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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.  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
4.  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
5.  Resistance of the Lichen Buellia frigida to Simulated Space Conditions during the Preflight Tests for BIOMEX—Viability Assay and Morphological Stability 
Astrobiology  2015;15(8):601-615.
Samples of the extremotolerant Antarctic endemite lichen Buellia frigida are currently exposed to low-Earth orbit–space and simulated Mars conditions at the Biology and Mars Experiment (BIOMEX), which is part of the ESA mission EXPOSE-R2 on the International Space Station and was launched on 23 July 2014. In preparation for the mission, several preflight tests (Experimental and Scientific Verification Tests, EVT and SVT) assessed the sample preparation and hardware integration procedures as well as the resistance of the candidate organism toward the abiotic stressors experienced under space and Mars conditions. Therefore, we quantified the post-exposure viability with a live/dead staining technique utilizing FUN-1 and confocal laser scanning microscopy (CLSM). In addition, we used scanning electron microscopy (SEM) to investigate putative patterns of morphological-anatomical damage that lichens may suffer under the extreme exposure conditions. The present results demonstrate that Buellia frigida is capable of surviving the conditions tested in EVT and SVT. The mycobiont showed lower average impairment of its viability than the photobiont (viability rates of >83% and >69%, respectively), and the lichen thallus suffered no significant damage in terms of thalline integrity and symbiotic contact. These results will become essential to substantiate and validate the results prospectively obtained from the returning space mission. Moreover, they will help assess the limits and limitations of terrestrial organisms under space and Mars conditions as well as characterize the adaptive traits that confer lichen extremotolerance. Key Words: Astrobiology—BIOMEX—EXPOSE-R2—Extremotolerance—Lichens. Astrobiology 15, 601–615.
PMCID: PMC4554929  PMID: 26218403
6.  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
7.  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
8.  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
9.  Zygomycetes in Vesicular Basanites from Vesteris Seamount, Greenland Basin – A New Type of Cryptoendolithic Fungi 
PLoS ONE  2015;10(7):e0133368.
Fungi have been recognized as a frequent colonizer of subseafloor basalt but a substantial understanding of their abundance, diversity and ecological role in this environment is still lacking. Here we report fossilized cryptoendolithic fungal communities represented by mainly Zygomycetes and minor Ascomycetes in vesicles of dredged volcanic rocks (basanites) from the Vesteris Seamount in the Greenland Basin. Zygomycetes had not been reported from subseafloor basalt previously. Different stages in zygospore formation are documented in the studied samples, representing a reproduction cycle. Spore structures of both Zygomycetes and Ascomycetes are mineralized by romanechite-like Mn oxide phases, indicating an involvement in Mn(II) oxidation to form Mn(III,VI) oxides. Zygospores still exhibit a core of carbonaceous matter due to their resistance to degradation. The fungi are closely associated with fossiliferous marine sediments that have been introduced into the vesicles. At the contact to sediment infillings, fungi produced haustoria that penetrated and scavenged on the remains of fragmented marine organisms. It is most likely that such marine debris is the main carbon source for fungi in shallow volcanic rocks, which favored the establishment of vital colonies.
