V. S. Soina

Preservation of cell structures in permafrost: a model for exobiology.

The present report is the first contribution toward a comprehensive fine-structural study of microbial cells from permafrost. Prokaryotes with a variety of cell wall types demonstrate high stability of cell structure after long-term cryopreservation in frozen soils and sediments of the Arctic. The surface capsular layers that were a salient feature of the cells both in situ and on nutrient media may be an adaptation to low temperature. To the extent that permafrost regions on Earth approximate Martian conditions, preservation of cell structure there can serve as the basis for predictions about preservation in Martian permafrost sediments.

Reproductive resting forms of Arthrobacter globiformis.

Submerged cultures ofArthrobacter globiformis grown in media unbalanced with respect to carbon and nitrogen sources were found to contain cells exhibiting features typical of resting forms: long-term viability, specific ultrastructure, dormant metabolism, and thermoresistance. Such cells were produced not only in the collection strain VKM B-l 112, but also in the Aglobiformis strains isolated from 2-to 3-million-year-old permafrost sediments.

Microorganisms and enzyme activity in permafrost after removal of long-term cold stress

Abstract Associations of immobilized microbial cells and organic-mineral complexes containing active enzymes are resistant to long-term (from tens of thousands to millions of years) effects of extremely low temperatures. This association enables the cells to restore their metabolic activity during permafrost thawing, because interactions with the heterogenous medium is made possible by the availability of active immobilized enzymes. The long-term effect of the cold probably favors an adaptational change of microbial metabolism that activates enzymes and cells during thawing.

Synthesis of Anabiosis Autoinducers by Non-Spore-Forming Bacteria as a Mechanism Regulating Their Activity in Soil and Subsoil Sedimentary Rocks

Non-spore-forming bacteria of the genera Arthrobacterand Micrococcus, isolated from permafrost subsoil, were found to produce greater amounts of the d1extracellular factor than closely related collection strains isolated from soil. The effect of this factor, responsible for cell transition to anabiosis, was not species-specific. Thus, the d1preparation isolated from the culture liquid of the permafrost isolate Arthrobacter globiformis245 produced an effect on the collection strain Arthrobacter globiformisB-1112 and also on Micrococcus luteusand Bacillus cereus.The d1preparation from the permafrost isolate of Arthrobacterdiffered from the chemical analogue of this factor, 4-n-hexylresorcinol, in the level of the induced cell response, which may have resulted from different cell sensitivity to various homologs of alkylhydroxybenzenes contained in the d1preparation. Thus, additional evidence was obtained indicating that autoregulation of bacterial growth and development is implemented at the level of intercellular interactions in microbial communities. Abundant production of the d1anabiosis-inducing factors by bacteria isolated from permafrost subsoil is probably a result of special antistress mechanisms responsible for the survival of these bacteria under extreme conditions of natural long-term cooling.

Reactivation of dormant and nonculturable bacterial forms from paleosoils and subsoil permafrost

Methods of reactivating the dormant forms (DFs) and nonculturable cells (NCs) of the bacterial communities of buried paleosoils and subsoil permafrost stored for long periods of time (thousands to millions of years), including completely sterile samples (CFU = 0), were developed. They were based on washing the DFs and NCs to remove anabiosis autoinducers (spore germination autoinhibitors) and introducing low molecular weight extracellular growth regulators of microbial or plant origin, such as alkylhydroxybenzenes of the alkylresorcinol subtype, indoleacetic acid, and wheat germ agglutinin. It was revealed that the dormant communities of permafrost and buried soils differed in their sensitivity to reactivating factors, probably due to different natural storage conditions of the tested soil substrates and the heterogeneity of dormant populations. The latter was confirmed by FISH (fluorescent in situ hybridization): applying the reactivation methods to the cells of the dormant permafrost community resulted in an increase in the number of metabolically active cells from 5 to 77% of their total number. In contrast, the addition of microbial anabiosis autoinducers (C12-AHB) to background surface soil and permafrost samples caused the transition of bacterial cells to the dormant or the nonculturable state, depending on the C12-AHB concentration and the sensitivity of the cells from the control soil or permafrost’ to it. The data obtained contribute to our knowledge concerning the role of intercellular communication factors and the survival of microorganisms under extreme environmental conditions.

