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1.  Protein patterns of black fungi under simulated Mars-like conditions 
Scientific Reports  2014;4:5114.
Two species of microcolonial fungi – Cryomyces antarcticus and Knufia perforans - and a species of black yeasts–Exophiala jeanselmei - were exposed to thermo-physical Mars-like conditions in the simulation chamber of the German Aerospace Center. In this study the alterations at the protein expression level from various fungi species under Mars-like conditions were analyzed for the first time using 2D gel electrophoresis. Despite of the expectations, the fungi did not express any additional proteins under Mars simulation that could be interpreted as stress induced HSPs. However, up-regulation of some proteins and significant decreasing of protein number were detected within the first 24 hours of the treatment. After 4 and 7 days of the experiment protein spot number was increased again and the protein patterns resemble the protein patterns of biomass from normal conditions. It indicates the recovery of the metabolic activity under Martian environmental conditions after one week of exposure.
PMCID: PMC4037706  PMID: 24870977
2.  Combined effects of agitation and aeration on the chitinolytic enzymes production by the Antarctic fungus Lecanicillium muscarium CCFEE 5003 
The Antarctic fungus Lecanicillium muscarium CCFEE 5003 is one of the most powerful chitinolytic organisms. It can produce high level of chitinolytic enzymes in a wide range of temperatures (5-30°C). Chitinolytic enzymes have lot of applications but their industrial production is still rather limited and no cold-active enzymes are produced. In view of massive production of L. muscarium chitinolytic enzymes, its cultivation in bioreactors is mandatory. Microbial cultivation and/or their metabolite production in bioreactors are sometime not possible and must be verified and optimized for possible exploitation. Agitation and aeration are the most important parameters in order to allow process up-scaling to the industrial level.
In this study, submerged cultures of L. muscarium CCFEE 5003 were carried out in a 2-L bench-top CSTR bioreactor in order to optimise the production of chitinolytic enzymes. The effect of stirrer speed (range 200-500 rpm) and aeration rate (range 0.5-1.5 vvm) combination was studied, by Response Surface Methodology (RSM), in a medium containing 1.0% yeast nitrogen base and 1% colloidal chitin. Optimization was carried out, within a "quadratic D-optimal" model, using quantitative and quantitative-multilevel factors for aeration and agitation, respectively. The model showed very good correlation parameters (R2, 0.931; Q2, 0.869) and the maximum of activity (373.0 U/L) was predicted at ca. 327 rpm and 1.1 vvm. However, the experimental data showed that highest activity (383.7 ± 7.8 U/L) was recorded at 1 vvm and 300 rpm. Evident shear effect caused by stirrer speed and, partially, by high aeration rates were observed. Under optimized conditions in bioreactor the fungus was able to produce a higher number of chitinolytic enzymes than those released in shaken flasks. In addition, production was 23% higher.
This work demonstrated the attitude of L. muscarium CCFEE 5003 to grow in bench-top bioreactor; outlined the strong influence of aeration and agitation on its growth and enzyme production and identified the optimal conditions for possible production at the industrial level.
PMCID: PMC3310808  PMID: 22270226
Chitinolytic enzymes production; Lecanicillium muscarium; Response Surface Methodology; Agitation and aeration
3.  Antarctic Epilithic Lichens as Niches for Black Meristematic Fungi 
Biology  2013;2(2):784-797.
Sixteen epilithic lichen samples (13 species), collected from seven locations in Northern and Southern Victoria Land in Antarctica, were investigated for the presence of black fungi. Thirteen fungal strains isolated were studied by both morphological and molecular methods. Nuclear ribosomal 18S gene sequences were used together with the most similar published and unpublished sequences of fungi from other sources, to reconstruct an ML tree. Most of the studied fungi could be grouped together with described or still unnamed rock-inhabiting species in lichen dominated Antarctic cryptoendolithic communities. At the edge of life, epilithic lichens withdraw inside the airspaces of rocks to find conditions still compatible with life; this study provides evidence, for the first time, that the same microbes associated to epilithic thalli also have the same fate and chose endolithic life. These results support the concept of lichens being complex symbiotic systems, which offer attractive and sheltered habitats for other microbes.
PMCID: PMC3960883  PMID: 24832808
black meristematic fungi; Dothideomycetes; Eurotiomycetes; lichen-associated fungi; phylogeny
4.  Microcolonial Fungi on Rocks: A Life in Constant Drought? 
Mycopathologia  2012;175(5-6):537-547.
