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At least 19 records

Dicyclohexylcarbodiimide-sensitive ATPase in Halobacterium saccharovorum

Membranes from Halobacterium saccharovorum contained a cryptic ATPase which required Mg(2+) or Mn(2+) and was activated by Triton X-100. The optimal pH for ATP hydrolysis was 9-10. ATP or GTP were hydrolyzed at the same rate while ITP, CTP, and UTP were hydrolyzed at about half that rate. The products of ATP hydrolysis were ADP and phosphate. The ATPase required high concentrations (3.5 M) of NaCl for maximum activity. ADP was a competitive inhibitor of the activity, with an apparent Ki of 50 micro-M. Dicyclohexylcarbodiimide (DCCD) inhibited ATP hydrolysis. The inhibition was marginal at the optimum pH of the enzyme. When the ATPase was preincubated with DCCD at varying pH values, but assayed at the optimal pH for activity, DCCD inhibition was observed to increase with increasing acidity of the preincubation medium. DCCD inhibition was also dependent on time of preincubation, and protein and DCCD concentrations. When preincubated at pH 6.0 for 4 h at a protein:DCCD ratio of 40 (w/w), ATPase activity was inhibited 90 percent.

Kristjansson, H.↗

ThermoBase: A Database of the Phylogeny and Physiology of Thermophilic and Hyperthermophilic Organisms

Thermophiles and hyperthermophiles are those organisms which grow at high temperature (> 40°C). The unusual properties of these organisms have received interest in multiple fields of biological research, and have found applications in biotechnology, especially in industrial processes. However, there are few listings of thermophilic and hyperthermophilic organisms and their relevant environmental and physiological data. Such repositories can be used to standardize definitions of thermophile and hyperthermophile limits and tolerances and would mitigate the need for extracting organism data from diverse literature sources across multiple, sometimes loosely related, research fields. Therefore, we have developed ThermoBase, a web-based and freely available database which currently houses comprehensive descriptions for 1238 thermophilic or hyperthermophilic organisms. ThermoBase reports taxonomic, metabolic, environmental, experimental, and physiological information in addition to literature resources. This includes parameters such as coupling ions for chemiosmosis, optimal pH and range, optimal temperature and range, optimal pressure, and optimal salinity. The database interface allows for search features and sorting of parameters. As such, it is the goal of ThermoBase to facilitate and expedite hypothesis generation, literature research, and understanding relating to thermophiles and hyperthermophiles within the scientific community in an accessible and centralized repository. ThermoBase is freely available online at the Astrobiology Habitable Environments Database (AHED; https://ahed.nasa.gov), at the Database Center for Life Science (TogoDB; http://togodb.org/db/thermobase), and in the S1 File.

Thermophiles↗

The Future of Astronaut Nutrition: Daily Production of Kefir Nutrient Packs in a Lunar Analog Mission

Astronaut nutrition will need to be supplemented with fresh nutrients and probiotics for long duration space travel. Kefir is a fermented milk beverage that provides probiotic bacteria, protein, calcium, and vitamin K. It has been historically cultured for thousands of years and is a modern-day health food. During the BioNutrients-2 experiment, one generation of kefir was cultured by astronauts on the ISS in fluorinated ethylene propylene (FEP) bags. This follow-on experiment aimed to test the hypothesis that kefir can be produced at ambient temperatures and safely passaged to make a series of nutrient packs in an analog environment. The Hawaiian Space Exploration Analog and Simulation (HI-SEAS) is a semi-controlled habitat located on the Mauna Loa volcano on the island of Hawaii at 2,500 m elevation. The recent EMMIHS (EuroMoonMars, International MoonBase Alliance, HI-SEAS) missions comprised of interdisciplinary, international crews for mock lunar missions. During a six-day mission in March 2024, crew members tested continuous passaging of kefir cultures (C-FIR commercial strain). After approximately 24 hours of growth, a portion of culture was used to seed the next generation of culture bags for four passages. A color board and pH indicator allowed the crew to easily determine when the culture had reached optimal pH. Laboratory analysis at NASA Ames Research Center showed that the pH met Kefir standards (<5). The yeast and lactic acid bacteria were in normal range and no contamination was detected in the final passage. By successfully accomplishing this experiment the team has demonstrated the ability to safely produce daily cultures of kefir in a microgravity applicable growth system.

