Gene replacement of adenylate kinase in the gram-positive thermophile Geobacillus stearothermophilus disrupts adenine nucleotide homeostasis and reduces cell viability

Thermophilic bacteria are of great value for industry and research communities. Unfortunately, the cellular processes and mechanisms of these organisms remain largely understudied. In the present study, we investigate how the inactivation of adenylate kinase (AK) affects the adenine nucleotide homeostasis of a gram-positive moderate thermophile, Geobacillus stearothermophilus strain NUB3621-R. AK plays a major role in the adenine nucleotide homeostasis of living cells and has been shown to be essential for the gram-negative mesophile Escherichia coli. To study the role of AK in the maintenance of adenylate energy charge (EC) and cell viability of G. stearothermophilus, we generated a recombinant strain of this organism in which its endogenous gene coding for the essential protein adenylate kinase (AK) has been replaced with the adk gene from the mesophile Bacillus subtilis. PCR, DNA sequencing and Southern analysis were performed to confirm proper gene replacement and preservation of neighboring genes. The highest growing temperature for recombinant cells was almost 20°C lower than for wild-type cells (56 vs. 75°C). This temperature-sensitive phenotype was secondary to heat inactivation of B. subtilis AK, as evidenced by enzyme activity assays and EC measurements. At higher temperatures (65°C), recombinant cells also had lower EC values (0.09) compared to wild-type cells (0.45), which reflects a disruption of adenine nucleotide homeostasis following AK inactivation.The authors would like to dedicate this paper to the memory of Dr. Neil Welker     

A strong correlation between the increase in number of proline residues and the rise in thermostability of five Bacillus oligo-1,6-glucosidases

Summary A p-nitrophenyl-α-d-glucopyranoside-hydrolysing oligo-1,6-glucosidase (dextrin 6-α-d-glucanohydrolase, EC 3.2.1.10) of Bacillus sp. KP 1071 capable of growing at 30°–66°C was purified to homogeneity. The molecular weight was estimated to be 62,000. The amino-terminal amino acid was methionine. The enzyme shared its antigenic groups in part with its homologous counterpart from Bacillus thermoglucosidasius KP 1006 (obligate thermophile), but did not at all with any one of oligo-1,6-glucosidases from Bacillus cereus ATCC 7064 (mesophile), Bacillus coagulans ATCC 7050 (facultative thermophile) and Bacillus flavocaldarius KP 1288 (extreme thermophile). A comparison of amino acid composition showed that the proline content increased greatly in a linearity with the rise in thermostability in the order, mesophile → facultative thermophile → KP 1071 → obligate thermophile → extreme thermophile enzymes. Presented at the Annual Meeting of the Agricultural Chemical Society of Japan, Kyoto, April 3, 1986     

Studies on interdomain interaction of 3-isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus, by constructing chimeric enzymes

In our previous study, we showed that a chimeric isopropylmalate dehydrogenase, 2T2M6T, between an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, isopropylmalate dehydrogenases (the name roughly denotes the primary structure; the first 20% from the N-terminal is coded by the thermophile leuB gene, next 20% by mesophile, and the rest by the thermophile gene) denatured in two steps with a stable intermediate, suggesting that in the chimera some of the interdomain interaction was lost by amino acid substitutions in the "2M" part. To identify the residues involved in the interdomain interactions, the first and the second halves of the 2M part of the chimera were substituted with the corresponding sequence of the thermophile enzyme. Both chimeras, 3T1M6T and 2T1M7T, apparently showed one transition in the thermal denaturation without any stable intermediate state, suggesting that the cooperativity of the conformational stability was at least partly restored by the substitutions. The present study also suggested involvement of one or more basic residues in the unusual stability of the thermophile enzyme. Received: September 29, 1998 / Accepted: June 25, 1999     

Production of a thermostable ATPase by the facultative thermophile,Bacillus coagulans

The properties of the ATPase in the facultative thermophile, Bacillus coagulans, grown at thermophilic or mesophilic temperatures were similar. Arrhenius plots did not show discontinuities indicative of thermoadaptation. Magnesium stimulation of the enzyme was dependant on the assay temperature but independant of the growth temperature. The ATPase in cells grown at 35°C or 55°C was equally thermostable at 65°C. In contrast, the ATPase from the mesophile, Bacillus megaterium (T _max=42°C) was completely inactivated at 55°C in 5 min.     

