The viking carbon assimilation experiments: interim report.

A synthesis of organic matter from atmospheric carbon monoxide or carbon dioxide, or both, appears to take place in the surface material of Mars at a low rate. The synthesis appears to be thermolabile and to be inhibited by moisture.

Sterile soil from Antarctica: organic analysis.

Soils from the dry-valley region of Antarctica can be sterile by the usual microbiological criteria and yet contain significant amounts of organic carbon. Examination of one such soil shows that the organic material is finely divided anthracite coal. These findings have significant implications for the biological exploration of Mars.

The viking biological investigation: preliminary results.

Three different types of biological experiments on samples of martian surface material ("soil") were conducted inside the Viking lander. In the carbon assimilation or pyrolytic release experiment, (14)CO(2) and (14)CO were exposed to soil in the presence of light. A small amount of gas was found to be converted into organic material. Heat treatment of a duplicate sample prevented such conversion. In the gas exchange experiment, soil was first humidified (exposed to water vapor) for 6 sols and then wet with a complex aqueous solution of metabolites. The gas above the soil was monitored by gas chromatography. A substantial amount of O(2) was detected in the first chromatogram taken 2.8 hours after humidification. Subsequent analyses revealed that significant increases in CO(2) and only small changes in N(2) had also occurred. In the labeled release experiment, soil was moistened with a solution containing several (14)C-labeled organic compounds. A substantial evolution of radioactive gas was registered but did not occur with a duplicate heat-treated sample. Alternative chemical and biological interpretations are possible for these preliminary data. The experiments are still in process, and these results so far do not allow a decision regarding the existence of life on the plonet Mars.

Carbon monoxide metabolism in roadside soils.

Air-dried soils which were equilibrated under relative humidities greater than 93% or moistened with liquid water showed marked increases in their capacities to oxidize CO to CO(2). Liquid water addition in excess of saturation resulted in lower CO oxidation rates, reflecting the limited diffusion of CO through the aqueous phase. After 35 days' storage under 100% relative humidity, the capacity for CO oxidation decreased to 21% of the value observed with a freshly collected sample. Incubation of this stored soil under an atmosphere containing 200 ppm of CO (250 mg/m) for 21 days resulted in a sevenfold increase in CO oxidation. A correlation was noted between the CO oxidative activity and the history of previous exposure of soils to high ambient levels of CO. The organisms responsible for CO oxidation apparently comprise a small fraction of the microbial population in the soils. With a roadside soil the oxidation of CO provided the driving force for the assimilation of CO(2). The stoichiometry of the oxidative and assimilatory reactions in soil was in the range of values reported from laboratory studies with CO chemoautotrophs (carboxydobacteria). It is proposed that the population and activity of CO-oxidizing microorganisms increase in response to increasing levels of CO in the environment.

Purification and reversible inactivation of the isocitrate dehydrogenase from an obligate halophile.

The nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase of Halobacterium cutirubrum is rapidly inactivated at low NaCl levels. As much as 75% of the initial activity can be restored by dialyzing the inactive enzyme against 4 m NaCl. A mixture of 4 mm isocitrate and 10 mm MnCl(2) gives the same protection as 4 m NaCl but does not replace the NaCl requirement for reactivation. The reactivated and native enzymes have identical sedimentation rates on sucrose gradients, electrophoretic mobilities on polyacrylamide gels, and elution rates from Sephadex G-200. However, there are distinct differences between the active and inactive forms of the enzyme. Compared with the active enzyme, the inactive protein has a lower sedimentation rate, a lower electrophoretic mobility, and a faster elution rate from Sephadex. These differences indicate that inactivation causes a major conformational change in the protein. Presumably, the removal of NaCl permits the enzyme to expand into a less dense, inactive form. The isocitrate dehydrogenase was purified 69-fold by a procedure involving the following steps. When the enzyme is selectively protected with isocitrate and MnCl(2) at low ionic strength, most of the contaminating proteins are precipitated with (NH(4))(2)SO(4) at 0.9 saturation. The enzyme in the supernatant fluid is then inactivated at low NaCl levels, precipitated with 0.5 saturated (NH(4))(2)SO(4), and reactivated with 4 m NaCl. Minor impurities are removed by gel filtration on Sephadex G-200. The resulting preparation is more than 95% pure as judged by disc electrophoresis.