Astronaut nutrition↗

Dissolution and reduction of magnetite by bacteria

Magnetite (Fe3O4) is an iron oxide of mixed oxidation state [Fe(II), Fe(III)] that contributes largely to geomagnetism and plays a significant role in diagenesis in marine and freshwater sediments. Magnetic data are the primary evidence for ocean floor spreading and accurate interpretation of the sedimentary magnetic record depends on an understanding of the conditions under which magnetite is stable. Though chemical reduction of magnetite by dissolved sulfide is well known, biological reduction has not been considered likely based upon thermodynamic considerations. This study shows that marine and freshwater strains of the bacterium Shewanella putrefaciens are capable of the rapid dissolution and reduction of magnetite, converting millimolar amounts to soluble Fe(II)in a few days at room temperature. Conditions under which magnetite reduction is optimal (pH 5-6, 22-37 degrees C) are consistent with an enzymatic process and not with simple chemical reduction. Magnetite reduction requires viable cells and cell contact, and it appears to be coupled to electron transport and growth. In a minimal medium with formate or lactate as the electron donor, more than 10 times the amount of magnetite was reduced over no carbon controls. These data suggest that magnetite reduction is coupled to carbon metabolism in S. putrefaciens. Bacterial reduction rates of magnetite are of the same order of magnitude as those estimated for reduction by sulfide. If such remobilization of magnetite occurs in nature, it could have a major impact on sediment magnetism and diagenesis.

Non-NASA Center↗

Thermococcus sulfurophilus sp. nov., a New Hyperthermophilic, Sulfur-Reducing Archaeon Isolated from Deep-Sea Hydrothermal Vent

A new hyperthermophilic, anaerobic, sulfur-reducing, organo-heterotrophic archaeon, strain OGL-20P, was isolated from "black smoker" chimney material at the Rainbow hydrothermal vent site in the Atlantic Ocean (36.2 N; 33.9 W). The cells of strain OGL-20P have irregular coccoid shape and are motile with a single flagellum. Growth occurs within pH range of 5.5-8.2 (optimal at pH 7.0-7.2), salinity range of 1-5% NaCl (optimal concentration 3% NaCl wt/vol), and temperature range of +55 C to +94 C (optimal growth at +83 C to +85 C). Strain OGL-20P is resistant to freezing (at -20 C). New isolate is strictly anaerobic with sulfur-type of respiration. A limited number of compounds are utilized as electron donors, including peptone, becto-tryptone, casamino-acids, and yeast extract but does not grow with separate amino acids. Sulfur and Iron can be used as electron acceptors; but not sulfate, sulfite, thiosulfate or nitrate. Strain OGL-20P is resistant to chloramphenicol, kanamycin, and gentamycin. Growth of str. OGL20P is inhibited by tetracyclin but not by Na2MoO4. The G+C content of DNA is 57.2 mol%. The 16S ribosomal RNA sequence analysis allows one to classify strain OGL-20P as a representative of a now species of Thermococcus genus. The name Thermococcus sulfurophilus op. nov., was suggested for the new isolate, type strain OGL-20P (sup T) (= ATCC BAA_394 (sup T) = DSM...(supT)).

Pikuta, Elena V.↗

Anaerovirgula multivorans gen. nov., sp. nov., a Novel Spore-Forming, Alkaliphilic Anaerobe Isolated from Owens Lake, California, USA

A novel, alkaliphilic, obligately anaerobic bacterium, strain SCAT, was isolated from mud sediments of a soda lake in California, USA. The rod-shaped cells were motile, Gram-positive, formed spores and were 0.4-0.5x2.5-5.0 micrometers in size. Growth occurred within the pH range 6.7-10.0 and was optimal at pH 8.5. The temperature range for growth was 10-45 degrees C, with optimal growth at 35 degrees C. NaCl was required for growth. Growth occurred at 0.5-9.0% (w/v) NaCl and was optimal at 1-2% (w/v). The novel isolate was a catalase-negative chemo-organoheterotroph that fermented sugars, proteolysis products, some organic and amino acids, glycerol, d-cellobiose and cellulose. It was also capable of growth by the Stickland reaction. Strain SCAT was sensitive to tetracycline, chloramphenicol, rifampicin and gentamicin, but it was resistant to ampicillin and kanamycin. The G+C content of the genomic DNA was 34.2 mol%. Major fatty acid components were C14:0, iso-C15:0, C16:1omega9c and C16:0. 16S rRNA gene sequence analysis of strain SCAT showed a similarity of approximately 97% with the type strains of Clostridium formicaceticum and Clostridium aceticum in clostridial cluster XI and a similarity of less than 94.2% to any other recognized Clostridium species and those of related genera in this cluster. Strain SCAT was clearly differentiated from C. formicaceticum and C. aceticum based on comparison of their phenotypic properties and fatty acid profiles, as well as low levels of DNA-DNA relatedness between strain SCAT and the type strains of these two species. Therefore, strain SCAT is considered to represent a novel species of a new genus, Anaerovirgula multivorans gen. nov., sp. nov., in clostridial cluster XI. The type strain is SCAT (=ATCC BAA-1084T=JCM 12857T=DSM 17722T=CIP 107910T).

