Unveiling metabolic pathways involved in the extreme desiccation tolerance of an Atacama cyanobacterium.

Gloeocapsopsis dulcis strain AAB1 is an extremely xerotolerant cyanobacterium isolated from the Atacama Desert (i.e., the driest and oldest desert on Earth) that holds astrobiological significance due to its ability to biosynthesize compatible solutes at ultra-low water activities. We sequenced and assembled the G. dulcis genome de novo using a combination of long- and short-read sequencing, which resulted in high-quality consensus sequences of the chromosome and two plasmids. We leveraged the G. dulcis genome to generate a genome-scale metabolic model (iGd895) to simulate growth in silico. iGd895 represents, to our knowledge, the first genome-scale metabolic reconstruction developed for an extremely xerotolerant cyanobacterium. The model's predictive capability was assessed by comparing the in silico growth rate with in vitro growth rates of G. dulcis, in addition to the synthesis of trehalose. iGd895 allowed us to explore simulations of key metabolic processes such as essential pathways for water-stress tolerance, and significant alterations to reaction flux distribution and metabolic network reorganization resulting from water limitation. Our study provides insights into the potential metabolic strategies employed by G. dulcis, emphasizing the crucial roles of compatible solutes, metabolic water, energy conservation, and the precise regulation of reaction rates in their adaptation to water stress.

Dark microbiome and extremely low organics in Atacama fossil delta unveil Mars life detection limits.

Identifying unequivocal signs of life on Mars is one of the most important objectives for sending missions to the red planet. Here we report Red Stone, a 163-100 My alluvial fan-fan delta that formed under arid conditions in the Atacama Desert, rich in hematite and mudstones containing clays such as vermiculite and smectites, and therefore geologically analogous to Mars. We show that Red Stone samples display an important number of microorganisms with an unusual high rate of phylogenetic indeterminacy, what we refer to as "dark microbiome", and a mix of biosignatures from extant and ancient microorganisms that can be barely detected with state-of-the-art laboratory equipment. Our analyses by testbed instruments that are on or will be sent to Mars unveil that although the mineralogy of Red Stone matches that detected by ground-based instruments on the red planet, similarly low levels of organics will be hard, if not impossible to detect in Martian rocks depending on the instrument and technique used. Our results stress the importance in returning samples to Earth for conclusively addressing whether life ever existed on Mars.

Fundamental Science and Engineering Questions in Planetary Cave Exploration.

Nearly half a century ago, two papers postulated the likelihood of lunar lava tube caves using mathematical models. Today, armed with an array of orbiting and fly-by satellites and survey instrumentation, we have now acquired cave data across our solar system-including the identification of potential cave entrances on the Moon, Mars, and at least nine other planetary bodies. These discoveries gave rise to the study of planetary caves. To help advance this field, we leveraged the expertise of an interdisciplinary group to identify a strategy to explore caves beyond Earth. Focusing primarily on astrobiology, the cave environment, geology, robotics, instrumentation, and human exploration, our goal was to produce a framework to guide this subdiscipline through at least the next decade. To do this, we first assembled a list of 198 science and engineering questions. Then, through a series of social surveys, 114 scientists and engineers winnowed down the list to the top 53 highest priority questions. This exercise resulted in identifying emerging and crucial research areas that require robust development to ultimately support a robotic mission to a planetary cave-principally the Moon and/or Mars. With the necessary financial investment and institutional support, the research and technological development required to achieve these necessary advancements over the next decade are attainable. Subsequently, we will be positioned to robotically examine lunar caves and search for evidence of life within Martian caves; in turn, this will set the stage for human exploration and potential habitation of both the lunar and Martian subsurface.

Emendation of the Coccoid Cyanobacterial Genus Gloeocapsopsis and Description of the New Species Gloeocapsopsis diffluens sp. nov. and Gloeocapsopsis dulcis sp. nov. Isolated From the Coastal Range of the Atacama Desert (Chile).