Temperature dependent dynamics in highly homologous adenylate kinases

To correlate the structural features of enzymes to temperature adaptation, we studied psychrophile, mesophile, and thermophile adenylate kinases as model enzymes using bioinformatics and computational tools. Phylogenetic analysis revealed that mesophile and thermophile variants are clustered in one stem of phylogenetic tree and are close to contemporary time, while psychrophile enzyme is more close to their common ancestor. This finding is in good agreement with the process of environmental changes from ice age toward current warm conditions on the earth. We also performed Molecular Dynamics simulation at corresponding temperatures of all enzyme variants including 308, 318, and 328 K. It was found that mesophile enzyme has no distinct deviation of Root Mean Square Deviation (RMSD) and Radius of Gyration (Rg) values from equilibrium states at operating temperature of thermophile enzyme as well as its own optimum temperature. However, psychrophile enzyme undergoes more fluctuations with higher amplitude of change; particularly at 328 K. It was also found that initial increasing of RMSD and Rg for Psychrophile enzyme at all temperatures is occurred gradually; while, the increment of this structural parameters for thermophile enzyme at 328 K is occurred in a highly cooperative and switching manner demonstrating snap structural change of thermophile enzyme in its own temperature. By analysis of Root Mean Square Fluctuation values at different temperatures, we identified two flexible fragments in adenylate kinases so that different dynamic behavior of these regions in mesophile enzyme against operating temperatures of psychrophile and thermophile variants is critical in compensation of flexibility challenges at respective temperatures.     

Substitution of aspartic acid with glutamic acid increases the unfolding transition temperature of a protein

Proteins from thermophiles are more stable than those from mesophiles. Several factors have been suggested as causes for this greater stability, but no general rule has been found. The amino acid composition of thermophile proteins indicates that the content of polar amino acids such as Asn, Gln, Ser, and Thr is lower, and that of charged amino acids such as Arg, Glu, and Lys is higher than in mesophile proteins. Among charged amino acids, however, the content of Asp is even lower in thermophile proteins than in mesophile proteins. To investigate the reasons for the lower occurrence of Asp compared to Glu in thermophile proteins, Glu was substituted with Asp in a hyperthermophile protein, MjTRX, and Asp was substituted with Glu in a mesophile protein, ETRX. Each substitution of Glu with Asp decreased the Tm of MjTRX by about 2 degrees C, while each substitution of Asp with Glu increased the Tm of ETRX by about 1.5 degrees C. The change of Tm destabilizes the MjTRX by 0.55 kcal/mol and stabilizes the ETRX by 0.45 kcal/mol in free energy.     

Crystal structure of a trimeric archaeal adenylate kinase from the mesophile Methanococcus maripaludis with an unusually broad functional range and thermal stability

The structure of the trimeric adenylate kinase from the Archaebacteria Methanococcus mariplaludis (AK_MAR) has been solved to 2.5‐Å resolution and the temperature dependent stability and kinetics of the enzyme measured. The K_M and V_max of AK_MAR exhibit only modest temperature dependence from 30°–60°C. Although M. mariplaludis is a mesophile with a maximum growth temperature of 43°C, AK_MAR has a very broad functional range and stability (T_m = 74.0°C) that are more consistent with a thermophilic enzyme with high thermostability and exceptional activity over a wide range of temperatures, suggesting that this microbe may have only recently invaded a mesophilic niche and has yet to fully adapt. A comparison of the Local Structural Entropy (LSE) for AK_MAR to the related adenylate kinases from the mesophile Methanococcus voltae and thermophile Methanococcus thermolithotrophicus show that changes in LSE are able to fully account for the intermediate stability of AK_MAR and highlights a general mechanism for protein adaptation in this class of enzymes. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Adenylate energy charge during batch culture of Thermoactinomyces vulgaris 42

