Elena V. Pikuta

Microbial Extremophiles at the Limits of Life

Prokaryotic extremophiles were the first representatives of life on Earth and they are responsible for the genesis of geological structures during the evolution and creation of all currently known ecosystems. Flexibility of the genome probably allowed life to adapt to a wide spectrum of extreme environments. As a result, modern prokaryotic diversity formed in a framework of physico-chemical factors, and it is composed of: thermophilic, psychrophilic, acidophilic, alkaliphilic, halophilic, barophilic, and radioresistant species. This artificial systematics cannot reflect the multiple actions of different environmental factors since one organism could unite characteristics of several extreme-groups. In this review we show the current status of studies in all fields of extremophiles and summarize the limits of life for different species of microbial extremophiles. We also discuss the finding of extremophiles from unusual places such as soils, and briefly review recent studies of microfossils in meteorites in the context of the significance of microbial extremophiles to Astrobiology.

Desulfotomaculum alkaliphilum sp. nov., a new alkaliphilic, moderately thermophilic, sulfate-reducing bacterium.

A new moderately thermophilic, alkaliphilic, sulfate-reducing, chemolithoheterotrophic bacterium, strain S1T, was isolated from a mixed cow/pig manure with neutral pH. The bacterium is an obligately anaerobic, non-motile, Gram-positive, spore-forming curved rod growing within a pH range of 8.0-9.15 (optimal growth at pH 8.6-8.7) and temperature range of 30-58 degrees C (optimal growth at 50-55 degrees C). The optimum NaCl concentration for growth is 0.1%. Strain S1T is an obligately carbonate-dependent alkaliphile. The G+C content of the DNA is 40.9 mol%. A limited number of compounds are utilized as electron donors, including H2+acetate, formate, ethanol, lactate and pyruvate. Sulfate, sulfite and thiosulfate, but not sulfur or nitrate, can be used as electron acceptors. Strain S1T is able to utilize acetate or yeast extract as sources of carbon. Analysis of the 16S rDNA sequence allowed strain S1T (= DSM 12257T) to be classified as a representative of a new species of the genus Desulfotomaculum, Desulfotomaculum alkaliphilum sp. nov.

Spirochaeta dissipatitropha sp. nov., an alkaliphilic, obligately anaerobic bacterium, and emended description of the genus Spirochaeta Ehrenberg 1835.

A novel obligately anaerobic, mesophilic, alkaliphilic spirochaete, strain ASpC2(T), was isolated from an anaerobic sediment of alkaline, hypersaline Owens Lake in California, USA. The Gram-negative cells are motile, helical in shape and 0.23 x 8.0-18.0 mum. Growth occurs within the following ranges: 13-41 degrees C, with optimal growth at 35 degrees C; 1-3 % (w/v) NaCl, with optimal growth at 2 % (w/v) NaCl; and pH 7.8-10.5, with optimal growth at pH 10.0. The novel isolate is strictly alkaliphilic and requires high concentrations of carbonate ions in the medium. It utilizes some sugars, some organic acids, some amino acids, Casamino acids, yeast extract and peptone. The main end products of glucose fermentation are CO(2) and acetate. Strain ASpC2(T) is resistant to kanamycin and rifampicin, but sensitive to ampicillin, chloramphenicol, gentamicin and tetracycline. The DNA G+C content of the new isolate is 43.8 mol%, its genome size is 6 x 10(8) Da and the melting temperature of its genomic DNA is 71 degrees C. DNA-DNA hybridization experiments demonstrated 46 % similarity with the phylogenetically most closely related species, Spirochaeta asiatica Z-7591(T). On the basis of physiological and molecular properties, the new isolate belongs taxonomically to a novel species within the genus Spirochaeta, for which the name Spirochaeta dissipatitropha sp. nov. is proposed (type strain, ASpC2(T)=ATCC BAA-1083(T)=JCM 12856(T)). S. dissipatitropha ASpC2(T) is the second strain in the genus (after Spirochaeta smaragdinae SEBR 4228(T)) that is able to use proteolysis products as the sole energy source, and additional tests have shown that other halo-alkaliphilic spirochaetes (Spirochaeta americana, Spirochaeta alkalica and Spirochaeta africana) are also able to grow on yeast extract alone; therefore, an emended description for the genus Spirochaeta is given.

