Thiosulfate- and sulfide-dependent pyridine nucleotide reduction and gluconeogenesis in intact Thiobacillus neapolitanus.

Nicotinamide adenine dinucleotide phosphate (reduced form) is formed more rapidly after the addition of thiosulfate to suspensions of intact Thiobacillus neapolitanus in the absence of CO(2) than nicotinamide adenine dinucleotide (reduced form). Measurement of acid-stable metabolites shows this phenomenon to be the result of rapid reoxidation of nicotinamide adenine dinucleotide (reduced form) by 3-phosphoglyceric acid and other oxidized intermediates, which are converted to triose and hexose phosphates, and that, in reality, the rate of nicotinamide adenine dinucleotide (oxidized form) reduction exceeds that of nicotinamide adenine dinucleotide phosphate (oxidized form) by approximately 4.5-fold. The overall rate of pyridine nucleotide reduction by thiosulfate (264 nmol per min per mg of protein) is in excess of that rate needed to sustain growth. Pyridine nucleotide reduction, adenosine triphosphate synthesis, and carbohydrate synthesis are prevented by the uncoupler m-Cl-Carbonylcyanide phenylhydrazone. Sodium amytal inhibits pyridine nucleotide reduction and carbohydrate synthesis are prevented by the uncoupler m-Cl-carbonylcyanide observations are reproduced when sulfide serves as the substrate. The rate of pyridine nucleotide anaerobic reduction with endogenous substrates or thiosulfate is less than 1% of the aerobic rate with thiosulfate. We conclude that the principal, if not the only, pathway of pyridine nucleotide reduction proceeds through an energy-dependent and amytal-sensitive step when either thiosulfate or sulfide is used as the substrate.

Antarctica as a Martian model.

Previous investigators have reported that the microbiological population of the glacier free valleys of southern Victorialand, Antarctica, is sparse, and that from about 10% of the soil samples examined no micro-organisms could be cultivated. Since these areas are assumed to be more favorable to the growth of terrestrial organisms than any Martian environment, the previous authors concluded that the probability of terrestrial organisms growing on Mars would therefore be so small that sterilization standards could be relaxed by many orders of magnitude. The unsuitability of the Antarctic environment to the multiplication of terrestrial micro-organisms was ascribed by them to limiting amounts of water. We have carried out a survey of a variety of environments in the dry valleys, ranging from mountain crests to valley floors. The main purpose of our investigation was the determination of active microbial multiplication in the soil. A series of techniques was employed which permitted the detection of bacterial growth in situ. All evidence points to an active growth of micro-organisms in the Antarctic soil in all locations which we examined. The measurements were supported by electron micrographs of soil films which showed colonial growth covering soil particles. These findings suggest that Antarctica does not serve as a useful model for the Martian environment in evaluating quarantine standards.

Biological instrumentation for the Viking 1975 mission to Mars.

A brief introduction is given on why Mars is of interest from a biological point of view, along with an overview of the Viking 1975 mission. Details are given about the four biology instruments aboard the spacecraft and the experiments for which they are to be used. These are: the carbon assimilation experiment to determine whether the soil is biologically active, by incubation in presence of 14C-labelled CO and CO2 (known to be present in the Martian atmosphere); the label release experiment to detect metabolic activity by the release of radioactive CO2, from 14C-labelled simple organic substrates; the gas exchange experiment to detect biological activity by repeated gas chromatography analysis of soil samples; the light scattering experiment, where increase of scattering and decrease of light transmission would indicate the growth of organisms. Examples are given of data obtained with terrestrial soils in these experiments.

Growth Inhibition in Thiobacillus neapolitanus by Histidine, Methionine, Phenylalanine, and Threonine.

Thiobacillus neapolitanus, a strict chemoautotroph, is sensitive to the addition of 10(-4)m methionine, histidine, threonine, or phenylalanine to the thiosulfate medium on which it grows. When histidine, threonine, or phenylalanine are added at the time of inoculation, spontaneous mutants tolerant to the three amino acids are selected. These mutants appear to result from a single genetic change; of 18 independently isolated histidine-tolerant mutants, all are also tolerant to phenylalanine and threonine. The uptake of (14)C-phenylalanine into exponentially growing cells of one such mutant is negligible in contrast with the uptake observed in the phenylalanine-sensitive parent. The addition of methionine to the medium slows growth, but spontaneous mutants are not selected. Inhibition of growth by these amino acids is observed only under conditions of amino acid imbalance; the addition of an equimolar mixture of 16 amino acids, in which each component is present at a concentration of 10(-3)m, causes no inhibition. Histidine and threonine inhibition may be released by equimolar amounts of any one of seven amino acids: serine, alanine, glycine, leucine, valine, tryptophan, or tyrosine; histidine inhibition is also released by isoleucine, and threonine inhibition by methionine. None of the inhibiting amino acids inhibits oxidation of thiosulfate in cell suspensions. A group of hexoses, pentoses, and Krebs cycle intermediates were tested for inhibition of growth or release of inhibition by histidine, phenylalanine, or threonine, but no effects, either inhibition or relief of inhibition, were found.

Yield coefficients of Thiobacillus neapolitanus in continuous culture.

Thiobacillus neapolitanus, when grown in continuous culture with thiosulfate limiting growth, possessed an apparent maximal molar growth yield of 8.0 g (dry weight) per mole of thiosulfate. The substrate requirement for energy of maintenance was the highest yet reported, amounting to 21.8 mmoles of thiosulfate per g per hr. The molar growth yield, corrected for this maintenance energy requirement, was 13.9 g (dry weight) per mole of thiosulfate. It was concluded that substrate-level phosphorylation during sulfite oxidation accounted for about 45% of the adenosine triphosphate (ATP) requirement for CO(2) assimilation and maintenance during growth on limiting thiosulfate, that three sites of energy conservation exist in the electron-transport chain terminating in oxygen, and that 7.8 moles of ATP are required to fix and assimilate 1 mole of CO(2) into cell material.