Black microcolonial fungi (MCF) and black yeasts are among the most stress-resistant eukaryotic organisms known on Earth. They mainly inhabit bare rock surfaces in hot and cold deserts of all regions of the Earth, but some of them have a close phylogenetic relation to human pathogenic black fungi which makes them important model organisms also with respect to clinical mycology. The environment of those fungi is especially characterized by extreme changes from humidity to long periods of desiccation and extreme temperature differences. A key to the understanding of MCF ecology is the question about metabolic activity versus dormancy in the natural environments. In this study, the time lag from the desiccated state to rehydration and full metabolic activity and growth was measured and defined in accordance with simulated environmental conditions. The ability to survive after desiccation and the speed of rehydration as well as changes of the whole cell protein pattern are demonstrated. Whereas both mesophilic strains—Exophiala jeanselmei and Knufia perforans (=Coniosporium perforans)—show a clear reaction toward desiccation by production of small proteins, Cryomyces antarcticus—the extremotolerant MCF—does not show any response to desiccation but seems just to down-regulate its metabolism. Data on intracellular sugar suggest that both trehalose and mannitol might play a cell protective role in those fungi.
Electronic supplementary material
The online version of this article (doi:10.1007/s11046-012-9592-1) contains supplementary material, which is available to authorized users.
PMCID: PMC3669513  PMID: 23073825
Black yeast; Anhydrobiosis; Two-dimensional gel electrophoresis; Protein profiling; Trehalose
5.  Nothing Special in the Specialist? Draft Genome Sequence of Cryomyces antarcticus, the Most Extremophilic Fungus from Antarctica 
PLoS ONE  2014;9(10):e109908.
The draft genome of the Antarctic endemic fungus Cryomyces antarcticus is presented. This rock inhabiting, microcolonial fungus is extremely stress tolerant and it is a model organism for exobiology and studies on stress resistance in Eukaryots. Since this fungus is a specialist in the most extreme environment of the Earth, the analysis of its genome is of important value for the understanding of fungal genome evolution and stress adaptation. A comparison with Neurospora crassa as well as with other microcolonial fungi shows that the fungus has a genome size of 24 Mbp, which is the average in the fungal kingdom. Although sexual reproduction was never observed in this fungus, 34 mating genes are present with protein homologs in the classes Eurotiomycetes, Sordariomycetes and Dothideomycetes. The first analysis of the draft genome did not reveal any significant deviations of this genome from comparative species and mesophilic hyphomycetes.
PMCID: PMC4190365  PMID: 25296285
6.  Microbial Diversity of Cryptoendolithic Communities from the McMurdo Dry Valleys, Antarctica 
In the McMurdo Dry Valleys of Antarctica, microorganisms colonize the pore spaces of exposed rocks and are thereby protected from the desiccating environmental conditions on the surface. These cryptoendolithic communities have received attention in microscopy and culture-based studies but have not been examined by molecular approaches. We surveyed the microbial biodiversity of selected cryptoendolithic communities by analyzing clone libraries of rRNA genes amplified from environmental DNA. Over 1,100 individual clones from two types of cryptoendolithic communities, cyanobacterium dominated and lichen dominated, were analyzed. Clones fell into 51 relatedness groups (phylotypes) with ≥98% rRNA sequence identity (46 bacterial and 5 eucaryal). No representatives of Archaea were detected. No phylotypes were shared between the two classes of endolithic communities studied. Clone libraries based on both types of communities were dominated by a relatively small number of phylotypes that, because of their relative abundance, presumably represent the main primary producers in these communities. In the lichen-dominated community, three rRNA sequences, from a fungus, a green alga, and a chloroplast, of the types known to be associated with lichens, accounted for over 70% of the clones. This high abundance confirms the dominance of lichens in this community. In contrast, analysis of the supposedly cyanobacterium-dominated community indicated, in addition to cyanobacteria, at least two unsuspected organisms that, because of their abundance, may play important roles in the community. These included a member of the α subdivision of the Proteobacteria that potentially is capable of aerobic anoxygenic photosynthesis and a distant relative of Deinococcus that defines, along with other Deinococcus-related sequences from Antarctica, a new clade within the Thermus-Deinococcus bacterial phylogenetic division.
PMCID: PMC165166  PMID: 12839754
7.  Polyextremotolerant black fungi: oligotrophism, adaptive potential, and a link to lichen symbioses 
Black meristematic fungi can survive high doses of radiation and are resistant to desiccation. These adaptations help them to colonize harsh oligotrophic habitats, e.g., on the surface and subsurface of rocks. One of their most characteristic stress-resistance mechanisms is the accumulation of melanin in the cell walls. This, production of other protective molecules and a plastic morphology further contribute to ecological flexibility of black fungi. Increased growth rates of some species after exposure to ionizing radiation even suggest yet unknown mechanisms of energy production. Other unusual metabolic strategies may include harvesting UV or visible light or gaining energy by forming facultative lichen-like associations with algae or cyanobacteria. The latter is not entirely surprising, since certain black fungal lineages are phylogenetically related to clades of lichen-forming fungi. Similar to black fungi, lichen-forming fungi are adapted to growth on exposed surfaces with low availability of nutrients. They also efficiently use protective molecules to tolerate frequent periods of extreme stress. Traits shared by both groups of fungi may have been important in facilitating the evolution and radiation of lichen-symbioses.
PMCID: PMC3492852  PMID: 23162543
melanin; oligotrophism; secondary metabolites; protective molecules; stress
8.  Investigating the Effects of Simulated Martian Ultraviolet Radiation on Halococcus dombrowskii and Other Extremely Halophilic Archaebacteria 
Astrobiology  2009;9(1):104-112.
