Organization and copy number of initiator tRNA genes in slow- and fast-growing mycobacteria

We have previously reported the isolation and characterization of a functional initiator tRNA gene,metA, and a second initiator tRNA-like sequence,metB, fromMycobacterium tuberculosis. Here we describe the fine mapping of the initiator tRNA gene locus of the avirulent (H37Ra) and virulent (H37Rv) strains ofM. tuberculosis. The genomic blot analyses show that the 1.7 kb (harbouringmetE) and the 6.0 kb BamHI (harbouringmetA) fragments are linked. Further, sequencing of a portion of the 6.0kb fragment, in conjunction with the sequence of the 1.7 kb fragment confirmed the presence of an IS6110 element in the vicinity ofmetB. The IS element is flanked by inverted (28 bp, with 3 contiguous mismatches in the middle) and direct (3 bp) repeats considered to be the hallmarks of IS6110 integration sites. The organization of the initiator tRNA gene locus is identical in both the H37Ra and H37Rv strains and they carry a single copy of the functional initiator tRNA gene. Interestingly, the fast growingMycobacterium smegmatis also bears a single initiator tRNA gene. This finding is significant in view of the qualitative differences in total tRNA pools and the copy number of rRNA genes in the fast- and slow-growing mycobacteria. Finally, we discuss hypotheses related to the origin ofmetB inM. tuberculosis.     

Ornithine lipid of Mycobacterium tuberculosis: its distribution in some slow- and fast-growing mycobacteria

An ornithine-amide lipid is present in Mycobacterium tuberculosis. Its structure was established by a combination of chemical analysis and mass spectrometry. 3-Hydroxyoctadecanoic and 3-hydroxyeicosanoic acids (and homologues) were found to be linked through an amide bond to the alpha-amino group of L-ornithine, the hydroxyl group of the fatty acid being esterified mainly by tuberculostearic acid (10-methyloctadecanoic acid). This ornithine-amide lipid was detected in several other slow-growing pathogenic mycobacteria by thin layer chromatography, but not in an avirulent strain (H37 Ra) of M. tuberculosis. In each case mass spectrometry showed that all the structures were identical, thus revising an earlier reported structure for the lipid from M. bovis.     

C-Terminal mutations in the chloroplast ATP synthase gamma subunit impair ATP synthesis and stimulate ATP hydrolysis

Two highly conserved amino acid residues, an arginine and a glutamine, located near the C-terminal end of the gamma subunit, form a "catch" by hydrogen bonding with residues in an anionic loop on one of the three catalytic beta subunits of the bovine mitochondrial F1-ATPase [Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628]. The catch is considered to play a critical role in the binding change mechanism whereby binding of ATP to one catalytic site releases the catch and induces a partial rotation of the gamma subunit. This role is supported by the observation that mutation of the equivalent arginine and glutamine residues in the Escherichia coli F1 gamma subunit drastically reduced all ATP-dependent catalytic activities of the enzyme [Greene, M. D., and Frasch, W. D. (2003) J. Biol. Chem. 278, 5194-5198]. In this study, we show that simultaneous substitution of the equivalent residues in the chloroplast F1 gamma subunit, arginine 304 and glutamine 305, with alanine decreased the level of proton-coupled ATP synthesis by more than 80%. Both the Mg2+-dependent and Ca2+-dependent ATP hydrolysis activities increased by more than 3-fold as a result of these mutations; however, the sulfite-stimulated activity decreased by more than 60%. The Mg2+-dependent, but not the Ca2+-dependent, ATPase activity of the double mutant was insensitive to inhibition by the phytotoxic inhibitor tentoxin, indicating selective loss of catalytic cooperativity in the presence of Mg2+ ions. The results indicate that the catch residues are required for efficient proton coupling and for activation of multisite catalysis when MgATP is the substrate. The catch is not, however, required for CaATP-driven multisite catalysis or, therefore, for rotation of the gamma subunit.     

