that the untreated water strains, the DSM strain and the E. coli strain were inactivated within 1 min of chlorine treatment. The strains from chlorinated water (TW11 and TW27) showed a different susceptibility to chlorine disinfection, the rate of inactivation being greater at pH 6 than at pH 8 for both strains. Under the standard conditions of temperature 21 °C, pH 7 and chlorine concentration 0·2 mg l⁻¹, an increase or decrease of approximately 1 log unit in the number of bacteria did not affect the kill rate of the strains TW11 and TW27.     

Modulation of membrane curvature by phosphatidic acid and lysophosphatidic acid

The local generation of phosphatidic acid plays a key role in the regulation of intracellular membrane transport through mechanisms which are largely unknown. Phosphatidic acid may recruit and activate downstream effectors, or change the biophysical properties of the membrane and directly induce membrane bending and/or destabilization. To evaluate these possibilities, we determined the phase properties of phosphatidic acid and lysophosphatidic acid at physiological conditions of pH and ion concentrations. In single-lipid systems, unsaturated phosphatidic acid behaved as a cylindrical, bilayer-preferring lipid at cytosolic conditions (37 °C, pH 7.2, 0.5 m m free Mg²⁺), but acquired a type-II shape at typical intra-Golgi conditions, a mildly acidic pH and submillimolar free Ca²⁺ (pH 6.6–5.9, 0.3 m m Ca²⁺). Lysophosphatidic acid formed type-I lipid micelles in the absence of divalent cations, but anhydrous cation-lysophosphatidic acid bilayer complexes in their presence. These data suggest a similar molecular shape for phosphatidic acid and lysophosphatidic acid at cytosolic conditions; however, experiments in mixed-lipid systems indicate that their shape is not identical. Lysophosphatidic acid stabilized the bilayer phase of unsaturated phosphatidylethanolamine, while the opposite effect was observed in the presence of phosphatidic acid. These results support the hypothesis that a conversion of lysophosphatidic acid into phosphatidic acid by endophilin or BARS (50 kDa brefeldin A ribosylated substrate) may induce negative spontaneous monolayer curvature and regulate endocytic and Golgi membrane fission. Alternative models for the regulation of membrane fission based on the strong dependence of the molecular shape of (lyso)phosphatidic acid on pH and divalent cations are also discussed.     

Respiration of steelhead trout sperm: sensitivity to pH and carbon dioxide

Steelhead trout Oncorhynchus mykiss sperm held in seminal plasma or sperm-immobilizing buffer (pH 8·6) at 10° C consumed O₂ at the rate of c . 2 nmol O₂ min⁻¹ 10⁻⁹ sperm; the rate of O₂ consumption was not different in sperm held for 4 or 24 h. Decreasing the extracellular pH from 8·5 to 7·5 either by diluting semen with buffer titrated with HCl or by increasing the partial pressure of CO₂ in the incubation atmosphere resulted in c . a 40% decrease in the rate of sperm respiration. The data did not, however, support the hypothesis that the precipitous reduction in the capacity for sperm motility that occurs as external pH is reduced is a result of a decrease in cellular metabolism. The rate of O₂ consumption of freshly collected semen from different males was not correlated to cellular ATP content or to the proportion of sperm that were motile upon activation; the initial ATP content and sperm motility were positively correlated. The rate of O₂ consumption was not significantly increased following sperm activation or by the addition of an uncoupler of oxidative phosphorylation, carbonyl cyanide p -trifluoromethoxyphenylhydrazone, suggesting that these sperm have little, if any, capacity for increased oxidative metabolism.