Astrobiology of Comets

We review the current state of knowledge concerning microbial extremophiles and comets and the potential significance of comets to Astrobiology. We model the thermal history of a cometary body, regarded as an assemblage of boulders, dust, ices and organics, as it approaches a perihelion distance of ~ 1AU. The transfer of incident energy from sunlight into the interior leads to the melting of near surface ices, some under stable porous crust, providing possible habitats for a wide range of microorganisms. We provide data concerning new evidence for indigenous microfossils in CI meteorites, which may be the remains of extinct cometary cores. We discuss the dominant microbial communities of polar sea-ice, Antarctic ice sheet, and cryoconite environments as possible analogs for microbial ecosystems that may grow in sub-crustal pools or in ice/water films in comets.

Proteocatella sphenisci gen. nov., sp. nov., a psychrotolerant, spore-forming anaerobe isolated from penguin guano

A novel, obligately anaerobic, psychrotolerant bacterium, designated strain PPP2T, was isolated from guano of the Magellanic penguin (Spheniscus magellanicus) in Chilean Patagonia. Cells were Gram-stain-positive, spore-forming, straight rods (0.7-0.8x3.0-5.0 microm) that were motile by means of peritrichous flagella. Growth was observed at pH 6.7-9.7 (optimum pH 8.3) and 2-37 degrees C (optimum 29 degrees C). Growth was observed between 0 and 4% (w/v) NaCl with optimum growth at 0.5% (w/v). Strain PPP2T was a catalase-negative chemo-organoheterotroph that was capable of fermentative metabolism. Peptone, bacto-tryptone, Casamino acids, oxalate, starch, chitin and yeast extract were utilized as substrates. The major metabolic products were acetate, butyrate and ethanol. Strain PPP2T was resistant to ampicillin, but sensitive to tetracycline, chloramphenicol, rifampicin, kanamycin, vancomycin and gentamicin. The DNA G+C content of strain PPP2T was 39.5 mol%. Phylogenetic analysis revealed that strain PPP2T was related most closely to Clostridium sticklandii SR (approximately 90% 16S rRNA gene sequence similarity). On the basis of phylogenetic analysis and phenotypic characteristics, strain PPP2T is considered to represent a novel species of a new genus, for which the name Proteocatella sphenisci gen. nov., sp. nov. is proposed. The type strain of Proteocatella sphenisci is PPP2T (=ATCC BAA-755T=JCM 12175T=CIP 108034T).

Microorganisms on comets, Europa, and the polar ice caps of Mars

Microbial extremophiles live on Earth wherever there is liquid water and a source of energy. Observations by ground-based observatories, space missions, and satellites have provided strong evidence that water ice exists today on comets, Europa, Callisto, and Ganymede and in the snow, permafrost, glaciers and polar ice caps of Mars. Studies of the cryoconite pools and ice bubble systems of Antarctica suggest that solar heating of dark rocks entrained in ice can cause localized melting of ice providing ideal conditions for the growth of microbial communities with the creation of micro-environments where trapped metabolic gasses produce entrained isolated atmospheres as in the Antarctic ice-bubble systems. It is suggested that these considerations indicate that several groups of microorganisms should be capable of episodic growth within liquid water envelopes surrounding dark rocks in cometary ices and the permafrost and polar caps of Mars. We discuss some of the types of microorganisms we have encountered within the permafrost and snow of Siberia, the cryoconite pools of Alaska, and frozen deep within the Antarctic ice sheet above Lake Vostok.

Anaerobic decomposition of cellulose by alkaliphilic microbial community of Owens Lake, California

The study of alkaliphilic microbial communities from anaerobic sediments of Owens and Mono Lakes in California has established the presence of active microbial cellulolytic processes in both studied lakes. The prior study of the microbial diversity of anaerobes in Mono Lake showed that the trophic chain of organic decomposition includes secondary anaerobes that previously were found to be unknown species (Spirochaeta americana, Tindallia californiensis, and Desulfonatronum thiodismutans). As we published earlier, the secondary anaerobes of Owens Lake morphologically were found to be very similar to those of Mono Lake. However, detailed comparison of the physiology and genetics has led to the conclusion that some links of organic decomposition in the trophic chain of the Owens Lake community are represented by a different unknown species. A new isolate of a sugarlytics free-living spirochete from Owens Lake ASpC2, which morphologically was similar to S. americana AspG1T isolated from Mono Lake, was found to have a different metabolic capacity such as the lack of capability to produce hydrogen during the fermentation of sugars. Furthermore, from the same microbial community of Owens Lake, another sugarlytics spore-forming alkaliphilic strain SCA was isolated in pure culture and described. Here we discuss the universal structure of the microbial community, types of microbial communities, review some hypothesis about Earths Primordial Ocean and relevant new discoveries about water on Mars. This paper also presents some of the characteristics of novel isolates from anaerobic sediments of Owens Lake as a unique relic ecosystem of Astrobiological significance, and describes the participation of these strains in the process of cellulose degradation.