The taxonomy of coccoid cyanobacteria, such as Chroococcidiopsis, Pleurocapsa, Chroococcus, Gloeothece, Gloeocapsa, Gloeocapsopsis, and the related recent genera Sinocapsa and Aliterella, can easily be intermixed when solely compared on a morphological basis. There is still little support on the taxonomic position of some of the addressed genera, as genetic information is available only for a fraction of species that have been described solely on morphology. Modern polyphasic approaches that combine classic morphological investigations with DNA-based molecular analyses and the evaluation of ecological properties can disentangle these easily confusable unicellular genera. By using such an approach, we present here the formal description of two novel unicellular cyanobacterial species that inhabit the Coastal Range of the Atacama Desert, Gloeocapsopsis dulcis (first reported as Gloeocapsopsis AAB1) and Gloeocapsopsis diffluens. Both species could be clearly separated from previously reported species by 16S rRNA and 16S-23S ITS gene sequencing, the resulting secondary structures, p-distance analyses of the 16S-23S ITS, and morphology. For avoiding further confusions emendation of the genus Gloeocapsopsis as well as epitypification of the type species Gloeocapsopsis crepidinum based on the strain LEGE06123 were conducted.

Inhabited subsurface wet smectites in the hyperarid core of the Atacama Desert as an analog for the search for life on Mars.

The modern Martian surface is unlikely to be habitable due to its extreme aridity among other environmental factors. This is the reason why the hyperarid core of the Atacama Desert has been studied as an analog for the habitability of Mars for more than 50 years. Here we report a layer enriched in smectites located just 30 cm below the surface of the hyperarid core of the Atacama. We discovered the clay-rich layer to be wet (a phenomenon never observed before in this region), keeping a high and constant relative humidity of 78% (aw 0.780), and completely isolated from the changing and extremely dry subaerial conditions characteristic of the Atacama. The smectite-rich layer is inhabited by at least 30 halophilic species of metabolically active bacteria and archaea, unveiling a previously unreported habitat for microbial life under the surface of the driest place on Earth. The discovery of a diverse microbial community in smectite-rich subsurface layers in the hyperarid core of the Atacama, and the collection of biosignatures we have identified within the clays, suggest that similar shallow clay deposits on Mars may contain biosignatures easily reachable by current rovers and landers.

Can Halophilic and Psychrophilic Microorganisms Modify the Freezing/Melting Curve of Cold Salty Solutions? Implications for Mars Habitability.

We present the hypothesis that microorganisms can change the freezing/melting curve of cold salty solutions by protein expression, as it is known that proteins can affect the liquid-to-ice transition, an ability that could be of ecological advantage for organisms on Earth and on Mars. We tested our hypothesis by identifying a suitable candidate, the well-known psycrophile and halotolerant bacteria Rhodococcus sp. JG3, and analyzing its response in culture conditions that included specific hygroscopic salts relevant to Mars-that is, highly concentrated magnesium perchlorate solutions of 20 wt % and 50 wt % Mg(ClO4)2 at both end members of the eutectic concentration (44 wt %)-and subfreezing temperatures (263 K and 253 K). Using a combination of techniques of molecular microbiology and aqueous geochemistry, we evaluated the potential roles of proteins over- or underexpressed as important players in different mechanisms for the adaptability of life to cold environments. We recorded the changes observed by micro-differential scanning calorimetry. Unfortunately, Rhodococcus sp. JG3 did not show our hypothesized effect on the melting characteristics of cold Mg-perchlorate solutions. However, the question remains as to whether our novel hypothesis that halophilic/psychrophilic bacteria or archaea can alter the freezing/melting curve of salt solutions could be validated. The null result obtained after analyzing just one case lays the foundation to continue the search for proteins produced by microorganisms that thrive in very cold, high-saline solutions, which would involve testing different microorganisms with different salt components. The immediate implications for the habitability of Mars are discussed.

Metabolomics as an Emerging Tool in the Search for Astrobiologically Relevant Biomarkers.

It is now routinely possible to sequence and recover microbial genomes from environmental samples. To the degree it is feasible to assign transcriptional and translational functions to these genomes, it should be possible, in principle, to largely understand the complete molecular inputs and outputs of a microbial community. However, gene-based tools alone are presently insufficient to describe the full suite of chemical reactions and small molecules that compose a living cell. Metabolomic tools have developed quickly and now enable rapid detection and identification of small molecules within biological and environmental samples. The convergence of these technologies will soon facilitate the detection of novel enzymatic activities, novel organisms, and potentially extraterrestrial life-forms on solar system bodies. This review explores the methodological problems and scientific opportunities facing researchers who hope to apply metabolomic methods in astrobiology-related fields, and how present challenges might be overcome.

Author Correction: Aeolian transport of viable microbial life across the Atacama Desert, Chile: Implications for Mars.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The beauty and the yeast: can the microalgae Dunaliella form a borderline lichen with Hortaea werneckii?