The isolation of viable extremely halophilic archaea from 250-million-year-old rock salt suggests the possibility of their long-term survival under desiccation. Since halite has been found on Mars and in meteorites, haloarchaeal survival of martian surface conditions is being explored. Halococcus dombrowskii H4 DSM 14522T was exposed to UV doses over a wavelength range of 200–400 nm to simulate martian UV flux. Cells embedded in a thin layer of laboratory-grown halite were found to accumulate preferentially within fluid inclusions. Survival was assessed by staining with the LIVE/DEAD kit dyes, determining colony-forming units, and using growth tests. Halite-embedded cells showed no loss of viability after exposure to about 21 kJ/m2, and they resumed growth in liquid medium with lag phases of 12 days or more after exposure up to 148 kJ/m2. The estimated D37 (dose of 37 % survival) for Hcc. dombrowskii was ≥ 400 kJ/m2. However, exposure of cells to UV flux while in liquid culture reduced D37 by 2 orders of magnitude (to about 1 kJ/m2); similar results were obtained with Halobacterium salinarum NRC-1 and Haloarcula japonica. The absorption of incoming light of shorter wavelength by color centers resulting from defects in the halite crystal structure likely contributed to these results. Under natural conditions, haloarchaeal cells become embedded in salt upon evaporation; therefore, dispersal of potential microscopic life within small crystals, perhaps in dust, on the surface of Mars could resist damage by UV radiation.
PMCID: PMC3182532  PMID: 19215203
Halococcus dombrowskii; Simulated martian UV radiation; LIVE/DEAD staining; Halite fluid inclusions; UV transmittance and reflectance; Desiccation
9.  The complete mitochondrial genome of the Antarctic springtail Cryptopygus antarcticus (Hexapoda: Collembola) 
BMC Genomics  2008;9:315.
Mitogenomics data, i.e. complete mitochondrial genome sequences, are popular molecular markers used for phylogenetic, phylogeographic and ecological studies in different animal lineages. Their comparative analysis has been used to shed light on the evolutionary history of given taxa and on the molecular processes that regulate the evolution of the mitochondrial genome. A considerable literature is available in the fields of invertebrate biochemical and ecophysiological adaptation to extreme environmental conditions, exemplified by those of the Antarctic. Nevertheless, limited molecular data are available from terrestrial Antarctic species, and this study represents the first attempt towards the description of a mitochondrial genome from one of the most widespread and common collembolan species of Antarctica.
In this study we describe the mitochondrial genome of the Antarctic collembolan Cryptopygus antarcticus Willem, 1901. The genome contains the standard set of 37 genes usually present in animal mtDNAs and a large non-coding fragment putatively corresponding to the region (A+T-rich) responsible for the control of replication and transcription. All genes are arranged in the gene order typical of Pancrustacea. Three additional short non-coding regions are present at gene junctions. Two of these are located in positions of abrupt shift of the coding polarity of genes oriented on opposite strands suggesting a role in the attenuation of the polycistronic mRNA transcription(s). In addition, remnants of an additional copy of trnL(uag) are present between trnS(uga) and nad1. Nucleotide composition is biased towards a high A% and T% (A+T = 70.9%), as typically found in hexapod mtDNAs. There is also a significant strand asymmetry, with the J-strand being more abundant in A and C. Within the A+T-rich region, some short sequence fragments appear to be similar (in position and primary sequence) to those involved in the origin of the N-strand replication of the Drosophila mtDNA.
The mitochondrial genome of C. antarcticus shares several features with other pancrustacean genomes, although the presence of unusual non-coding regions is also suggestive of molecular rearrangements that probably occurred before the differentiation of major collembolan families. Closer examination of gene boundaries also confirms previous observations on the presence of unusual start and stop codons, and suggests a role for tRNA secondary structures as potential cleavage signals involved in the maturation of the primary transcript. Sequences potentially involved in the regulation of replication/transcription are present both in the A+T-rich region and in other areas of the genome. Their position is similar to that observed in a limited number of insect species, suggesting unique replication/transcription mechanisms for basal and derived hexapod lineages. This initial description and characterization of the mitochondrial genome of C. antarcticus will constitute the essential foundation prerequisite for investigations of the evolutionary history of one of the most speciose collembolan genera present in Antarctica and other localities of the Southern Hemisphere.