Formation of resting forms of arthrobacter globiformis in autolyzing cell suspensions

Under conditions of the spontaneous or induced autolysis of thick cell suspensions,Arthrobacter globiformis strains produced cells exhibiting features typical of resting microbial forms. The number of viable resting cells was greater under conditions of induced rather than spontaneous autolysis. The thermoresistance of the resting cells of A.globiformis strains isolated from 2-to 3 million-year-old permafrost was higher than that of the collectionA. globiformis strain.

Formation of resting cells by non-spore-forming microorganisms as a strategy of long-term survival in the environment

Non-spore-forming bacteria of the genera Micrococcus and Arthrobacter, including the isolates from permafrost sediments, were found to be able to form cystlike cells under special conditions. Cystlike cells maintained the viability during long-term storage (for up to several years), had undetectable respiratory activity and the elevated resistance to heating and other unfavorable conditions, possessed the specific fine structure and morphology, and were formed in the life cycles of the microorganism. These properties allow cystlike cells to be attributed to a new type of resting microbial forms. Furthermore, the distinctive feature of resting cystlike cells was their low P/S ratios and high Ca/K ratios in comparison to vegetative cells as shown by X-ray microanalysis. The experimentally obtained bacterial cystlike cells with thickened and laminated cell walls and altered texture of the cytoplasm were similar to the cells abundant in native microbial populations isolated from permafrost sediments and ancient soils of the Kolyma lowland (Siberia, Russia). Due to the inherent elevated resistance to adverse conditions and maintenance of viability for prolonged periods, resting cystlike cells are likely to ensure long-term survival of non-spore-forming bacteria in cold environments.

Micro-organisms and biomarkers in permafrost

Studies of microorganisms populating subsoil layers skipped the latent period (individual communications in the first half of this century), passed through the lagphase of accumulation of reliable information and overcoming of sound scepsis and reached eventually the stage of exponential growth. Today, we can state with certainty the ever-increasing interest shown by not only biologists but also experts from various natural sciences in the progress of studies in this field. This interest is related to changes in our concepts of extreme habitats and extremophilia of microorganisms under conditions of a heterophase medium. It becomes obvious that the overwhelming majority of microorganisms exist under the ‘extreme’ conditions of lowered and often negative temperatures, high pressure, low concentrations of accessible nutrients, oxygen, physiological dryness, etc. It becomes clear that the reserves of biomass in the interior of the earth abounding in ‘extreme’ habitats are many times the overall biomass of the surface and subsurface soil layers. The subject of this discussion is metabolic activity and, naturally, the possibility of cell adaptation to such a medium, i.e. in essence, the advisability of the ecological term ‘extreme medium’ in its application to viable microorganisms populating these biotopes under long-term stability of medium parameters.

Viable bacteria, methane and high ice content in antarctic permafrost: Relevance to Mars

1Florida State Univ., Tallahassee, FL 32306-2043, USA, FAX: 1-904-644-9829, email: friedm@bio.fsu.edu; 2Lab. Soil Cryol., Russian Acad. Sei., Puschino, Moscow Region 142292, RUSSIA, FAX: 7-096-779-0532, e-mail: gilichin@issp.sherpukov.su; aByrd Polar Res. Ctr., Ohio State Univ., Columbus, OH 43210-1002, USA, FAX: 1614-292-4697, e-mail: wilsongs@geology.ohio-state.edu, 4Dept. Soil Biol., Moscow State Univ., 119899 Moscow, RUSSIA, e-mail: rott@soina.msk.ru; 5Dept. Oceanogr., Florida State University, Tallahassee, FL 32306-3015, USA, FAX: 1-904-644-2581, e-mail: jchanton@mailer.fsu.edu; ~Department of Biology, Florida A. and M. Univ., Tallahassee, FL 32307, USA, FAX: 1-904-561-2996, e-mail: friedm@bio.fsu.edu; 7NASA-Ames Res. Ctr., Mail Stop 245-3, Moffett Field, CA 93035, USA, FAX: 1415-604-6776, e-mail: mckay@galileo.arc.nasa.gov.

Role of cell differentiation in high tolerance by prokaryotes of long-term preservation in permafrost

Abstract The effect of low temperature on the cell structure of bacteria isolated from permafrost results in structural changes leading to cell differentiation into types of resting cells rangign from spores showing a high endogenous dormancy to typical “dormant” cells of non-spore-forming bacteria showing exogenous dormancy, which is considered to be less highly resistant to extreme conditions in laboratory experiments. In permafrost, dormant cells of non-spore-forming bacteria may demonstrate considerable resistance to long-term freezing and as a result a higher survival level than spore-forming bacteria.