Pikuta, Elena V.↗

Initial blood storage experiment

The design of the Initial Blood Storage Experiment (IBSE) was based upon a carefully controlled comparison between identical sets of human blood cell suspensions - red cells, white cell, and platelets - one set of which was transported aboard the Columbia on a 6 day 11 hour mission, and the other held on the ground. Both sets were carried inside stainless steel dewars within specially fabricated flight hardware. Individual bags of cell suspensions were randomly assigned with respect to ground vs orbit status, dewar chamber, and specific location within the dewar. To foster optimal preservation, each cell type was held under specific optimal conditions of pH, ionic strength, solute concentration, gas tension, and temperature. An added variable in this initial experiment was provided by the use of three different polymer/plasticizer formulations for the sealed bags which held the blood cells. At termination of the experiment, aliquots of the suspensions, identified only by code, were distributed to be assayed. Assays were selected to constitute a broad survey of cellular properties and thereby maximize the chances of detection of gravitational effects. A total of 74 different outcome measurements were reported for statistical analysis. When the measurements were completed, the results were entered into the IBSE data base, at which time the data were matched with the original blood bag numbers to determine their status with respect to polymer/plasticizer type, orbit status (orbit or ground), and storage position within the experimental hardware. The data were studied by analysis of variance. Initially, type of bag and orbital status were main factors; later more detailed analyses were made on specific issues such as position in the hardware and specific plastic. If the analysis of variance indicated a statistical significance at the 5 percent level the corresponding p-value was reported.

Surgenor, Douglas MACN.↗

The Effect of Temperature and Solution pH on Tetragonal Lysozyme Nucleation Kinetics

Part of the challenge of macromolecular crystal growth for structure determination is obtaining an appropriate number of crystals with a crystal volume suitable for x-ray analysis. In this respect an understanding of the effect of solution conditions on macromolecule nucleation rates is advantageous. This study investigated the effects of supersaturation, temperature and pH on the nucleation rate of tetragonal lysozyme crystals. Batch crystallization plates were prepared at given solution concentrations and incubated at set temperatures over one week. The number of crystals per well with their size and axial ratios were recorded and correlated with solution conditions, Duplicate experiments indicate the reproducibility of the technique, Crystal numbers were found to increase with increasing supersaturation and temperature. The most significant variable however, was pH, where crystal numbers changed by two orders of magnitude over the pH range 4.0 to 5.2. Crystal size varied also with solution conditions, with the largest crystals being obtained at pH 5.2. Having optimized the crystallization conditions, a batch of crystals were prepared under exactly the same conditions and fifty of these crystals were analyzed by x-ray techniques. The results indicate that even under the same crystallization conditions, a marked variation in crystal properties exists.

Judge, Russell A.↗

The Effect of Temperature and Solution pH on the Nucleation of Tetragonal Lysozyme Crystals

Part of the challenge of macromolecular crystal growth for structure determination is obtaining crystals with a volume suitable for x-ray analysis. In this respect an understanding of the effect of solution conditions on macromolecule nucleation rates is advantageous. This study investigated the effects of supersaturation, temperature, and pH on the nucleation rate of tetragonal lysozyme crystals. Batch crystallization plates were prepared at given solution concentrations and incubated at set temperatures over 1 week. The number of crystals per well with their size and axial ratios were recorded and correlated with solution conditions. Crystal numbers were found to increase with increasing supersaturation and temperature. The most significant variable, however, was pH; crystal numbers changed by two orders of magnitude over the pH range 4.0-5.2. Crystal size also varied with solution conditions, with the largest crystals obtained at pH 5.2. Having optimized the crystallization conditions, we prepared a batch of crystals under the same initial conditions, and 50 of these crystals were analyzed by x-ray diffraction techniques. The results indicate that even under the same crystallization conditions, a marked variation in crystal properties exists.