Regulation of glutamine synthetase. V. Partial purification and properties of glutamine synthetase from Bacillus licheniformis.

The glutamine synthetase of Bacillus licheniformis has been obtained at about 15% purity. Sucrose gradient centrifugation gave a molecular weight value of approximately 612,000. Both l- and d-glutamate can be utilized as substrates in the biosynthetic reaction, although the l isomer was five times more active. The requirement for adenosine triphosphate (ATP) can be partially replaced by guanosine or inosine triphosphates, but not by cytidine or uridine triphosphates. The Mn(++) was required for activity, and the requirement cannot be satisfied with Mg(++). Maximal activity of the biosynthetic reaction was observed when ATP and Mn(++) were present in equimolar amounts. An excess of either reactant gave less activity. However, other purine and pyrimidine nucleotides, when added in combination with ATP, can partially substitute for ATP in attaining the equimolar ratio of nucleotide to Mn(++). A complex of ATP and Mn(++) is the preferred form of substrate. The B. licheniformis enzyme catalyzes the glutamyl transfer reaction but at a much slower rate than the Escherichia coli glutamine synthetase. Either adenosine diphosphate (ADP) or ATP can activate the glutamotransferase, although ADP is more active.

Regulation of glutamine synthetase. VI. Interactions of inhibitors for Bacillus licheniformis glutamine synthetase.

The relationships of five feedback inhibitors for the Bacillus licheniformis glutamine synthetase were investigated. The inhibitors were distinguishable by differences in their competitive relationship for the substrates of the enzyme. Mixtures of l-glutamine and adenosine-5'-monophosphate (AMP) or histidine and AMP caused synergistic inhibition of glutamine synthesis. Histidine, alanine, and glycine acted antagonistically toward the l-glutamine inhibition. Alanine acted antagonistically toward the glycine and histidine inhibitions. Independence of inhibitory action was observed with the other pairs of effectors. Possible mechanisms by which the inhibitors may interact to control glutamine synthesis are discussed. The low rate of catalysis of the glutamyl transfer reaction by the B. licheniformis glutamine synthetase can be attributed to the fact that l-glutamine serves both as a substrate and an inhibitor for the enzyme. Effectors which act antagonistically toward the l-glutamine inhibition stimulated glutamotransferase activity. The stimulation was not observed when d-glutamine was used as substrate for the glutamyl transfer reaction.

Nature of the inactivation of the isocitrate dehydrogenase from an obligate halophile.

The nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase (ICDH) of Halobacterium cutirubrum is rapidly inactivated at low NaCl levels. From sucrose gradient analysis, it was estimated that the active ICDH has an S(20,w) of 5.3 and a molecular weight of 75,000. The inactivation by removal of NaCl causes an unfolding of the protein yielding a less-compact conformer with an S(20,w) of 2.0. This inactivation apparently causes internal sulfhydryl groups to be exposed. Over 90% of the initial activity can be restored by dialyzing the inactivated ICDH against 4 m NaCl, provided that the exposed sulfhydryl groups are protected with dithiothreitol. The ICDH is permanently inactivated when the sulfhydryl groups are oxidized or alkylated. The alkylation of the inactive ICDH was demonstrated by treatment with (14)C-N-ethyl maleimide. Sucrose gradient analysis showed that (14)C was bound to a protein with sedimentation properties identical to that of reversibly inactivated ICDH, i.e., an S(20,w) of 2.0. Much less (14)C was bound when active ICDH was treated with (14)C-N-ethyl maleimide. The H. cutirubrum ICDH resembles other bacterial isocitrate dehydrogenases in being susceptible to concerted feedback inhibition by oxalacetate and glyoxalate.