Lichenized fungi usually develop complex, stratified morphologies through an intricately balanced living together with their algal partners, but several species are known to form only more or less loose associations with algae. These borderline lichens are still little explored although they could inform us about early stages of lichen evolution. We studied the association of the extremely halotolerant fungus Hortaea werneckii with the alga Dunaliella atacamensis, discovered in a cave in the Atacama Desert (Chile), and with D. salina, common inhabitant of saltern brines. D. atacamensis forms small colonies, in which cells of H. werneckii can be frequently observed, while such interaction has not been observed with D. salina. As symbiotic interactions between Dunaliella and Hortaea have not been reported, we performed a series of co-cultivation experiments to inspect whether these species could interact and develop more distinct lichen-like symbiotic structures. We set up co-cultures between axenic strains of Hortaea werneckii (isolated both from Mediterranean salterns and from the Atacama cave) and isolates of D. atacamensis (from the Atacama cave) and D. salina (isolated from Mediterranean salterns). Although we used different growth media and cultivation approaches, bright field and SEM microscopy analyses did not indicate any mutual effects in these experiments. We discuss the implications for fungal algal interactions along the transition from algal exploiters to lichen symbioses.

Aeolian transport of viable microbial life across the Atacama Desert, Chile: Implications for Mars.

Here we inspect whether microbial life may disperse using dust transported by wind in the Atacama Desert in northern Chile, a well-known Mars analog model. By setting a simple experiment across the hyperarid core of the Atacama we found that a number of viable bacteria and fungi are in fact able to traverse the driest and most UV irradiated desert on Earth unscathed using wind-transported dust, particularly in the later afternoon hours. This finding suggests that microbial life on Mars, extant or past, may have similarly benefited from aeolian transport to move across the planet and find suitable habitats to thrive and evolve.

The extremely halotolerant black yeast Hortaea werneckii - a model for intraspecific hybridization in clonal fungi.

The polymorphic black yeast Hortaea werneckii (Capnodiales, Ascomycota) is extremely halotolerant (growth from 0 to 30% [w/v] NaCl) and has been extensively studied as a model for halotolerance in Eukaryotes for over two decades. Its most frequent sources are hypersaline environments and adjacent sea-water habitats in temperate, subtropical and tropical climates. Although typically saprobic, H. werneckii can also act as a commensal coloniser on human skin, causing tinea nigra on hands and soles. Here, we report that addition of NaCl to culture media expands the growth range of H. werneckii to 37 °C, which explains its colonisation of human skin, with its increased salinity. The morphological and physiological plasticity/ versatility of H. werneckii indicate that a species complex might be involved. This was investigated in this polyphasic taxonomic analysis based on the global diversity of H. werneckii strains collected from hypersaline environments, and from humans and animals. Analysis of D1/D2domains of 28S and internal transcribed spacer rDNA revealed 10 and 17 genotypes, respectively, that were not always compliant. The genotypes have global distributions. Human and environmental strains with the same genotypes are intermingled. Due to the limited number of phylogenetically informative characters in the ribosomal DNA dataset, the partial genes encoding for ß-tubulin (BTB) and mini-chromosome maintenance protein (MCM7) were also sequenced. The use of these genes was hampered by ambiguous sequences obtained by Sanger sequencing, as a consequence of the diploid and highly heterozygous genome of many H. werneckii strains. Analysis of the BTB and MCM7 genes showed that in some cases two copies of the gene from the same genome are positioned in distant phylogenetic clusters of the intraspecific gene tree. Analysis of whole-genome sequences of selected H. werneckii strains generally confirmed the phylogenetic distances estimated on the basis of ribosomal genes, but also showed substantial reticulation within the phylogenetic history of the strains. This is in line with the hypothesis that the diploid genomes of H. werneckii were formed by hybridizations, which have sometimes occurred between relatively divergent strains.

Draft Genome Sequence of the Extremely Desiccation-Tolerant Cyanobacterium Gloeocapsopsis sp. Strain AAB1.

Gloeocapsopsis sp. strain AAB1 is an extremely desiccation-tolerant cyanobacterium isolated from translucent quartz stones from the Atacama Desert (Chile). Here, we report its draft genome sequence, which consists of 137 contigs with an ∼5.4-Mb genome size. The annotation revealed 5,641 coding DNA sequences, 38 tRNA genes, and 5 rRNA genes.

The Hyperarid Core of the Atacama Desert, an Extremely Dry and Carbon Deprived Habitat of Potential Interest for the Field of Carbon Science.