Judge, Russell A.↗

The catalysis of nucleotide polymerization by compounds of divalent lead

The nonenzymatic, nontemplate catalysis of nucleotide polymerization by Pb(2+) ions, a possible prebiotic catalyst, is reported. Adenosine and uridine phosphoimidazoles were reacted in buffered solutions of lead salts and products were analyzed by means of paper chromatography and electrophoresis. In the presence of Pb(2+) ion at pH 8.0 and 7.0 the reaction is found to progress rapidly with excellent yields of oligomers, with optimal yields observed at pH 8.0. Little temperature dependence in the range 0 to 30 C is observed, however hydrolysis of the reaction products is minimal when the reaction is carried out at 0 C. Results show that the yield of oligomers is insensitive to mixing or the source of lead ions, indicating that naturally occurring minerals or precipitates could be a source of Pb(2+) ions under prebiotic conditions.

Sleeper, H. L.↗

RNA-Cleaving DNA Enzymes with Altered Regio- or Enantioselectivity

In vitro evolution methods were used to obtain DNA enzymes that cleave either a 2',5' - phosphodiester following a wibonucleotide or a 3',5' -phosphodiester following an L-ribonucleotide. Both enzymes can operate in an intermolecular reaction format with multiple turnover. The DNA enzyme that cleaves a 2',5' -phosphodiester exhibits a k(sub cat) of approx. 0.01/ min and catalytic efficiency, k(sub cat)/k(sub m) of approx. 10(exp 5)/ M min. The enzyme that cleaves an L-ribonudeotide is about 10-fold slower and has a catalytic efficiency of approx. 4 x 10(exp 5)/ M min. Both enzymes require a divalent metal cation for their activity and have optimal catalytic rate at pH 7-8 and 35-50 C. In a comparison of each enzyme s activity with either its corresponding substrate that contains an unnatural ribonudeotide or a substrate that instead contains a standard ribonucleotide, the 2',5' -phosphodiester-deaving DNA enzyme exhibited a regioselectivity of 6000- fold, while the L-ribonucleotide-cleaving DNA enzyme exhibited an enantioselectivity of 50-fold. These molecules demonstrate how in vitro evolution can be used to obtain regio- and enantioselective catalysts that exhibit specificities for nonnatural analogues of biological compounds.

Ordoukhanian, Phillip↗

A DNA enzyme with N-glycosylase activity

In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 10(6)-fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an N-glycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.

Non-NASA Center↗

Basal glycogenolysis in mouse skeletal muscle: in vitro model predicts in vivo fluxes

A previously published mammalian kinetic model of skeletal muscle glycogenolysis, consisting of literature in vitro parameters, was modified by substituting mouse specific Vmax values. The model demonstrates that glycogen breakdown to lactate is under ATPase control. Our criteria to test whether in vitro parameters could reproduce in vivo dynamics was the ability of the model to fit phosphocreatine (PCr) and inorganic phosphate (Pi) dynamic NMR data from ischemic basal mouse hindlimbs and predict biochemically-assayed lactate concentrations. Fitting was accomplished by optimizing four parameters--the ATPase rate coefficient, fraction of activated glycogen phosphorylase, and the equilibrium constants of creatine kinase and adenylate kinase (due to the absence of pH in the model). The optimized parameter values were physiologically reasonable, the resultant model fit the [PCr] and [Pi] timecourses well, and the model predicted the final measured lactate concentration. This result demonstrates that additional features of in vivo enzyme binding are not necessary for quantitative description of glycogenolytic dynamics.

Non-NASA Center↗

Addendum: Development of a preprototype times wastewater recovery subsystem

The results of the second generation operational improvements and the TIMES (Thermoelectric Integrated Membrane Evaporation Subsystem) 2 study are covered. Areas covered in the second generation operational improvements are improved temperature control, water quality improvements, subsytem operational improvements, solid handling improvements, wastewater pretreatment optimization, and membrane rejuvenation concepts. The task for the TIMES 2 study are thermoelectric regenerator improvement, recycle loop pH operational criteria, recycle loop component optimization, and hollow fiber membrane evaporator improvement. Results are presented and conclusions are drawn from both studies.