PMCID: PMC2483729  PMID: 18593463
10.  The origin and emergence of life under impact bombardment 
Craters formed by asteroids and comets offer a number of possibilities as sites for prebiotic chemistry, and they invite a literal application of Darwin's ‘warm little pond’. Some of these attributes, such as prolonged circulation of heated water, are found in deep-ocean hydrothermal vent systems, previously proposed as sites for prebiotic chemistry. However, impact craters host important characteristics in a single location, which include the formation of diverse metal sulphides, clays and zeolites as secondary hydrothermal minerals (which can act as templates or catalysts for prebiotic syntheses), fracturing of rock during impact (creating a large surface area for reactions), the delivery of iron in the case of the impact of iron-containing meteorites (which might itself act as a substrate for prebiotic reactions), diverse impact energies resulting in different rates of hydrothermal cooling and thus organic syntheses, and the indiscriminate nature of impacts into every available lithology—generating large numbers of ‘experiments’ in the origin of life. Following the evolution of life, craters provide cryptoendolithic and chasmoendolithic habitats, particularly in non-sedimentary lithologies, where limited pore space would otherwise restrict colonization. In impact melt sheets, shattered, mixed rocks ultimately provided diverse geochemical gradients, which in present-day craters support the growth of microbial communities.
PMCID: PMC1664688  PMID: 17008223
organics; colonization; thermophiles; asteroid; crater
11.  Biomass of the cryptoendolithic microbiota from the Antarctic desert. 
Extractable lipid phosphate was used to determine the biomass of the cryptoendolithic microbiota that colonizes sandstone rocks in the Ross Desert region of Antarctica. The mean amount of lipid phosphate was 0.053 micromole/cm2 (n = 9), which equals 2.54 g of C per m2 (range, 1.92 to 3.26 g of C per m2) of biomass in the biotic zone of these rocks. The turnover of phospholipids was comparable to that of temperate sediments and soils (t1/2, 6 to 9 days) at 0 degrees C and a light intensity of 305 micromoles of photons per m2 per s, indicating that this was a good method to measure viable biomass. The biomass was 0.3 to 9.6% of the total carbon content of the biotic zone and was about 2 orders of magnitude smaller than the epilithic lichen dry weight at a location some 7 degrees north in latitude. The cryptoendolithic microbiota had a uniform density throughout the biotic zone under the rock surface. The results indicate that the cryptoendolithic microbial biomass is small but viable in this unique, extreme ecosystem.
PMCID: PMC202579  PMID: 11536603
12.  Effects of Late-Cenozoic Glaciation on Habitat Availability in Antarctic Benthic Shrimps (Crustacea: Decapoda: Caridea) 
PLoS ONE  2012;7(9):e46283.
Marine invertebrates inhabiting the high Antarctic continental shelves are challenged by disturbance of the seafloor by grounded ice, low but stable water temperatures and variable food availability in response to seasonal sea-ice cover. Though a high diversity of life has successfully adapted to such conditions, it is generally agreed that during the Last Glacial Maximum (LGM) the large-scale cover of the Southern Ocean by multi-annual sea ice and the advance of the continental ice sheets across the shelf faced life with conditions, exceeding those seen today by an order of magnitude. Conditions prevailing at the LGM may have therefore acted as a bottleneck event to both the ecology as well as genetic diversity of today's fauna. Here, we use for the first time specific Species Distribution Models (SDMs) for marine arthropods of the Southern Ocean to assess effects of habitat contraction during the LGM on the three most common benthic caridean shrimp species that exhibit a strong depth zonation on the Antarctic continental shelf. While the shallow-water species Chorismus antarcticus and Notocrangon antarcticus were limited to a drastically reduced habitat during the LGM, the deep-water shrimp Nematocarcinus lanceopes found refuge in the Southern Ocean deep sea. The modeling results are in accordance with genetic diversity patterns available for C. antarcticus and N. lanceopes and support the hypothesis that habitat contraction at the LGM resulted in a loss of genetic diversity in shallow water benthos.
PMCID: PMC3459913  PMID: 23029463
13.  Ionizing Radiation: how fungi cope, adapt, and exploit with the help of melanin 
Current opinion in microbiology  2008;11(6):525-531.
Life on Earth has always existed in the flux of ionizing radiation. However, fungi seem to interact with the ionizing radiation differently from other Earth’s inhabitants. Recent data show that melanized fungal species like those from Chernobyl’s reactor respond to ionizing radiation with enhanced growth. Fungi colonize space stations and adapt morphologically to extreme conditions. Radiation exposure causes upregulation of many key genes, and an inducible microhomology-mediated recombination pathway could be a potential mechanism of adaptive evolution in eukaryotes. The discovery of melanized organisms in high radiation environments, the space stations, Antarctic mountains, and in the reactor cooling water combined with phenomenon of ‘radiotropism’ raises the tantalizing possibility that melanins have functions analogous to other energy harvesting pigments such as chlorophylls.
PMCID: PMC2677413  PMID: 18848901
14.  The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica 
Brazilian Journal of Microbiology  2011;42(3):937-947.
The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island) and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano) soil, soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia), Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula, and Sporidiobolus, and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species. Several species isolated in our study have been previously reported to be Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7% produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4°C and 20°C, indicating that they could be metabolically active in the sampled substrates.
PMCID: PMC3768797  PMID: 24031709
yeasts; Antarctica; diversity; extracellular enzymes; mycosporines
15.  Gone with the currents: lack of genetic differentiation at the circum-continental scale in the Antarctic krill Euphausia superba 
BMC Genetics  2011;12:32.