Cholesterol is accumulated by mycobacteria but its degradation is limited to non-pathogenic fast-growing mycobacteria

In this report we show that fast-growing non-pathogenic mycobacteria degrade cholesterol from liquid media, and are able to grow on cholesterol as a sole carbon source. In contrast, slow-growing mycobacteria, including pathogenic Mycobacterium tuberculosis and bacillus Calmette-Guérin (BCG), do not degrade and use cholesterol as a carbon source. Nevertheless, pathogenic mycobacteria are able to uptake, modify, and accumulate cholesterol from liquid growth media, and form a zone of clearance around a colony when plated on solid media containing cholesterol. These data suggest that cholesterol may have a role in mycobacterial infection other than its use as carbon source.     

ATP synthase: what we know about ATP hydrolysis and what we do not know about ATP synthesis

In ATP synthase, X-ray structures, demonstration of ATP-driven gamma-subunit rotation, and tryptophan fluorescence techniques to determine catalytic site occupancy and nucleotide binding affinities have resulted in pronounced progress in understanding ATP hydrolysis, for which a mechanism is presented here. In contrast, ATP synthesis remains enigmatic. The molecular mechanism by which ADP is bound in presence of a high ATP/ADP concentration ratio is a fundamental unknown; similarly P(i) binding is not understood. Techniques to measure catalytic site occupancy and ligand binding affinity changes during net ATP synthesis are much needed. Relation of these parameters to gamma-rotation is a further goal. A speculative model for ATP synthesis is offered.     

Synthesis and hydrolysis of ATP by the mitochondrial ATP synthase

A brief summary of the factors that control synthesis and hydrolysis of ATP by the mitochondrial H+-ATP synthase is made. Particular emphasis is placed on the role of the natural ATPase inhibitor protein. It is clear from the existing data obtained with a number of agents that there is no correlation between variations of the rate of ATP hydrolysis and ATP synthesis as driven by respiration. The mechanism by which each condition differentially affects the two activities is not entirely known. For the case of the natural ATPase inhibitor protein, it appears that the protein controls the kinetics of the enzyme. This control seems essential for achieving maximal accumulation of ATP during electron transport in systems that contain relatively high concentrations of ATP.     

Adsorption of slow- and fast-growing rhizobia to soybean and cowpea roots

Roots of soybean (Glycine max [L.] Merr. cv Hardee) and cowpea (Vigna unguiculata [L.] Walp. cv Pink Eye Purple Hull) were immersed in suspensions containing 10⁴Rhizobium cells per milliliter of a nitrogen-free solution. After 30 to 120 minutes the roots were rinsed, and the distal 2-centimeter segments excised and homogenized. Portions of the homogenates then were plated on a yeast-extract mannitol medium for bacterial cell counts. The adsorption capacities of four slow-growing rhizobia and a fast-growing R. meliloti strain varied considerably. Adsorption was independent of plant species and of the abilities of the Rhizobium strains to infect and nodulate. R. lupini 96B9 had the greatest adsorption capacity, and Rhizobium sp. 3G4b16 the least. Rhizobium sp. 229, R. japonicum 138, and R. meliloti 102F51 were intermediate, except on cowpea, where the adsorption of strain 102F51 was similar to that of strain 3G4b16. The initial adsorption rates of bacteria cultured in synthetic media and in the presence of soybean roots were about the same. Addition of soybean lectin to the bacterial inoculum failed to influence initial adsorption rates. Both treatments, however, reduced the numbers of bacteria that bound after incubation with roots for 120 minutes. The relationship between the logarithm of the number of strain 138 cells bound per soybean root segment and the logarithm of the density of bacteria in the inoculum was linear over five orders of magnitude. Binding of strain 138 to soybean roots was greatest at room temperature (27°C) and substantially attenuated at both 4 and 37°C. Although R. lupini 96B9 strongly rejected a model hydrophobic plastic surface, there were no simple correlations between bacterial binding to model hydrophobic and hydrophilic plastic surfaces and bacterial adsorption to roots.     