PMCID: PMC4504512  PMID: 26181773
10.  Phylogenetic and Functional Substrate Specificity for Endolithic Microbial Communities in Hyper-Arid Environments 
Under extreme water deficit, endolithic (inside rock) microbial ecosystems are considered environmental refuges for life in cold and hot deserts, yet their diversity and functional adaptations remain vastly unexplored. The metagenomic analyses of the communities from two rock substrates, calcite and ignimbrite, revealed that they were dominated by Cyanobacteria, Actinobacteria, and Chloroflexi. The relative distribution of major phyla was significantly different between the two substrates and biodiversity estimates, from 16S rRNA gene sequences and from the metagenomic data, all pointed to a higher taxonomic diversity in the calcite community. While both endolithic communities showed adaptations to extreme aridity and to the rock habitat, their functional capabilities revealed significant differences. ABC transporters and pathways for osmoregulation were more diverse in the calcite chasmoendolithic community. In contrast, the ignimbrite cryptoendolithic community was enriched in pathways for secondary metabolites, such as non-ribosomal peptides (NRP) and polyketides (PK). Assemblies of the metagenome data produced population genomes for the major phyla found in both communities and revealed a greater diversity of Cyanobacteria population genomes for the calcite substrate. Draft genomes of the dominant Cyanobacteria in each community were constructed with more than 93% estimated completeness. The two annotated proteomes shared 64% amino acid identity and a significantly higher number of genes involved in iron update, and NRPS gene clusters, were found in the draft genomes from the ignimbrite. Both the community-wide and genome-specific differences may be related to higher water availability and the colonization of large fissures and cracks in the calcite in contrast to a harsh competition for colonization space and nutrient resources in the narrow pores of the ignimbrite. Together, these results indicated that the habitable architecture of both lithic substrates- chasmoendolithic versus cryptoendolithic – might be an essential element in determining the colonization and the diversity of the microbial communities in endolithic substrates at the dry limit for life.
PMCID: PMC4784552  PMID: 27014224
endoliths; cyanobacteria; extreme environment; Atacama Desert; hyper-arid environment; metagenomics
11.  Adaptation strategies of endolithic chlorophototrophs to survive the hyperarid and extreme solar radiation environment of the Atacama Desert 
The Atacama Desert, northern Chile, is one of the driest deserts on Earth and, as such, a natural laboratory to explore the limits of life and the strategies evolved by microorganisms to adapt to extreme environments. Here we report the exceptional adaptation strategies of chlorophototrophic and eukaryotic algae, and chlorophototrophic and prokaryotic cyanobacteria to the hyperarid and extremely high solar radiation conditions occurring in this desert. Our approach combined several microscopy techniques, spectroscopic analytical methods, and molecular analyses. We found that the major adaptation strategy was to avoid the extreme environmental conditions by colonizing cryptoendolithic, as well as, hypoendolithic habitats within gypsum deposits. The cryptoendolithic colonization occurred a few millimeters beneath the gypsum surface and showed a succession of organized horizons of algae and cyanobacteria, which has never been reported for endolithic microbial communities. The presence of cyanobacteria beneath the algal layer, in close contact with sepiolite inclusions, and their hypoendolithic colonization suggest that occasional liquid water might persist within these sub-microhabitats. We also identified the presence of abundant carotenoids in the upper cryptoendolithic algal habitat and scytonemin in the cyanobacteria hypoendolithic habitat. This study illustrates that successful lithobiontic microbial colonization at the limit for microbial life is the result of a combination of adaptive strategies to avoid excess solar irradiance and extreme evapotranspiration rates, taking advantage of the complex structural and mineralogical characteristics of gypsum deposits—conceptually called “rock's habitable architecture.” Additionally, self-protection by synthesis and accumulation of secondary metabolites likely produces a shielding effect that prevents photoinhibition and lethal photooxidative damage to the chlorophototrophs, representing another level of adaptation.
PMCID: PMC4564735  PMID: 26441871
Atacama Desert; carotenoids; endolithic chlorophototrophs; extreme environment; gypsum; scytonemin
12.  Extremotolerant fungi from alpine rock lichens and their phylogenetic relationships 
Fungal Diversity  2015;76:119-142.