Southern Ocean fauna represent a significant amount of global biodiversity, whose origin may be linked to glacial cycles determining local extinction/eradication with ice advance, survival of refugee populations and post-glacial re-colonization. This pattern implies high potential for differentiation in benthic shelf species with limited dispersal, yet consequences for pelagic organisms are less clear. The present study investigates levels of genetic variation and population structure of the Antarctic krill Euphausia superba using mitochondrial DNA and EST-linked microsatellite markers for an unprecedentedly comprehensive sampling of its populations over a circum-Antarctic range.
MtDNA (ND1) sequences and EST-linked microsatellite markers indicated no clear sign of genetic structure among populations over large geographic scales, despite considerable power to detect differences inferred from forward-time simulations. Based on ND1, few instances of genetic heterogeneity, not significant after correction for multiple tests, were detected between geographic or temporal samples. Neutrality tests and mismatch distribution based on mtDNA sequences revealed strong evidence of past population expansion. Significant positive values of the parameter g (a measure of population growth) were obtained from microsatellite markers using a coalescent-based genealogical method and suggested a recent start (60 000 - 40 000 years ago) for the expansion.
The results provide evidence of lack of genetic heterogeneity of Antarctic krill at large geographic scales and unequivocal support for recent population expansion. Lack of genetic structuring likely reflects the tight link between krill and circum-Antarctic ocean currents and is consistent with the hypothesis that differentiation processes in Antarctic species are largely influenced by dispersal potential, whereas small-scale spatial and temporal differentiation might be due to local conditions leading to genetic patchiness. The signal of recent population growth suggests differential impact of glacial cycles on pelagic Antarctic species, which experienced population expansion during glaciations with increased available habitat, versus sedentary benthic shelf species.
EST-linked microsatellites provide new perspectives to complement the results based on mtDNA and suggest that data-mining of EST libraries will be a useful approach to facilitate use of microsatellites for additional species.
PMCID: PMC3095564  PMID: 21486439
16.  Diversity and Distribution of Deep-Sea Shrimps in the Ross Sea Region of Antarctica 
PLoS ONE  2014;9(7):e103195.
Although decapod crustaceans are widespread in the oceans, only Natantia (shrimps) are common in the Antarctic. Because remoteness, depth and ice cover restrict sampling in the South Ocean, species distribution modelling is a useful tool for evaluating distributions. We used physical specimen and towed camera data to describe the diversity and distribution of shrimps in the Ross Sea region of Antarctica. Eight shrimp species were recorded: Chorismus antarcticus; Notocrangon antarcticus; Nematocarcinus lanceopes; Dendrobranchiata; Pasiphaea scotiae; Pasiphaea cf. ledoyeri; Petalidium sp., and a new species of Lebbeus. For the two most common species, N. antarcticus and N. lanceopes, we used maximum entropy modelling, based on records of 60 specimens and over 1130 observations across 23 sites in depths from 269 m to 3433 m, to predict distributions in relation to environmental variables. Two independent sets of environmental data layers at 0.05° and 0.5° resolution respectively, showed how spatial resolution affected the model. Chorismus antarcticus and N. antarcticus were found only on the continental shelf and upper slopes, while N. lanceopes, Lebbeus n. sp., Dendrobranchiata, Petalidium sp., Pasiphaea cf. ledoyeri, and Pasiphaea scotiae were found on the slopes, seamounts and abyssal plain. The environmental variables that contributed most to models for N. antarcticus were depth, chlorophyll-a concentration, temperature, and salinity, and for N. lanceopes were depth, ice concentration, seabed slope/rugosity, and temperature. The relative ranking, but not the composition of these variables changed in models using different spatial resolutions, and the predicted extent of suitable habitat was smaller in models using the finer-scale environmental layers. Our modelling indicated that shrimps were widespread throughout the Ross Sea region and were thus likely to play important functional role in the ecosystem, and that the spatial resolution of data needs to be considered both in the use of species distribution models.
PMCID: PMC4106907  PMID: 25051333
17.  Isolation of Novel Extreme-Tolerant Cyanobacteria from a Rock-Dwelling Microbial Community by Using Exposure to Low Earth Orbit▿ † 
Many cyanobacteria are known to tolerate environmental extremes. Motivated by an interest in selecting cyanobacteria for applications in space, we launched rocks from a limestone cliff in Beer, Devon, United Kingdom, containing an epilithic and endolithic rock-dwelling community of cyanobacteria into low Earth orbit (LEO) at a height of approximately 300 kilometers. The community was exposed for 10 days to isolate cyanobacteria that can survive exposure to the extreme radiation and desiccating conditions associated with space. Culture-independent (16S rRNA) and culture-dependent methods showed that the cyanobacterial community was composed of Pleurocapsales, Oscillatoriales, and Chroococcales. A single cyanobacterium, a previously uncharacterized extremophile, was isolated after exposure to LEO. We were able to isolate the cyanobacterium from the limestone cliff after exposing the rock-dwelling community to desiccation and vacuum (0.7 × 10−3 kPa) in the laboratory. The ability of the organism to survive the conditions in space may be linked to the formation of dense colonies. These experiments show how extreme environmental conditions, including space, can be used to select for novel microorganisms. Furthermore, it improves our knowledge of environmental tolerances of extremophilic rock-dwelling cyanobacteria.