Dehner, G. F.↗

Comparison of the surface charge behavior of commercial silicon nitride and silicon carbide powders

The adsorption and desorption of protons from aqueous solution onto the surfaces of a variety of commercial silicon carbide and silicon nitride powders has been examined using a surface titration methodology. This method provides information on some colloidal characteristics, such as the point of zero charge (pzc) and the variation of proton adsorption with dispersion pH, useful for the prediction of optimal ceramic-processing conditions. Qualitatively, the magnitude of the proton adsorption from solution reveals small differences among all of the materials studied. However, the results show that the pzc for the various silicon nitride powders is affected by the powder synthesis route. Complementary investigations have shown that milling can also act to shift the pzc exhibited by silicon nitride powder. Also, studies of the role of the electrolyte in the development of surface charge have indicated no evidence of specific adsorption of ammonium ion on either silicon nitride or silicon carbide powders.

Whitman, Pamela K.↗

The Effect of Solution Parameters on Lysozyme Nucleation Rates and Crystal Quality

In the pursuit of strongly diffracting high quality macromolecule crystals of suitable volume, this study investigates how the formation of macromolecules in solution and their growth characteristics effect crystal volume and diffracting quality. We systematically investigated the effect of solution conditions on lysozyme nucleation rates and the volume of crystals produced. Batch crystallization plates were used in combination with a video microscope system to measure nucleation rates and crystal volume. As expected from classical nucleation theory, crystal numbers were found to increase with increases in temperature and supersaturation. Small changes in solution pH, at constant supersaturation values were found, however, to dramatically effect the number of crystals nucleated in the wells varying from 1000s to 10s in the pH range 4.0 to 5.2. Having optimized the conditions required to produce an appropriate number of crystals of a suitable volume for X-ray analysis, a large number of uniform crystals were produced under exactly the same conditions. In the X-ray analysis of more than 50 such crystals there was found a wide variation in crystal lattice parameters and data quality. The variation in X-ray quality crystal samples is thought to be related to the growth rate variation caused by growth rate dispersion seen in lysozyme crystal growth experiments.

Judge, R. A.↗

Psychrotolerant Anaerobes from Lake Podprudnoe, Antarctica and Penguin Spheniscus demersus Colony, South Africa

The study of a sample collected from a wind-made ice sculpture near Lake Podprudnoe, Antarctica led to the isolation of the psychrotolerant strain ISLP-3. Cells of the new isolate are vibrio-shaped that measure 0.5 x 1.0-3.0 micron in size. Growth occurs within the temperature range 5-35 C with the optimum at 22 C. Salinity range for growth is 0-2 % NaCl with the optimum at 0.25 %. The new isolate grows within a pH range from 6.0 to 9.5 with the optimum at 7.5. Strain ISLP-3 is saccharolytic, growing on the following substrates: D-glucose, D-ribose, D-fructose, D-arabinose, maltose, sucrose, D-trehalose, D-mannose, D-cellobiose, lactose, starch, chitin, triethylamine, N-acetylglucosamine, and urea. The best growth occurred on D-cellobiose. An environmental sample of pond water near a colony of the endemic species of African penguins, Spheniscus demersus, was collected in February 2008 and delivered directly to the Astrobiology laboratory at NSSTC. The microbiological study of this sample led to the isolation of two psychrotolerant strains ARHSd-7G and ARHSd-9G. Both strains are strictly anaerobic bacteria and are able to grow at high pH and low temperatures. The cells of strain ARHSd-7G are motile, vibrio-shaped, spore-forming cells. Optimal growth of this strain occurs at 30 C, 3 % NaCl, and pH 8.9. The isolate ARHSd-7G combines sugarlytic and proteolytic metabolisms, growing on some proteolysis products including peptone and yeast extract and a number of sugars. The second isolate, ARHSd-9G, exhibits thin, elongated rods that measure 0.4 x 3-5 micron. The cells are motile and spore-forming. Optimal growth of strain ARHSd-9G occurs at 30 C, 1.75 % NaCl, and pH 8.5. The strain ARHSd-9G is sugarlytic, growing well on substrates such as D-glucose, sucrose, D-cellobiose, maltose, fructose, D-mannose, and trehalose (the only exception is positive growth on yeast extract). In this report, the physiological and morphological characteristics of the novel psychrotolerant, alkaliphilic, and neutrophilic isolates from the Antarctica 2008 expedition will be discussed.

Guisler, Melissa↗