Fungi other than the lichen mycobiont frequently co-occur within lichen thalli and on the same rock in harsh environments. In these situations dark-pigmented mycelial structures are commonly observed on lichen thalli, where they persist under the same stressful conditions as their hosts. Here we used a comprehensive sampling of lichen-associated fungi from an alpine habitat to assess their phylogenetic relationships with fungi previously known from other niches. The multilocus phylogenetic analyses suggest that most of the 248 isolates belong to the Chaetothyriomycetes and Dothideomycetes, while a minor fraction represents Sordariomycetes and Leotiomycetes. As many lichens also were infected by phenotypically distinct lichenicolous fungi of diverse lineages, it remains difficult to assess whether the culture isolates represent these fungi or are from additional cryptic, extremotolerant fungi within the thalli. Some of these strains represent yet undescribed lineages within Chaethothyriomycetes and Dothideomycetes, whereas other strains belong to genera of fungi, that are known as lichen colonizers, plant and human pathogens, rock-inhabiting fungi, parasites and saprotrophs. The symbiotic structures of the lichen thalli appear to be a shared habitat of phylogenetically diverse stress-tolerant fungi, which potentially benefit from the lichen niche in otherwise hostile habitats.
Electronic supplementary material
The online version of this article (doi:10.1007/s13225-015-0343-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4739527  PMID: 26877720
Black fungi; Endolichenic; Symbioses; Lichenicolous; Life style; Phylogeny
13.  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
14.  Simulations of gravitational stress on normovolemic and hypovolemic men and women 
Earth based simulations of physiologic responses to space mission activities are needed to develop prospective countermeasures. To determine whether upright lower body positive pressure (LBPP) provides a suitable space mission simulation, we investigated cardiovascular responses of normovolemic and hypovolemic, men and women, to supine and orthostatic stress, induced by head-up tilt (HUT) and upright LBPP, representing standing in lunar, Martian and Earth’s gravities.
Six men and six women were tested in normovolemic and hypovolemic (furosemide, intravenous, 0.5 mg/kg) conditions. Continuous electrocardiogram, blood pressure, segmental bioimpedance and stroke volume (echocardiography) were recorded supine and at lunar, Martian and Earth’s gravities (10°, 20°, 80° HUT vs. 20%, 40%, 100% body weight upright LBPP), respectively. Cardiovascular responses were assessed from mean values, spectral powers and spontaneous baroreflex parameters.
Hypovolemia reduced plasma volume by ~10% and stroke volume by ~25% at supine and increasing orthostatic stress resulted in further reductions. Upright LBPP induced more plasma volume losses at simulated lunar and Martian gravities compared with HUT, while both techniques induced comparable central hypovolemia at each stress. Cardiovascular responses to orthostatic stress were comparable between HUT and upright LBPP in both normovolemic and hypovolemic conditions, however, hypovolemic blood pressure was greater during standing at 100% body weight compared to 80° HUT due to a greater increase of total peripheral resistance.
HUT and upright LBPP induced comparable cardiovascular responses, supporting the use of upright LBPP as a potential model to simulate activity in lunar and Martian gravities.
PMCID: PMC4376358  PMID: 24754201
Blood pressure regulation; hypovolemia; baroreflex
15.  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
16.  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
17.  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
18.  Characterization of Aspergillus fumigatus Isolates from Air and Surfaces of the International Space Station 
mSphere  2016;1(5):e00227-16.
As durations of manned space missions increase, it is imperative to understand the long-term consequence of microbial exposure on human health in a closed human habitat. To date, studies aimed at bacterial and fungal contamination of space vessels have highlighted species compositions biased toward hardy, persistent organisms capable of withstanding harsh conditions. In the current study, we assessed traits of two independent Aspergillus fumigatus strains isolated from the International Space Station. Ubiquitously found in terrestrial soil and atmospheric environments, A. fumigatus is a significant opportunistic fungal threat to human health, particularly among the immunocompromised. Using two well-known clinical isolates of A. fumigatus as comparators, we found that both ISS isolates exhibited normal in vitro growth and chemical stress tolerance yet caused higher lethality in a vertebrate model of invasive disease. These findings substantiate the need for additional studies of physical traits and biological activities of microbes adapted to microgravity and other extreme extraterrestrial conditions.