Correlations between ATP hydrolysis, ATP synthesis, generation and utilization of ΔpH in mitochondrial ATPase-ATP synthase

Using fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine induced either by ATP hydrolysis in the ATPase-ATP synthase complex or by succinate oxidation in inverted submitochondrial particles, correlations have been established between ATP hydrolysis, ATP synthesis and the generation and utilization of ΔpH. The results obtained are best explained in terms of local circuits of protons.     

Influence of the redox state and the activation of the chloroplast ATP synthase on proton-transport-coupled ATP synthesis/hydrolysis

The membrane-bound ATP synthase from chloroplasts can occur in different redox and activation states. In the absence of reductants the enzyme usually is oxidized and inactive, E^ox_i. Illumination in the presence of dithiothreitol leads to an active, reduced enzyme, E^red_a. If this form is stored in the dark in the presence of dithiothreitol an inactive, reduced enzyme E^red_i is formed. The rates of ATP synthesis and ATP hydrolysis catalyzed by the different enzyme species are measured as a function of ΔpH (Δψ = 0 mV). The ΔpH was generated with an acid-base transition using a rapid-mixing quenched flow apparatus. The following results were obtained. (1) The oxidized ATP synthase catalyzes high rates of ATP synthesis, v^ox_max = 400 ATP per CF₀F₁ per s. The half-maximal rate is obtained at ΔpH = 3.4. (2) The active, reduced ATP synthase catalyzes high rates of ATP synthesis, v^red_max = 400 ATP per CF₀F₁ per s. The half-maximal rate is obtained at ΔpH = 2.7. It catalyzes also high rates of ATP hydrolysis v^red_max = −90 ATP per CF₀F per s at ΔpH = 0. (3) The inactive species (both oxidized and reduced) catalyze neither ATP synthesis nor ATP hydrolysis. The activation/inactivation of the reduced enzyme is completely reversible. (4) The activation of the reduced, inactive enzyme is measured as a function of ΔpH by measuring the rate of ATP hydrolysis catalyzed by the active species. Half-maximal activation is observed at ΔpH = 2.2. (5) On the basis of these results a reaction scheme is proposed relating the redox reaction, the activation and the catalytic reaction of the chloroplast ATP synthase.     

Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNAala T-loops in slow- and fast-growing mycobacteria

Background  

Mycobacteriophage Ms6 integrates into Mycobacterium smegmatis and M. bovis BCG chromosome at the 3' end of tRNA^ala genes. Homologous recombination occurs between the phage attP core and the attB site located in the T-loop. Integration-proficient vectors derived from Ms6 are useful genetic tools, but their insertion sites in the BCG chromosome remain poorly defined. The primary objective of this study was to identify Ms6 target genes in M. smegmatis and BCG. We then aimed to modify the attP site in Ms6-derived vectors, to switch integration to other tRNA^ala loci. This provided the basis for the development of recombinant M. bovis BCG strains expressing several reporter genes inserted into different tRNA^ala genes.     

ATP synthesis by F-type ATP synthase is obligatorily dependent on the transmembrane voltage.

ATP synthase is the universal enzyme that manufactures cellular ATP using the energy stored in a transmembrane ion gradient. This energy gradient has two components: the concentration difference (DeltapH or DeltapNa(+)) and the electrical potential difference DeltaPsi, which are thermodynamically equivalent. However, they are not kinetically equivalent, as the mitochondrial and bacterial ATP synthases require a transmembrane potential, DeltaPsi, but the chloroplast enzyme has appeared to operate on DeltapH alone. Here we show that, contrary to the accepted wisdom, the 'acid bath' procedure used to study the chloroplast enzyme develops not only a DeltapH but also a membrane potential, and that this potential is essential for ATP synthesis. Thus, for the chloroplast and other ATP synthases, the membrane potential is the fundamental driving force for their normal operation. We discuss the biochemical reasons for this phenomenon and a model that is consistent with these new experimental facts.     