Applicability of radiotracer methods of measuring(14)CO 2 assimilation for determining microbial activity in soil including a newin situ method.

The applicability of two methods (pyrolysis gas chromatography and acidification-wet oxidation) for determining(14)CO2 incorporation into soil microorganisms was investigated. Both methods were able to distinguish biologically incorporated(14)C from abiotically adsorbed(14)C, but to varying degrees, there being a larger carryover of abiotic(14)C into the organic fraction and a higher percentage of assimilated(14)C in the organic fraction with the wet oxidation method. Using(14)C assimilation as a measure, it was possible to determine microbial activities in soils of diverse properties under a variety of conditions, including polar soils under harsh environmental conditions. Both light and dark(14)CO2 fixation was measurable.(14)CO2 assimilation was not always proportional to the enumerable microorganisms. A new design for measurement of microbial activityin situ enabled measurement of total C influx (primary productivity) into soils with minimal perturbation to the natural soil ecosystem.

Regulation of glutamine synthetase. II. Patterns of feedback inhibition in microorganisms.

The feedback inhibition of glutamine synthetase was investigated by use of partially purified enzyme preparations from Salmonella typhimurium, Micrococcus sodonensis, Pseudomonas fluorescens, Bacillus cereus, Bacillus licheniformis, Clostridium pasteurianum, Rhodospirillum rubrum, Neurospora crassa, Candida utilis, and Chlorella pyrenoidosa. Inhibition analyses indicated that the enzyme of each organism can be effectively regulated with mixtures of end products from the diverse pathways of glutamine metabolism. When tested individually, tryptophan, histidine, alanine, glycine, glutamine, 5'-adenylate (AMP), cytidine-5'-triphosphate, carbamyl phosphate, and glucosamine-6-phosphate gave limited inhibition. In most cases, the inhibitors were independent in their action, and cumulative degrees of inhibition were obtained with mixtures of these end products. In contrast, with the glutamine synthetases of the two Bacillus species, the simultaneous presence of AMP and histidine (or AMP and glutamine) gave inhibition greater than the sum of the amounts of inhibition caused by either inhibitor alone. Also, alanine and carbamyl phosphate acted synergistically to inhibit the enzyme from N. crassa. The remarkable similarity in the overall patterns of end-product inhibition observed with the enzymes from different sources indicates that these diverse organisms have evolved comparable mechanisms for the regulation of glutamine metabolism. Nevertheless, the enzymes from different sources do differ significantly in their physical and catalytic properties, as was demonstrated by dissimilarities in their purification behaviors, specificity for nucleotide substrate, ability to catalyze the glutamyl transfer reaction, and ability to utilize Mn(++) and Mg(++) as activators for the biosynthetic reaction.

Bacteriorhodopsin formation in Halobacterium halobium.

Systematic examinations were made of factors influencing bacteriorhodopsin formation during the growth of Halobacterium halobium. Light-induced adenosine triphosphate (ATP) production and [14C]proline uptake were used as measures of functional ability of the purple membrane. Maximum bacteriorhodopsin formation occurred under growth conditions of illumination and limited aeration. The purple membrane -ATP production system did not confer an appreciable growth advantage. Growth in the dark or with adequate aeration partially suppressed bacteriorhodopsin formation and the effects were additive. Nicotine effectively inhibited bacteriorhodopsin formation. A rapid synthesis of functional pigment occurred when washed suspensions of cells which had been grown under illumination with nicotine present were incubated under dark, aerobic conditions. The alleviation of this nicotine inhibition was not blocked by chloramphenicol or bacitracin. Bacteriorhodopsin formation was negligible when washed suspensions of cells from dark, limited aeration or light, adequate aeration cultures were incubated in the light with limited aeration. A nutritionally complex medium was needed to elicit appreciable bacteriorhodopsin formation by the cells from the dark or adequately aerated cultures. Bacitracin partially inhibited this bacteriorhodopsin formation by cells form the light, adequately aerated culture.