One mission of the Microbial Observatory Experiments on the International Space Station (ISS) is to examine the traits and diversity of fungal isolates to gain a better understanding of how fungi may adapt to microgravity environments and how this may affect interactions with humans in a closed habitat. Here, we report an initial characterization of two isolates, ISSFT-021 and IF1SW-F4, of Aspergillus fumigatus collected from the ISS and a comparison to the experimentally established clinical isolates Af293 and CEA10. Whole-genome sequencing of ISSFT-021 and IF1SW-F4 showed 54,960 and 52,129 single nucleotide polymorphisms, respectively, compared to Af293, which is consistent with observed genetic heterogeneity among sequenced A. fumigatus isolates from diverse clinical and environmental sources. Assessment of in vitro growth characteristics, secondary metabolite production, and susceptibility to chemical stresses revealed no outstanding differences between ISS and clinical strains that would suggest special adaptation to life aboard the ISS. Virulence assessment in a neutrophil-deficient larval zebrafish model of invasive aspergillosis revealed that both ISSFT-021 and IF1SW-F4 were significantly more lethal than Af293 and CEA10. Taken together, these genomic, in vitro, and in vivo analyses of two A. fumigatus strains isolated from the ISS provide a benchmark for future investigations of these strains and for continuing research on specific microbial isolates from manned space environments.
IMPORTANCE As durations of manned space missions increase, it is imperative to understand the long-term consequence of microbial exposure on human health in a closed human habitat. To date, studies aimed at bacterial and fungal contamination of space vessels have highlighted species compositions biased toward hardy, persistent organisms capable of withstanding harsh conditions. In the current study, we assessed traits of two independent Aspergillus fumigatus strains isolated from the International Space Station. Ubiquitously found in terrestrial soil and atmospheric environments, A. fumigatus is a significant opportunistic fungal threat to human health, particularly among the immunocompromised. Using two well-known clinical isolates of A. fumigatus as comparators, we found that both ISS isolates exhibited normal in vitro growth and chemical stress tolerance yet caused higher lethality in a vertebrate model of invasive disease. These findings substantiate the need for additional studies of physical traits and biological activities of microbes adapted to microgravity and other extreme extraterrestrial conditions.
PMCID: PMC5082629  PMID: 27830189
Aspergillus fumigatus; International Space Station; SNP analysis; secondary metabolites; virulence
19.  Niche Filtering of Bacteria in Soil and Rock Habitats of the Colorado Plateau Desert, Utah, USA 
A common feature of microbial colonization in deserts is biological soil crusts (BSCs), and these comprise a complex community dominated by Cyanobacteria. Rock substrates, particularly sandstone, are also colonized by microbial communities. These are separated by bare sandy soil that also supports microbial colonization. Here we report a high-throughput sequencing study of BSC and cryptoendolith plus adjacent bare soil communities in the Colorado Plateau Desert, Utah, USA. Bare soils supported a community with low levels of recoverable DNA and high evenness, whilst BSC yielded relatively high recoverable DNA, and reduced evenness compared to bare soil due to specialized crust taxa. The cryptoendolithic community displayed the greatest evenness but the lowest diversity, reflecting the highly specialized nature of these communities. A strong substrate-dependent pattern of community assembly was observed, and in particular cyanobacterial taxa were distinct. Soils were virtually devoid of photoautotrophic signatures, BSC was dominated by a closely related group of Microcoleus/Phormidium taxa, whilst cryptoendolithic colonization in sandstone supported almost exclusively a single genus, Chroococcidiopsis. We interpret this as strong evidence for niche filtering of taxa in communities. Local inter-niche recruitment of photoautotrophs may therefore be limited and so communities likely depend significantly on cyanobacterial recruitment from distant sources of similar substrate. We discuss the implication of this finding in terms of conservation and management of desert microbiota.
PMCID: PMC5035745  PMID: 27725810
biological soil crust; cryptoendolith; Cyanobacteria; desert; Utah
20.  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
21.  Photosynthetic Carbon Incorporation and Turnover in Antarctic Cryptoendolithic Microbial Communities: Are They the Slowest-Growing Communities on Earth? 