Differential effects of triphenyltin and 8-azido-ATP on the ATP synthesis, ATP-Pi exchange, and ATP hydrolysis in liposomes containing ATP synthase and bacteriorhodopsin

The ATP hydrolysis activity of purified ATP synthase reconstituted in liposomes was inhibited by triphenyltin in a manner different from that of other thiol-specific reagents. In liposomes containing ATP synthase and bacteriorhodopsin, ATP hydrolysis and ATP-Pi exchange were inhibited by triphenyltin to a greater extent than the ATP synthesis, in contrast to what was found with an F1-specific inhibitor, 8-azido-ATP. The possibility is discussed that ATP hydrolysis and ATP synthesis are differently coupled to proton conduction through F0.     

Stereochemical reaction mechanism formulations for enzyme-catalyzed pyrophosphate hydrolysis,ATP hydrolysis,and ATP synthesis

Fundamental concepts pertaining to the stereochemistry paths of polar additionelimination (nucleophilic substitution) reactions at phosphate phosphorus centers are reviewed and employed to analyze ¹⁸O exchange reactions catalyzed by inorganic pyrophosphatase and mitochondrial ATP synthetase. The analysis suggests reasonable choices for the stereochemistry path of the ¹⁸O exchanges. This, in turn, permits reasonable choices for the stereochemistry paths of hydrolysis of pyrophosphate catalyzed by pyrophosphatase and of hydrolysis and synthesis of ATP catalyzed by ATP synthetase.     

Characterization of phosphorus uptake in slow- and fast-growing southern pine seedlings grown in solution culture

The effects of low-P growth conditions on growth and net P acquisition were examined in two species of pine that are indigenous to P-deficient soils of the Atlantic Coastal Plain: pond pine (Pinus serotina Michx.), a moderately-fast growing pine, and a slow-growing seed source of loblolly pine P. taeda L.) from Texas. Short-term ³²P uptake experiments were conducted using intact nonmycorrhizal seedlings that had been grown for 7 weeks in continuously-flowing solution culture at 5 or 100 µM P. Growth and P uptake of pond pine were more responsive to a higher P supply than the slow-growing loblolly pine. Pond pine seedlings in the 100 µM P treatment were twice the size of those grown in 5 µM P and accumulated almost five times as much seedling P. In contrast, seedling biomass of loblolly pine increased by only 8% under high-P growth conditions, and seedlings accumulated twice as much P, reflecting the higher P concentrations in shoot and root tissues. Although rates of unidirectional influx of ³²P were 22 and 61% higher under low-P growth conditions in pond and loblolly pine, respectively, net uptake rates in seedlings from the 5 µM P treatment were over three times those of seedlings grown in 100 µM P. These results suggest that unidirectional efflux out of the root was controlling net uptake of P as much, if not more, than unidirectional influx. Efflux of³² P out of root tissue, particularly older tissue, decreased in seedlings grown under low-P conditions, possibly due to a reduction in the size of the phosphorus pool available for efflux, i.e. the soluble P_i pool. Over 75% of the total root P in both loblolly and pond pine seedlings grown in 100 µM P treatment was present as organic P, suggesting that organic P, particularly phytate, may represent important storage pools in roots of woody species. Within each species, higher rates of influx and net uptake in seedlings from the low-P treatment were associated with lower P concentrations in shoot and root tissue, and shoot FW:root FW ratios. Efflux may represent a short-term means of regulating net P uptake, while the demand for P created by growth and storage may represent a long-term regulation.     

Kinetic modeling of ATP synthesis by ATP synthase and its mechanistic implications

Based on the torsional mechanism of ATP synthesis by ATP synthase, a kinetic scheme has been developed in this work. The scheme considers adenine nucleotide transport, binding of substrates ADP and P(i), unbinding of product ATP, and ATP synthesis. This kinetic scheme has been analyzed mathematically, and a kinetic model has been obtained to explain the experimentally observed hyperbolic Michaellian dependence of the rate of ATP synthesis on the ADP concentration by ATP synthase under physiological steady-state operating conditions. The principal results of the kinetic model have been compared with the experimental data and an estimate of the enzymological kinetic parameters V(max), K(M), and K(I) has been determined. Mechanistic implications arising from further analysis of the kinetic model have been discussed. These biological implications provide deep insight into the sequence of events leading to ATP synthesis.