The adenylate energy charge of thermophilic actinomycete Thermoactinomyces vulgaris 42 cells growing exponentially on mineral medium with glucose and casein was around 0.95. After the glucose exhausion, the energy charge fell steadily to a value of 0.5 and remained constant for at least 5 h. The ATP content was found to be a suitable gowth monitoring parameter for Thermoactinomyces vulgaris 42.Abbreviations TCA trichloroacetic acid     

Adenine nucleotide content and energy charge of Bacillus megaterium during batch growth in low-phosphate medium

Abstract The effect of a low phosphate concentration on intracellular adenine nucleotides, oxygen consumption and poly-β-hydroxybutyric acid synthesis, was investigated with batch cultures of Bacillus megaterium . At low phosphate concentrations the cells contained much larger amounts of poly-β-hydroxybutyric acid, but displayed lower adenylate energy charge and oxygen uptake than did control cells. The ratio of ATP to ADP was much greater in the control cells. The levels of ATP and AMP were lower in low-phosphate cells.     

The effect of season and location on phosphoadenylate concentrations and adenylate energy charge in two species of freshwater clams

Summary Concentrations of phosphoadenylate nucleotides and the adenylate energy charge ((ATP+1/2ADP)/(ATP+ADP+AMP)) have been suggested as sensitive integrating measures of the energy state of organisms. This synoptic study investigated the seasonal and spatial variation of phosphoadenylate concentrations and AEC in two freshwater bivalve molluscs, the paper-shell clam, Anodonta imbecillis and the asian clam, Corbicula fluminea. Concentrations of all three adenylates, as well as the total adenylate concentration and adenylate energy charge of both species varied seasonally. These fluctuations were closely related to reproductive periods in both species. Total adenylate concentrations and ATP concentrations were slightly negatively correlated with shell length in A. imbecillis but the ADP and AMP concentrations and AEC were not significantly correlated with shell length. In C. fluminea the AEC was negatively correlated were positively correlated with shell length. Neither species exhibited significant differences in AEC between two collection locations. When C. fluminea collected from the Savannah River were acclimated and fed in the laboratory their AEC increased significantly.     

Adenine nucleotides and the xanthophyll cycle in leaves

The relationships among the leaf adenylate energy charge, the xanthophyll-cycle components, and photosystem II (PSII) fluorescence quenching were determined in leaves of cotton (Gossypium hirsutum L. cv. Acala) under different leaf temperatures and different intercellular CO₂ concentrations (C_i). Attenuating the rate of photosynthesis by lowering the C_i at a given temperature and photon flux density increased the concentration of high-energy adenylate phosphate bonds (adenylate energy charge) in the cell by restricting ATP consumption (A.M. Gilmore, O. Björkman 1994, Planta 192, 526–536). In this study we show that decreases in photosynthesis and increases in the adenylate energy charge at steady state were both correlated with decreases in PSII photo-chemical efficiency as determined by chlorophyll fluorescence analysis. Attenuating photosynthesis by decreasing C_i also stimulated violaxanthin-de-epoxidation-dependent nonradiative dissipation (NRD) of excess energy in PSII, measured by nonphotochemical fluorescence quenching. However, high NRD levels, which indicate a large trans-thylakoid proton gradient, were not dependent on a high adenylate energy charge, especially at low temperatures. Moreover, dithiothreitol at concentrations sufficient to fully inhibit violaxanthin de-epoxidation and strongly inhibit NRD, affected neither the increased adenylate energy charge nor the decreased PSII photo-chemical efficiency that result from inhibiting photosynthesis. The build-up of a high adenylate energy charge in the light that took place at low C_i and low temperatures was accompanied by a slowing of the relaxation of non-photochemical fluorescence quenching after darkening. This slowly relaxing component of nonphotochemical quenching was also correlated with a sustained high adenylate energy charge in the dark. These results indicate that hydrolysis of ATP that accumulated in the light may acidify the lumen and thus sustain the level of NRD for extended periods after darkening the leaf. Hence, sustained nonphotochemical quenching often observed in leaves subjected to stress, rather than being indicative of photoinhibitory damage, apparently reflects the continued operation of NRD, a photoprotective process.Abbreviations A antheraxanthin - adenylate kinase (myokinase), ATP:AMPphosphotransferase - C_i intercellular CO₂ concentration - DPS de-epoxidation state of violaxanthin, ([Z+A]/[V+A+Z]) - DTT dithiothreitol - pH trans-thylakoid proton gradient - [2ATP+ADP] - F steady-state fluorescence in the presence of NRD - F_M maximal fluorescence in the absence of NRD - F_M maximal fluorescence in the presence of NRD - NRD nonradiative energy dissipation - PET photosynthetic electron transport rate - PFD photon flux density - _PSII photon yield of PSII photochemistry at the actual reduction state in the light or dark - Q_A the primary electron acceptor of PSII - [ATP+ADP+AMP] - SV_N Stern-Volmer nonphotochemical quenching - V violaxanthin - Z zeaxanthin We thank Connie Shih for skillful assistance in growing plants and for conducting HPLC analyses. A Carnegie Institution Fellowship to A.G. is also gratefully acknowledged.     