The main forms of terrestrial life in the cold, desolate Ross Desert of Antarctica are lichen-dominated or cyanobacterium-dominated cryptoendolithic (hidden in rock) microbial communities. Though microbial community biomass (as measured by extractable lipid phosphate) was well within the range of values determined for other microbial communities, community lipid carbon turnover times (calculated from community lipid biomass, rates of community photosynthetic carbon incorporation into lipids versus temperature, and the in situ temperature record) were among the longest on Earth (ca. 20,000 years). When the temperature is above freezing and moisture is present, moderate rates of photosynthesis can be measured. Lichen communities had a psychrophilic temperature response (maximal rate of 4.5 ng of C h-1 m-2 at 10°C) while cyanobacteria communities had maximal rates at 20 to 30°C (3 ng of C h-1 m-2). These extraordinarily slowly growing communities were not nutrient limited. No significant changes in photosynthetic metabolism were observed upon additions of 100 nM to 1 mM nitrate, ammonium, phosphate, and manganese. These simple, tenacious microbial communities demonstrate strategies of survival under conditions normally considered too extreme for life.
PMCID: PMC183568  PMID: 16348539
22.  A rock-inhabiting ancestor for mutualistic and pathogen-rich fungal lineages 
Studies in Mycology  2008;61:111-119.
Rock surfaces are unique terrestrial habitats in which rapid changes in the intensity of radiation, temperature, water supply and nutrient availability challenge the survival of microbes. A specialised, but diverse group of free-living, melanised fungi are amongst the persistent settlers of bare rocks. Multigene phylogenetic analyses were used to study relationships of ascomycetes from a variety of substrates, with a dataset including a broad sampling of rock dwellers from different geographical locations. Rock-inhabiting fungi appear particularly diverse in the early diverging lineages of the orders Chaetothyriales and Verrucariales. Although these orders share a most recent common ancestor, their lifestyles are strikingly different. Verrucariales are mostly lichen-forming fungi, while Chaetothyriales, by contrast, are best known as opportunistic pathogens of vertebrates (e.g. Cladophialophora bantiana and Exophiala dermatitidis, both agents of fatal brain infections) and saprophytes. The rock-dwelling habit is shown here to be key to the evolution of these two ecologically disparate orders. The most recent common ancestor of Verrucariales and Chaetothyriales is reconstructed as a non-lichenised rock-inhabitant. Ancestral state reconstructions suggest Verrucariales as one of the independent ascomycetes group where lichenisation has evolved on a hostile rock surface that might have favored this shift to a symbiotic lifestyle. Rock-inhabiting fungi are also ancestral to opportunistic pathogens, as they are found in the early diverging lineages of Chaetothyriales. In Chaetothyriales and Verrucariales, specific morphological and physiological traits (here referred to as extremotolerance) evolved in response to stresses in extreme conditions prevailing on rock surfaces. These factors facilitated colonisation of various substrates including the brains of vertebrates by opportunistic fungal pathogens, as well as helped establishment of a stable lichen symbiosis.
PMCID: PMC2610302  PMID: 19287533
Evolution of rock-dwelling habit; evolution of lichenisation; multigene phylogeny; ancestral state reconstruction; Verrucariales and Chaetothyriales (Chaetothyriomycetidae, Eurotiomycetes)
23.  Diversity and distribution of lichen-associated fungi in the Ny-Ålesund Region (Svalbard, High Arctic) as revealed by 454 pyrosequencing 
Scientific Reports  2015;5:14850.
