On methods of detection of extraterrestrial life.

New methods have been developed for detecting microbial growth from the microflora of desert soils. The first is a polarimetric method in which the fall in optical activity due to assimilation of D-glucose is followed. Detectable changes with desert soils were seen in a few hours, and the method can be employed with small amounts of material. The second method is the release of heat from metabolizable substrates as measured by a microcalorimeter. In the presence of glucose a characteristic response from desert soils was found within 24 hours.

[Dynamics of the formation of extracellular protease and the ATP pool in a thermophilic strain of Bacillus brevis]. / Dinamika obrazovaniia vnekletochnoi proteazy i ATF-pul termofil'nogo shtamma Bacillus brevis

The dynamics of varying ATP concentration was studied in the cells of the thermophilic strain of Bacillus brevis 224 synthesizing a neutral exoprotease. Some irregularities during the primary and secondary growth of the culture corresponded to the changes in the amount of ATP. A sharp decrease (by a factor of ca. 100) in the level of ATP pool was detected in the bacterial cells after 2.5 hours of their exponential growth; their growth ceased and the synthesis of protease was depressed. A decrease in the rate of the enzyme synthesis coincides with an increase in the concentration of ATP in the cells.

The influence of ultra-high vacuum on crystalline enzymes.

The exploration of physical conditions in space is closely related to the study of the influence of space factors on biological objects. The study of high vacuum is of particular importance because its effects are as yet imperfectly understood. Micro-organisms are suitable models for these investigations. It has already been found that in reference to high vacuum, micro-organisms may be divided into two groups: resistant and nonresistant forms. Bacterial and actinomycetal spores and conidia of Aspergillus are classified as the resistant forms. Parallel with these, rod-shaped non-sporeforming bacteria, yeasts and conidia of Penicillium, were found to be non-resistant forms. The mechanism of resistance and death of micro-organisms under high vacuum is still not clearly understood. Because of this the investigation employing such important macromolecules as crystalline cytochrome C, catalase, peroxidase and ATP have been carried out. These compounds were exposed to a high vacuum (10(-8)-10(-9) mm Hg) over a period of 72 hours. Then the weighed samples taken from treated specimens and controls were dissolved firstly in a phosphate buffer solution (pH=6.8-7.2) and were then diluted by this buffer up to a concentration of 10(-9) mg ml. The solutions obtained were investigated by the chemiluminescent method. It was found that high vacuum effects gave rise to a slight decrease (up 7%) in enzyme activity in concentrated solutions and to a significant inactivation (up to 30%) in diluted ones.

Detection of iron-porphyrin proteins with a biochemiluminescent method in search of extraterrestrial life.

Iron-porphyrin proteins (catalase, peroxidase, hemoglobin, cytochrome C) represent an important group of redoxenzymes which have vitally important functions in micro-organisms. A biochemiluminescent method was employed for the detection of iron-porphyrin proteins. The reaction of luminol oxidation with H2O2 is accompanied by chemiluminescence. The rate of hydrogen peroxide decomposition increased 10(5)-10(7) -fold in the presence of the above enzymes as compared with ferrous (or ferric) ions. Possible application of this reaction for the detection of iron-porphyrin proteins of microbial origin was studied. Other authors have suggested this reaction for the detection of extraterrestrial life. Kinetics of the above reaction in the presence of iron-porphyrin proteins were shown to differ both in amplitude and duration of the signal from the pattern observed in the presence of non-hemin catalysts. The reaction pattern in the presence of mixed-soil populations is similar to those observed with pure bacterial cultures and individual iron-porphyrin proteins. Photometric tests revealed that among preparations studied the addition of 0.01% lysozyme was the most effective in destroying cell walls in microbial populations. However, removal of cell walls is not a necessary prerequisite for the detection of iron porphyrin since, for effective luminol oxidation with H2O2 the medium should be kept at pH 12.0. Pretreatment of microbial suspensions with ultrasound increased 2-fold the total signal due to iron porphyrins. The above method gives a reproducible signal indicating the presence of iron porphyrins when sterile nutrient media were innoculated with desert soil samples (Repeteck, Kara-Kum) and incubated for 13 hr. The device was able to detect the presence of no less than 10(5) - 10(6) cells per ml. The addition of limonite (Fe2O3 X nH2O) does not result in the appearance of an appreciable signal in the luminol + H2O2 system.

Exobiology and the effect of physical factors on micro-organisms.

A study of the action of different physical factors on micro-organisms is necessary for a further development of exobiology. The action of temperature on crystalline preparations of catalase and peroxidase was studied by means of oscillographic polarography. A determination of the height of polarographic waves at the decrease of temperature from 20 degrees C to 0 degrees C has shown that structural elements of the peroxidase molecule connected with the enzymatic activity are more stable with the decrease of temperature cf. catalase. A relative resistance of the dehydrogenase activity in Az. vinelandii cells to high vacuum was found. Incubation of azotobacter cells under vacuum of 10(-9) mm Hg during 72 hr did not decrease the activity of alcohol and succinic dehydrogenase. Bac. cereus spores can be preserved from bactericidal UV action by thin films of chrome. The thickness of chrome film being 200-670 angstroms, spores are killed by a dose of 7.8 x 10(7) erg/cm2 at 253.7 microns wave length. Spores covered by chrome film thicker than 800 angstroms remain alive after this treatment. Investigations carried out with an 'Artificial Mars' camera led to the following results. The growth of Bac. megaterium on liquid growth media in this camera ceases as a result of UV rays killing all cells after 3 weeks. Untreated bacteria grow in the camera for a long time. Spore-forming bacteria isolated from the sand of the Kara-Kum Desert grow in ground limonite (with the addition of 2% garden soil) having maximum hygroscopic humidity (3.8%). Freezing and thawing (from -60 degrees C to +25 degrees C) corresponding to day temperature deviations on Mars, low pressure (P=10 mm Hg) and the composition of the atmosphere (CO2-50%, N2-40%, Ar-10%) do not influence the growth of xerophylic bacteria under study. Humidity is the main factor limiting the growth of micro-organisms under 'Artificial Mars' conditions. According to the further development of the microbiological meteorite analysis methods, samples of rocks and stone meteorites were sterilized, incubated in the desert or on a snow surface in the Arctic and after different times (from 100 days to 7 months), investigated. In all cases, microbes were found only on the sample surfaces, whereas 1 cm from the surface and in the central parts micro-organism were completely absent. Hence, microbiological analysis of central parts of meteorites fallen in the Arctic or during dry periods of the year in the desert can give reliable results.