Changes in the adenine nucleotide content of cysts of Globodera rostochiensis associated with the hatching of juveniles

The content of AMP, ADP and ATP within single cysts of Globodera rostochiensis (60–80 fig dry weight) was determined as ATP to an accuracy of ± 10^-11 mol by a bioluminescent technique, after microenzymic methods had been used to phosphorylate AMP and ADP to ATP. Results for a total of 120 cysts showed that a change occurs in the adenylate energy charge of their contents after they have been exposed to potato root diffusate. Cysts in water had a mean adenylate energy charge of 0–63 (s.E. ± 0–04), but a randomly selected group of cysts after 24 h treatment with potato root diffusate had a significantly lower mean of 0–49±0–04. In a second, similar experiment, cysts in diffusate for only 8 h had an energy charge of 0*55 ± 0–03, but this value was not significantly less than the corresponding mean of o-6i ±0–03 of cysts that remained in water. The results indicate an effect on the metabolism of the unhatched juveniles that occurs too soon after the addition of diffusate to be directly due to any increase in locomotor activity. Apparently, the primary action of the hatching factor had affected many juveniles within 24 h of the addition of potato root diffusate to the cyst.     

ATP-induced Changes in Energy Status and Membrane Integrity of Harvested Litchi Fruit and its Relation to Pathogen Resistance

Litchi is a subtropical fruit of high commercial value on the international market but the fruit deteriorates rapidly after harvest due to rot development caused by Peronophythora litchii. To investigate the role of energy metabolism during disease development on harvested litchi fruit, fruits were dipped into solutions of either 0 or 1.0 mm adenosine triphosphate (ATP) for 3 min before being inoculated with Peronophythora litchii or not. Fruit were then stored for 6 days at 25°C and 90–100% relative humidity. Significant reductions in pericarp browning and disease severity and significant delays in membrane permeability and malondialdehyde (MDA) content were found in ATP‐treated and P. litchii‐inoculated fruit. Higher ATP concentrations and adenylate energy charge (EC) were observed in ATP‐treated fruit. In addition, lower activities of phospholipase D, acid phosphatase and lipoxygenase enzymes involved in membrane lipid peroxidation and hydrolysis were recorded in ATP‐treated fruit. Thus, treatment with ATP maintained higher energy levels, inhibited activities of the membrane hydrolysis‐related enzymes, reduced membrane lipid peroxidation and helped maintain membrane integrity of the harvested litchi fruit at the early stage of storage, which could account for the inhibition of disease development of P. litchii‐inoculated fruit.     