PMCID: PMC2849240  PMID: 20154120
18.  A Conspicuous Clay Ovoid in Nakhla: Evidence for Subsurface Hydrothermal Alteration on Mars with Implications for Astrobiology 
Astrobiology  2014;14(8):651-693.
A conspicuous biomorphic ovoid structure has been discovered in the Nakhla martian meteorite, made of nanocrystalline iron-rich saponitic clay and amorphous material. The ovoid is indigenous to Nakhla and occurs within a late-formed amorphous mesostasis region of rhyolitic composition that is interstitial to two clinopyroxene grains with Al-rich rims, and contains acicular apatite crystals, olivine, sulfides, Ti-rich magnetite, and a new mineral of the rhoenite group. To infer the origin of the ovoid, a large set of analytical tools was employed, including scanning electron microscopy and backscattered electron imaging, wavelength-dispersive X-ray analysis, X-ray mapping, Raman spectroscopy, time-of-flight secondary ion mass spectrometry analysis, high-resolution transmission electron microscope imaging, and atomic force microscope topographic mapping. The concentric wall of the ovoid surrounds an originally hollow volume and exhibits internal layering of contrasting nanotextures but uniform chemical composition, and likely inherited its overall shape from a preexisting vesicle in the mesostasis glass. A final fibrous layer of Fe-rich phases blankets the interior surfaces of the ovoid wall structure. There is evidence that the parent rock of Nakhla has undergone a shock event from a nearby bolide impact that melted the rims of pyroxene and the interstitial matter and initiated an igneous hydrothermal system of rapidly cooling fluids, which were progressively mixed with fluids from the melted permafrost. Sharp temperature gradients were responsible for the crystallization of Al-rich clinopyroxene rims, rhoenite, acicular apatites, and the quenching of the mesostasis glass and the vesicle. During the formation of the ovoid structure, episodic fluid infiltration events resulted in the precipitation of saponite rinds around the vesicle walls, altered pyrrhotite to marcasite, and then isolated the ovoid wall structure from the rest of the system by depositing a layer of iron oxides/hydroxides. Carbonates, halite, and sulfates were deposited last within interstitial spaces and along fractures. Among three plausible competing hypotheses here, this particular abiotic scenario is considered to be the most reasonable explanation for the formation of the ovoid structure in Nakhla, and although compelling evidence for a biotic origin is lacking, it is evident that the martian subsurface contains niche environments where life could develop. Key Words: Biomorph—Clays—Search for life (biosignatures)—Martian meteorites—Hydrothermal systems. Astrobiology 14, 651–693.
PMCID: PMC4126275  PMID: 25046549
19.  Persistence of Biomarker ATP and ATP-Generating Capability in Bacterial Cells and Spores Contaminating Spacecraft Materials under Earth Conditions and in a Simulated Martian Environment▿  
Applied and Environmental Microbiology  2008;74(16):5159-5167.
Most planetary protection research has concentrated on characterizing viable bioloads on spacecraft surfaces, developing techniques for bioload reduction prior to launch, and studying the effects of simulated martian environments on microbial survival. Little research has examined the persistence of biogenic signature molecules on spacecraft materials under simulated martian surface conditions. This study examined how endogenous adenosine-5′-triphosphate (ATP) would persist on aluminum coupons under simulated martian conditions of 7.1 mbar, full-spectrum simulated martian radiation calibrated to 4 W m−2 of UV-C (200 to 280 nm), −10°C, and a Mars gas mix of CO2 (95.54%), N2 (2.7%), Ar (1.6%), O2 (0.13%), and H2O (0.03%). Cell or spore viabilities of Acinetobacter radioresistens, Bacillus pumilus, and B. subtilis were measured in minutes to hours, while high levels of endogenous ATP were recovered after exposures of up to 21 days. The dominant factor responsible for temporal reductions in viability and loss of ATP was the simulated Mars surface radiation; low pressure, low temperature, and the Mars gas composition exhibited only slight effects. The normal burst of endogenous ATP detected during spore germination in B. pumilus and B. subtilis was reduced by 1 or 2 orders of magnitude following, respectively, 8- or 30-min exposures to simulated martian conditions. The results support the conclusion that endogenous ATP will persist for time periods that are likely to extend beyond the nominal lengths of most surface missions on Mars, and planetary protection protocols prior to launch may require additional rigor to further reduce the presence and abundance of biosignature molecules on spacecraft surfaces.
PMCID: PMC2519281  PMID: 18567687
20.  Purification and characterization of pepsins A1 and A2 from the Antarctic rock cod Trematomus bernacchii 
The FEBS journal  2007;274(23):6152-6166.