Energy coupling in the active transport of proline and glutamate by the photosynthetic halophile Ectothiorhodospira halophila.

When illuminated, washed cell suspensions of Ectothiorhodospira halophila carry out a concentrative uptake of glutamate or proline. Dark-exposed cells accumulate glutamate but not proline. Proline transport was strongly inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP), a proton permeant that uncouples photophosphorylation, and by 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO), an inhibitor of photosynthetic electron transport. A stimulation of proline uptake was effected by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane adenosine triphosphatase (ATPase) which catalyzes the phosphorylation. These findings suggest that the driving force for proline transport is the proton-motive force established during photosynthetic electron transport. Glutamate uptake in the light was inhibited by CCCP and HQNO, but to a lesser extent than was the proline system. DCCD caused a mild inhibition of glutamate uptake in the light, but strongly inhibited the uptake by dark-exposed cells. CCCP strongly inhibited glutamate uptake in the dark. The light-dependent transport of glutamate is apparently driven by the proton-motive force established during photosynthetic electron transport. Hydrolysis of adenosine triphosphate (ATP) by membrane ATPase apparently establishes the proton-motive force to drive the light-independent transport. These conclusions were supported by demonstrating that light- or dark-exposed cells accumulate [3H]triphenylmethylphosphonium, a lipid-soluble cation. Several lines of indirect evidence indicated that the proline system required higher levels of energy than did the glutamate system(s). This could explain why ATP hydrolysis does not drive proline transport in the dark. Membrane vesicles were prepared by the sonic treatment of E. halophila spheroplasts. The vesicles contained active systems for the uptake of proline and glutamate.

Metal-induced inhibition of anaerobic metabolism of volatile fatty acids and hydrogen.

The effects of copper (Cu), chromium (Cr), cadmium (Cd), lead (Pb) and zinc (Zn) on the biotransformation of organic acids (acetate, propionate and butyrate) and H2 were assessed in serum-bottle microcosms. Experiments were performed over a range of metal concentrations (20-200 mg/l) using biomass from an anaerobic bioreactor fed continuously with ethanol distillery waste as inoculum. In general, the added metals inhibited the biotransformation of organic acids with increasing metal concentration. However, the extent of inhibition varied for the different biotransformations and for the different metals tested. For example, the concentration of CuCl2 effecting a 50% reduction in the rate constant for biotransformation of acetate, propionate and butyrate was 60, 75 and 30 mg/l, respectively. Cu and Cr (VI) were the most inhibitory metals in organic acid transformation, whereas Pb was the least toxic. The rate of biotransformation of acetate was reduced by half at Cu and Cr concentrations of 60 and 40 mg/l respectively, whereas Cd, Pb, and Zn concentrations of 160 to 200 mg/l had little effect. The activities of hydrogenotrophic methanogens were much less affected by the same metals and metal concentrations.

Ultraviolet-gas phase and -photocatalytic synthesis from CO and NH3.

The major photoproduct obtained on irradiation of gaseous NH3 and CO mixtures is ammonium cyanate; lesser amounts of urea, biurea, biuret semi-carbazide, formamide and cyanide were observed. The formation of the major gas phase photolysis product may be rationalized by the following reaction sequence: (see article). Urea is probably formed from NH4NCO in a thermal reaction while formamide may result from the disproportionation of NH2CO. Photocatalytic syntheses of 14C-urea, -formamide, and -formadehyde are effected by irradiation of 14CO and NH3 in the presence of Vycor, silica gel, or volcanic ash shale surfaces. These syntheses are catalyzed by ultraviolet wavelengths longer than those absorbed by the gaseous reactants. The syntheses are also effected when the surface material is first irradiated in the presence of CO followed by a dark incubation with NH3. Apparently, the initiating step is a light dependent formation of a reactive form of CO on the surface. A discussion is given on the possible contribution of these reactions to the abiotic synthesis of organic nitrogen compounds on Mars, on the primitive Earth and in interstellar space.