Relation between energy production and adenine nucleotide metabolism in human blood platelets

The relation between ATP production and adenine nucleotide metabolism was investigated in human platelets which were starved by incubation in glucose-free, CN^?-containing medium and subsequently incubated with different amounts of glucose. In the absence of mitochondrial energy production (blocked by CN^?) and glycogen catabolism (glycogen almost completely consumed during starvation), lactate production increased proportionally with increasing amounts of glucose. The generated ATP was almost completely consumed in the various ATP-consuming processes in the cell except for a fixed portion (about 7%) that was reserved for restoration of the adenylate energy charge. During the first 10 min after glucose addition, the adenine nucleotide pool remained constant. Thereafter, when the glycolytic flux, measured as lactate formation, was more than 3.5 μmol · min^?1 · 10^?11 cells, the pool increased slightly by resynthesis from hypoxanthine-inosine and then stabilized; at a lower flux the pool decreased and metabolic ATP and energy charge declined to values found during starvation. Between moments of rising and falling adenylate energy charges, periods of about 10 min remained in which the charge was constant and ATP supply and demand had reached equilibrium. This enabled comparison between the adenylate energy charge and ATP regeneration velocity. A linear relation was obtained for charge values between 0.4 and 0.85 and ATP regeneration rates between 0.6 and 3.5 ATP equiv. · min^?1 · 10^?11 cells. These data indicate that in starved platelets ATP regeneration velocity and energy charge are independent and that each appears to be subject to the availability of extracellular substrate.     

Adenylate energy charge of rat and human cultured hepatocytes

Summary A simple and rapid method for the assay of adenine nucleotides (ATP, ADP, and AMP) was established to evaluate the adenylate energy charge (ATP+ADP/2)/(ATP+ADP+AMP) of cultured hepatocytes. The effects of inhibitors of glycolysis, fatty acid oxidation, or oxidative phosphorylation on the energy charge were examined. The energy charges of cultured hepatocytes in rats and human were almost identical and were maintained at a high level between 6 and 24 h after changing the media (rat: 0.908±0.008n=9, human: 0.918±0.014n=6, mean ± SD). Inhibition of glycolysis with sodium fluoride or oxidative phosphorylation with antimycin A irreversibly reduced both the adenine nucleotide contents and the energy charge. However, the inhibition of fatty acid oxidation with 2-tetradecylglycidic acid did not affect the nucleotide contents, and the energy charge only decreased transiently to recover within 8 h. When the inhibitor of oxidative phosphorylation was removed, the recovery in the energy charge preceded the recovery in the adenine nucleotide contents. These findings suggest that the adenylate energy charge is a more sensitive measure of the changes in energy metabolism than the adenine nucleotide contents. Furthermore, energy charge regulates adenine nucleotide contents in cultured hepatocytes. It is important to confirm that the high energy charge of the cultured hepatocytes is maintained when these cells are used for metabolic studies.     

Purification and characterization of Bacillus coagulans oligo-1,6-glucosidase

A p-nitrophenyl-alpha-D-glucopyranoside-hydrolyzing oligo-1,6-glucosidase of Bacillus coagulans ATCC 7050 (facultative thermophile) was purified to homogeneity. The relative molecular mass, Stokes radius, sedimentation coefficient at 20 degrees C in water, molecular absorption coefficient at 280 nm and pH 6.8, and isoelectric point were estimated as 60 000, 3.29 nm, 4.8 X 10(-13) s, 1.34 X 10(5) M-1 cm-1, and 4.3, respectively. The amino-terminal amino acid was threonine. There was no common antigenic group between the enzyme and each of its homologous counterparts from Bacillus cereus ATCC 7064 (mesophile) and Bacillus thermoglucosidasius KP 1006 (obligate thermophile). These oligo-1,6-glucosidases strongly resembled one another in their amino acid composition, except that the proline content increased with the elevation of thermostability in the order, mesophile----facultative thermophile----obligate thermophile enzymes.     