Growth of the facultative anaerobes from Antarctica, Alaska, and Patagonia at low temperatures

Psychrotolerance, as an adaptation for surviving in extreme environments, is widespread among mesophilic microorganisms. Physico-chemical factors such as pressure, red-ox potential, pH and salinity could significantly alter the features of ecosystems by providing liquid water at subzero temperatures. Furthermore, organisms can respond to temperature changes by several known mechanisms, including changing the conformation capacities of constitutional proteins or by the synthesis of mucopolysaccharides around the cell wall and membrane. Such protective mechanisms make it possible for cells to not only passively survive low temperatures in a state of anabiosis, but also to be capable of actively metabolizing substrates and reproducing normally. The physiological and biochemical characteristics of the species, as well as genetics, could be remarkably changed due to adaptation and surviving in extreme environments. The cold shock genes of some of the studied strains of psychrotolerant facultative anaerobes were reported previously. In this paper we present experimental data for psychrotolerant, non spore-forming, facultative anaerobes isolated from geographically different cold regions of our planet. We show the growth response on changing from anaerobic conditions to aerobic with cultivation at low temperatures.

Raineyella antarctica gen. nov., sp. nov., a psychrotolerant, d-amino-acid-utilizing anaerobe isolated from two geographic locations of the Southern Hemisphere

A Gram-stain-positive bacterium, strain LZ-22T, was isolated from a rhizosphere of moss Leptobryum sp. collected at the shore of Lake Zub in Antarctica. Cells were motile, straight or pleomorphic rods with sizes of 0.6-1.0×3.5-10 µm. The novel isolate was a facultatively anaerobic, catalase-positive, psychrotolerant mesophile. Growth was observed at 3-41 °C (optimum 24-28 °C), with 0-7 % (w/v) NaCl (optimum 0.25 %) and at pH 4.0-9.0 (optimum pH 7.8). The quinone system of strain LZ-22T possessed predominately menaquinone MK-9(H4). The genomic G+C content was 70.2 mol%. Strain 10J was isolated from a biofilm of sediment microbial fuel cell, in Uruguay and had 99 % 16S rRNA gene sequence similarity to strain LZ-22T. DNA-DNA-hybridization values of 84 % confirmed that both strains belonged to the same species. Both strains grew on sugars, proteinaceous compounds, and some amino- and organic acids. Strain LZ-22T uniquely grew on D-enantiomers of histidine and valine while neglecting growth on L-enantiomers. Both strains were sensitive to most of the tested antibiotics but resistant to tested nitrofurans and sulfanilamides. Phylogenetic analyses of the 16S rRNA gene sequences indicated that the strains were related to members of the family Propionibacteriaceae (~93-94 % 16S rRNA gene sequence similarity) with formation of a separate branch within the radiation of the genera Granulicoccus and Luteococcus. Based on phenotypic and genotypic characteristics, we propose the affiliation of both strains into a novel species of a new genus. The name Raineyella antarctica gen. nov., sp. nov. is proposed for the novel taxon with the type strain LZ-22T (=ATCC TSD-18T=DSM 100494T=JCM 30886T).

Overview of Archaea

Archaea were separated from Eubacteria after discovery of their specifics in cell outer membrane that usually not affected by common antibiotics. Phylogenetic analysis introduced by Karl Wöese supported this separation. Presently, only two phyla Crenarchaeota and Euryarchaeota include the valid representatives. Another three phyla that were proposed based on the sequence analysis of environmental samples, do not contain validly published species, and for this reason they are not included in this review. The phylum Euryarchaeota currently includes eight classes and ten orders, while the Crenarchaeota phylum contains the only class with five orders. Members of the phyla Crenarchaeota have two or three family B and no family D DNA polymerases, but members of the Euryarchaeota contain the only family B polymerases and the only family D polymerases, and it is still not clear, which is the main functional enzyme in the replication process. In this article, we are present an update and comparative analysis for this domain, discussing unique features of this group and Evolution, estimating their physiology within the matrix of physic-chemical factors, and outlining future perspectives in their study. Rules of the diagonal for the diagrams with all Archaea are presented and discussed.