The Antarctic notothenioid Trematomus bernacchii (rock cod) lives at a constant mean temperature of −1.9 °C. Gastric digestion under these conditions relies on the proteolytic activity of aspartic proteases such as pepsin. To understand the molecular mechanisms of Antarctic fish pepsins, T. bernacchii pepsins A1 and A2 were cloned, overexpressed in E. coli, purified and characterized with a number of biochemical and biophysical methods. The properties of these two Antarctic isoenzymes were compared to porcine pepsin and found to be unique in a number of ways. Fish pepsins were found to be more temperature sensitive, generally less active at lower pH and more sensitive to inhibition by pepstatin than the mesophilic counterpart. The specificity of Antarctic fish pepsins was similar but not identical to pig pepsin, likely owing to changes in the sequence of fish enzymes near the active site. Gene duplication of Antarctic rock cod pepsins is the likely mechanism for adaptation to the harsh temperature environment in which these enzymes must function.
PMCID: PMC2533623  PMID: 17976195
Rock cod; aspartic proteases; specificity; cold-adapted protein
21.  JAXA protein crystallization in space: ongoing improvements for growing high-quality crystals 
Journal of Synchrotron Radiation  2013;20(Pt 6):968-973.
The Japan Aerospace Exploration Agency’s ‘high-quality protein crystal growth’ project is introduced. If crystallization conditions were carefully fixed in ground-based experiments, high-quality protein crystals grew in microgravity in many experiments on the International Space Station, especially when a highly homogeneous protein sample and a viscous crystallization solution were employed.
The Japan Aerospace Exploration Agency (JAXA) started a high-quality protein crystal growth project, now called JAXA PCG, on the International Space Station (ISS) in 2002. Using the counter-diffusion technique, 14 sessions of experiments have been performed as of 2012 with 580 proteins crystallized in total. Over the course of these experiments, a user-friendly interface framework for high accessibility has been constructed and crystallization techniques improved; devices to maximize the use of the microgravity environment have been designed, resulting in some high-resolution crystal growth. If crystallization conditions were carefully fixed in ground-based experiments, high-quality protein crystals grew in microgravity in many experiments on the ISS, especially when a highly homogeneous protein sample and a viscous crystallization solution were employed. In this article, the current status of JAXA PCG is discussed, and a rational approach to high-quality protein crystal growth in microgravity based on numerical analyses is explained.
PMCID: PMC3795566  PMID: 24121350
microgravity; protein crystal; JAXA; counter-diffusion; Japan Experiment Module ‘Kibo’; protein depletion zone; impurity depletion zone
22.  Effect of Shadowing on Survival of Bacteria under Conditions Simulating the Martian Atmosphere and UV Radiation▿ †  
Spacecraft-associated spores and four non-spore-forming bacterial isolates were prepared in Atacama Desert soil suspensions and tested both in solution and in a desiccated state to elucidate the shadowing effect of soil particulates on bacterial survival under simulated Martian atmospheric and UV irradiation conditions. All non-spore-forming cells that were prepared in nutrient-depleted, 0.2-μm-filtered desert soil (DSE) microcosms and desiccated for 75 days on aluminum died, whereas cells prepared similarly in 60-μm-filtered desert soil (DS) microcosms survived such conditions. Among the bacterial cells tested, Microbacterium schleiferi and Arthrobacter sp. exhibited elevated resistance to 254-nm UV irradiation (low-pressure Hg lamp), and their survival indices were comparable to those of DS- and DSE-associated Bacillus pumilus spores. Desiccated DSE-associated spores survived exposure to full Martian UV irradiation (200 to 400 nm) for 5 min and were only slightly affected by Martian atmospheric conditions in the absence of UV irradiation. Although prolonged UV irradiation (5 min to 12 h) killed substantial portions of the spores in DSE microcosms (∼5- to 6-log reduction with Martian UV irradiation), dramatic survival of spores was apparent in DS-spore microcosms. The survival of soil-associated wild-type spores under Martian conditions could have repercussions for forward contamination of extraterrestrial environments, especially Mars.
PMCID: PMC2258572  PMID: 18083857
23.  Meristematic cell proliferation and ribosome biogenesis are decoupled in diamagnetically levitated Arabidopsis seedlings 
BMC Plant Biology  2013;13:124.
Cell growth and cell proliferation are intimately linked in the presence of Earth’s gravity, but are decoupled under the microgravity conditions present in orbiting spacecraft. New technologies to simulate microgravity conditions for long-duration experiments, with stable environmental conditions, in Earth-based laboratories are required to further our understanding of the effect of extraterrestrial conditions on the growth, development and health of living matter.