In vitro adenine nucleotide catabolism in African catfish spermatozoa

It has been shown recently that African catfish (Clarias gariepinus) spermatozoa possess relatively low ATP content and low adenylate energy charge (AEC). One of the possible explanations for this phenomenon is that the spermatozoa actively catabolize adenine nucleotides. A relatively high rate of such catabolism could then contribute to the low ATP concentration and low adenylate energy charge observed in the spermatozoa in vitro. To check this hypothesis, we investigated ATP content and adenine nucleotide catabolism in African catfish spermatozoa stored at 4 °C in the presence of glycine as an energetic substrate. Our results indicate that the storage of African catfish sperm at 4 °C in the presence of glycine causes time-dependent ATP depletion. In contrast to ATP, the AMP content increases significantly during the same period of sperm storage, while the ADP increases only slightly. Moreover, a significant increase of inosine and hypoxanthine content was also found. Hypoxanthine was accumulated in the storage medium, but xanthine was found neither in spermatozoa nor in the storage medium. It indicates that hypoxanthine is not converted to xanthine, probably due to lack of xanthine oxidase activity in catfish spermatozoa. Present results suggest that adenine nucleotides may be converted to hypoxanthine according to the following pathway: ATP→ADP→AMP (adenosine/IMP)→inosine→hypoxanthine. Moreover, hypoxanthine seems to be the end product of adenine nucleotide catabolism in African catfish spermatozoa. In conclusion, our results suggest that a relatively high rate of adenine nucleotide catabolism contributes to the low ATP concentration and low adenylate energy charge observed in African catfish spermatozoa in vitro.     

Structures and analysis of highly homologous psychrophilic, mesophilic, and thermophilic adenylate kinases

The crystal structures of adenylate kinases from the psychrophile Bacillus globisporus and the mesophile Bacillus subtilis have been solved and compared with that from the thermophile Bacillus stearothermophilus. This is the first example we know of where a trio of protein structures has been solved that have the same number of amino acids and a high level of identity (66-74%) and yet come from organisms with different operating temperatures. The enzymes were characterized for their own thermal denaturation and inactivation, and they exhibited the same temperature preferences as their source organisms. The structures of the three highly homologous, dynamic proteins with different temperature-activity profiles provide an opportunity to explore a molecular mechanism of cold and heat adaptation. Their analysis suggests that the maintenance of the balance between stability and flexibility is crucial for proteins to function at their environmental temperatures, and it is achieved by the modification of intramolecular interactions in the process of temperature adaptation.     

Adenylate nucleotide levels and energy charge in Arthrobacter crystallopoietes during growth and starvation

The adenylate nucleotide concentrations, based on internal water space, were determined in cells of Arthrobacter crystallopoietes during growth and starvation and the energy charge of the cells was calculated. The energy charge of spherical cells rose during the first 10 h of growth, then remained nearly constant for as long as 20 h into the stationary phase. The energy charge of rod-shaped cells rose during the first 4 h of growth, then remained constant during subsequent growth and decreased in the stationary growth phase. Both spherical and rod-shaped cells excreted adenosine monophosphate but not adenosine triphosphate or adenosine diphosphate during starvation. The intracellular energy charge of spherical cells declined during the initial 10 h and then remained constant for 1 week of starvation at a value of 0.78. The intracellular energy charge of rod-shaped cells declined during the first 24 h of starvation, remained constant for the next 80 h, then decreased to a value of 0.73 after a total of 168 h starvation. Both cell forms remained more than 90% viable during this time. Addition of a carbon and energy source to starving cells resulted in an increase in the ATP concentration and as a result the energy charge increased to the same levels as found during growth.Nonstandard Abbreviations cGMP 3,5 guanosine monophosphate - ppGpp guanosine tetraphosphate - MS mineral salts - HC casein hydrolysate - TEA triethanolamine buffer - Pi inorganic phosphate     

Charges of nicotinamide adenine nucleotides and adenylate energy charge as regulatory parameters of the metabolism in Escherichia coli.

Methods for measurements of catabolic reduction charge (defined as NADH/(NADH+NAD+)) and anabolic reduction charge (defined as NADPH/(NADPH + NADP+)) are described using [14C]nicotinamide labeling of Escherichia coli cultures. Together with these parameters the adenylate energy charge (ATP + 1/2ADP)/(ATP + ADP + AMP) was measured using labeling with [2-3H]adenine. These three charges were found under different exponential growth conditions to have values independent of the growth conditions: catabolic reduction charge, 0.05; anabolic reduction charge, 0.45; and adenylate energy charge, 0.9. The charges were examined during interruption of growth primarily affecting catabolism, respiration, or anabolism, leading to changes of the charges. The changes of charges are evaluated as a possible regulation of the metabolic rates utilizing or producing the nucleotides by their respective charges.