This study assessed the diversity and distribution of fungal communities associated with seven lichen species in the Ny-Ålesund Region (Svalbard, High Arctic) using Roche 454 pyrosequencing with fungal-specific primers targeting the internal transcribed spacer (ITS) region of the ribosomal rRNA gene. Lichen-associated fungal communities showed high diversity, with a total of 42,259 reads belonging to 370 operational taxonomic units (OTUs) being found. Of these OTUs, 294 belonged to Ascomycota, 54 to Basidiomycota, 2 to Zygomycota, and 20 to unknown fungi. Leotiomycetes, Dothideomycetes, and Eurotiomycetes were the major classes, whereas the dominant orders were Helotiales, Capnodiales, and Chaetothyriales. Interestingly, most fungal OTUs were closely related to fungi from various habitats (e.g., soil, rock, plant tissues) in the Arctic, Antarctic and alpine regions, which suggests that living in association with lichen thalli may be a transient stage of life cycle for these fungi and that long-distance dispersal may be important to the fungi in the Arctic. In addition, host-related factors shaped the lichen-associated fungal communities in this region. Taken together, these results suggest that lichens thalli act as reservoirs of diverse fungi from various niches, which may improve our understanding of fungal evolution and ecology in the Arctic.
PMCID: PMC4604449  PMID: 26463847
24.  Detection of Macromolecules in Desert Cyanobacteria Mixed with a Lunar Mineral Analogue After Space Simulations 
In the context of future exposure missions in Low Earth Orbit and possibly on the Moon, two desert strains of the cyanobacterium Chroococcidiopsis, strains CCMEE 029 and 057, mixed or not with a lunar mineral analogue, were exposed to fractionated fluencies of UVC and polychromatic UV (200–400 nm) and to space vacuum. These experiments were carried out within the framework of the BIOMEX (BIOlogy and Mars EXperiment) project, which aims at broadening our knowledge of mineral-microorganism interaction and the stability/degradation of their macromolecules when exposed to space and simulated Martian conditions. The presence of mineral analogues provided a protective effect, preserving survivability and integrity of DNA and photosynthetic pigments, as revealed by testing colony-forming abilities, performing PCR-based assays and using confocal laser scanning microscopy. In particular, DNA and pigments were still detectable after 500 kJ/m2 of polychromatic UV and space vacuum (10−4 Pa), corresponding to conditions expected during one-year exposure in Low Earth Orbit on board the EXPOSE-R2 platform in the presence of 0.1 % Neutral Density (ND) filter. After exposure to high UV fluencies (800 MJ/m2) in the presence of minerals, however, altered fluorescence emission spectrum of the photosynthetic pigments were detected, whereas DNA was still amplified by PCR. The present paper considers the implications of such findings for the detection of biosignatures in extraterrestrial conditions and for putative future lunar missions.
PMCID: PMC4669540  PMID: 25351683
Astrobiology; Extreme environments; Expose-R2; Biosignatures; Lunar regolith
25.  Expression at 279 K, purification, crystallization and preliminary X-ray crystallographic analysis of a novel cold-active β-1,4-d-mannanase from the Antarctic springtail Cryptopygus antarcticus  
A cold-active β-1,4-d-mannanase from the Antarctic springtail C. antarcticus (CaMan) has been expressed, purified and crystallized. The CaMan crystal belonged to space group P212121, with unit-cell parameters a = 73.40, b = 83.81, c = 163.55 Å, and diffracted to 2.35 Å resolution. A 2.40 Å resolution data set was also collected from a CaMan–mannopentaose complex crystal.
The CaMan gene product from Cryptopygus antarcticus, which belongs to the glycoside hydrolase family 5 type β-1,4-d-mannanases, has been crystallized using a precipitant solution consisting of 0.1 M Tris–HCl pH 8.5, 25%(w/v) polyethylene glycol 3350 by the microbatch crystallization method at 295 K. The CaMan protein crystal belonged to space group P212121, with unit-cell parameters a = 73.40, b = 83.81, c = 163.55 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be about 61.29%. CaMan–mannopentaose (M5) complex crystals that were isomorphous to the CaMan crystals were obtained using the same mother liquor containing 1 mM M5.
PMCID: PMC3758150  PMID: 23989150
β-1,4-d-mannanase; Cryptopygus antarcticus; glycoside hydrolase family 5

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