We studied the response of transgenic seedlings of Arabidopsis thaliana, containing either the CycB1-GUS proliferation marker or the DR5-GUS auxin-mediated growth marker, to diamagnetic levitation in the bore of a superconducting solenoid magnet. As a control, a second set of seedlings were exposed to a strong magnetic field, but not to levitation forces. A third set was exposed to a strong field and simulated hypergravity (2 g). Cell proliferation and cell growth cytological parameters were measured for each set of seedlings. Nucleolin immunodetection was used as a marker of cell growth. Collectively, the data indicate that these two fundamental cellular processes are decoupled in root meristems, as in microgravity: cell proliferation was enhanced whereas cell growth markers were depleted. These results also demonstrated delocalisation of auxin signalling in the root tip despite the fact that levitation of the seedling as a whole does not prevent the sedimentation of statoliths in the root cells.
In our model system, we found that diamagnetic levitation led to changes that are very similar to those caused by real- [e.g. on board the International Space Station (ISS)] or mechanically-simulated microgravity [e.g. using a Random Positioning Machine (RPM)]. These changes decoupled meristematic cell proliferation from ribosome biogenesis, and altered auxin polar transport.
PMCID: PMC3847623  PMID: 24006876
24.  A comparison between the 2010 and 2005 basic life support guidelines during simulated hypogravity and microgravity 
Current 2010 terrestrial (1Gz) CPR guidelines have been advocated by space agencies for hypogravity and microgravity environments, but may not be feasible. The aims of this study were to (1) evaluate rescuer performance over 1.5 min of external chest compressions (ECCs) during simulated Martian hypogravity (0.38Gz) and microgravity (μG) in relation to 1Gz and rest baseline and (2) compare the physiological costs of conducting ECCs in accordance with the 2010 and 2005 CPR guidelines.
Thirty healthy male volunteers, ranging from 17 to 30 years, performed four sets of 30 ECCs for 1.5 min using the 2010 and 2005 ECC guidelines during 1Gz, 0.38Gz and μG simulations (Evetts-Russomano (ER) method), achieved by the use of a body suspension device. ECC depth and rate, range of elbow flexion, post-ECC heart rate (HR), minute ventilation (VE), peak oxygen consumption (VO2peak) and rate of perceived exertion (RPE) were measured.
All volunteers completed the study. Mean ECC rate was achieved for all gravitational conditions, but true depth during simulated microgravity was not sufficient for the 2005 (28.5 ± 7.0 mm) and 2010 (32.9 ± 8.7 mm) guidelines, even with a mean range of elbow flexion of 15°. HR, VE and VO2peak increased to an average of 136 ± 22 bpm, 37.5 ± 10.3 L·min−1, 20.5 ± 7.6 mL·kg−1·min−1 for 0.38Gz and 161 ± 19 bpm, 58.1 ± 15.0 L·min−1, 24.1 ± 5.6 mL·kg−1·min−1 for μG from a baseline of 84 ± 15 bpm, 11.4 ± 5.9 L·min−1, 3.2 ± 1.1 mL·kg−1·min-1, respectively. RPE was the only variable to increase with the 2010 guidelines.
No additional physiological cost using the 2010 basic life support (BLS) guidelines was needed for healthy males performing ECCs for 1.5 min, independent of gravitational environment. This cost, however, increased for each condition tested when the two guidelines were compared. Effective ECCs were not achievable for both guidelines in simulated μG using the ER BLS method. This suggests that future implementation of an ER BLS in a simulated μG instruction programme as well as upper arm strength training is required to perform effective BLS in space.
PMCID: PMC3710155  PMID: 23849595
Basic life support; CPR guidelines; Hypogravity; Microgravity
25.  Resistance of Bacterial Endospores to Outer Space for Planetary Protection Purposes—Experiment PROTECT of the EXPOSE-E Mission 
Astrobiology  2012;12(5):445-456.
Spore-forming bacteria are of particular concern in the context of planetary protection because their tough endospores may withstand certain sterilization procedures as well as the harsh environments of outer space or planetary surfaces. To test their hardiness on a hypothetical mission to Mars, spores of Bacillus subtilis 168 and Bacillus pumilus SAFR-032 were exposed for 1.5 years to selected parameters of space in the experiment PROTECT during the EXPOSE-E mission on board the International Space Station. Mounted as dry layers on spacecraft-qualified aluminum coupons, the “trip to Mars” spores experienced space vacuum, cosmic and extraterrestrial solar radiation, and temperature fluctuations, whereas the “stay on Mars” spores were subjected to a simulated martian environment that included atmospheric pressure and composition, and UV and cosmic radiation. The survival of spores from both assays was determined after retrieval. It was clearly shown that solar extraterrestrial UV radiation (λ≥110 nm) as well as the martian UV spectrum (λ≥200 nm) was the most deleterious factor applied; in some samples only a few survivors were recovered from spores exposed in monolayers. Spores in multilayers survived better by several orders of magnitude. All other environmental parameters encountered by the “trip to Mars” or “stay on Mars” spores did little harm to the spores, which showed about 50% survival or more. The data demonstrate the high chance of survival of spores on a Mars mission, if protected against solar irradiation. These results will have implications for planetary protection considerations. Key Words: Planetary protection—Bacterial spores—Space experiment—Simulated Mars mission. Astrobiology 12, 445–456.
PMCID: PMC3371261  PMID: 22680691

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