Photocatalytic Production of Organic Compounds from CO and H(2)O in a Simulated Martian Atmosphere.

[(14)C]CO(2) and [(14)C]organic compounds are formed when a mixture of [(14)C]CO and water vapor diluted in [(12)C]CO(2) or N(2) is irradiated with ultraviolet light in the presence of soil or pulverized vycor substratum. The [(14)C]CO(2) is recoverable from the gas phase, the [(14)C]organic products from the substratum. Three organic products have been tentatively identified as formaldehyde, acetaldehyde, and glycolic acid. The relative yields of [(14)C]CO(2) and [(14)C]organics are wavelength- and surface-dependent. Conversion of CO to CO(2) occurs primarily at wavelengths shorter than 2000 A, apparently involves the photolysis of water, and is inhibited by increasing amounts of vycor substratum. Organic formation occurs over a broad spectral ranger below 3000 A and increases with increasing amounts of substratum. It is suggested that organic synthesis results from adsorption of CO and H(2)O on surfaces, with excitation of one or both molecules occurring at wavelengths longer than those absorbed by the free gases. This process may occur on Mars and may have been important on the primitive earth.

Energy coupling in the active transport of amino acids by bacteriohodopsin-containing cells of Halobacterium holobium.

Growth of Halobacterium halobium under illumination with limiting aeration induces bacteriorhodopsin formation and renders the cells capable of photophosphorylation. Cells depleted of endogenous reserves by a starvation treatment were used to investigate the means by which energy is coupled to the active transport of [14C]proline, -leucine, and -histidine. Proline was readily accumulated by irradiated cells under anaerobiosis even when the photophosphorylation was abolished by the adenosine triphosphatase inhibitor N,N'-dicyclohexylcarbodimiide (DCCD). The uptake of proline in the dark was limited except when the cells were allowed to accumulate adenosine 5'-triphosphate (ATP) by prior light exposure or by the oxidation of glycerol. DCCD inhibited this dark uptake. These findings essentially support Mitchell's chemiosmotic theory of active transport. The driving force is apparently the proton-motive force developed when protons are extruded from irradiated bacteriorhodopsin or by the dydrolysis of ATP by membrane adenosine triphosphatase. Carbonylcyanide m-chlorophenylhydrazone (CCCP), a proton permeant known to abolish membrane potential, was a strong inhibitor of proline uptake. Leucine transport was also apparently driven by proton-motive force, although its kinetic properties differed from the proline system. Histidine transport is apparently not a chemiosmotic system. Dark- or light-exposed cells show comparable initial rats of histidine uptake, and these processes were only partially inhibited by DCCD or CCCP. The histidine system apparently does not utilize ATP per se since comparable rates of uptake were exhibited by cells of differing intracellular ATP levels. Irradiated cells did effect a greater total accumulation of histidine than dark-exposed cells. These findings suggest that ATP is needed for sustained transport.

Measurement of CO(2) Assimilation in Soils: an Experiment for the Biological Exploration of Mars.

A method is described for the measurement of CO(2) assimilation by microorganisms in soils. A determination involves exposing soil to CO(2), pyrolyzing the exposed soil, trapping the organic pyrolysis products on a column of firebrick coated with CuO, combusting the trapped organics by heating, and measuring the radioactivity in the CO(2) produced in the combustion. The detection of significant levels of C in the trapped organic fraction appears to be an unambiguous indication of biological activity. The CO(2) which is adsorbed or exchanged into soils by nonbiological processes does not interfere. The method easily detects the CO(2) fixed by 10 to 10 algae after light exposure for 3 to 24 hr. Assimilation of C is also demonstrable in dark-exposed soils containing 10 to 10 heterotrophic bacteria. Possible applications of the method in the biological exploration of Mars are discussed.