The Atacama Desert in Chile is the driest and oldest desert on Earth, also considered one of the best Mars analog models. Here, several heterotrophic microbial communities have been discovered in its driest regions, with the ones present in the soil subsurface being one of the most interesting due to its existence in a habitat with almost no water available and almost undetectable organic carbon sources. Our recent discovery of the driest site of the Atacama known to date (and the heterotrophic microbial species that are able to survive in this site) reaffirms the opportunity to better characterize the physiological and molecular mechanisms that these species use to detect, mobilize, incorporate and use carbon under these extremely harsh conditions. Here we summarize what has been reported up to date on the organic carbon concentrations in different sites of the hyperarid core of the Atacama Desert, proposing that due to the meager amounts of carbon and extremely dry conditions, the microbial communities of the hyperarid core of the Atacama Desert may be of interest for the field of carbon science.

Aspergillus atacamensis and A. salisburgensis: two new halophilic species from hypersaline/arid habitats with a phialosimplex-like morphology.

Halophilic fungal strains isolated from historical wooden staircase in a salt mine in Austria, and from wall biofilm and soil of a cave in the Coastal Range of the hyperarid Atacama Desert in Chile were characterised and described newly as Aspergillus salisburgensis and Aspergillus atacamensis. Morphological characters including solitary phialides producing solitary conidia and conidia in chains and/or heads suggested affinity to Aspergillus subgenus Polypaecilum. Strains required salt for growth, grew optimally on media with 10-25% NaCl and at 15-28 °C. These values are similar to those observed for Aspergillus salinarus comb. nov. (Phialosimplex salinarum), while the ex-type strains of Aspergillus sclerotialis, Aspergillus chlamydosporus and Aspergillus caninus (all belonging to Aspergillus subgen. Polypaecilum) grew optimally at 0-5% NaCl and showed fastest growth at 28-37 °C. Phylogenetic analyses on the basis of rDNA sequences, RAPD-PCR fingerprint patterns, and cellobiohydrolase gene (cbh-I) polymorphism clustered the strains into three groups and supported their taxonomic recognition as A. salinarus, A. atacamensis and A. salisburgensis. On the basis of phylogenetic inferences, also Sagenomella keratitidis is newly combined as Aspergillus keratitidis and inferred as a species of Aspergillus subgenus Polypaecilum.

Extremely high UV-C radiation resistant microorganisms from desert environments with different manganese concentrations.

Desiccation resistance and a high intracellular Mn/Fe ratio contribute to ionizing radiation resistance of Deinococcus radiodurans. We hypothesized that this was a general phenomenon and thus developed a strategy to search for highly radiation-resistant organisms based on their natural environment. While desiccation is a typical feature of deserts, the correlation between radiation resistance and the intracellular Mn/Fe ratio of indigenous microorganisms or the Mn/Fe ratio of the environment, has not yet been described. UV-C radiation is highly damaging to biomolecules including DNA. It was used in this study as a selective tool because of its relevance to early life on earth, high altitude aerobiology and the search for life beyond Earth. Surface soil samples were collected from the Sonoran Desert, Arizona (USA), from the Atacama Desert in Chile and from a manganese mine in northern Argentina. Microbial isolates were selected after exposure to UV-C irradiation and growth. The isolates comprised 28 genera grouped within six phyla, which we ranked according to their resistance to UV-C irradiation. Survival curves were performed for the most resistant isolates and correlated with their intracellular Mn/Fe ratio, which was determined by ICP-MS. Five percent of the isolates were highly resistant, including one more resistant than D. radiodurans, a bacterium generally considered the most radiation-resistant organism, thus used as a model for radiation resistance studies. No correlation was observed between the occurrence of resistant microorganisms and the Mn/Fe ratio in the soil samples. However, all resistant isolates showed an intracellular Mn/Fe ratio much higher than the sensitive isolates. Our findings could represent a new front in efforts to harness mechanisms of UV-C radiation resistance from extreme environments.

Discovery and microbial content of the driest site of the hyperarid Atacama Desert, Chile.

The Atacama Desert is the driest and oldest desert on Earth. Eleven years ago, the Yungay region was established as the driest site of this hyperarid desert and also close to the dry limit for life on Earth. Since then, much has been published about the extraordinary characteristics of this site and its pertinence as a Mars analogue model. However, as a result of a more systematic search in the Atacama here, we describe a new site, María Elena South (MES), which is much drier than Yungay. The mean atmospheric relative humidity (RH) at MES was 17.3%, with the RH of its soils remaining at a constant 14% at the depth of 1 m, a value that matches the lowest RH measurements taken by the Mars Science Laboratory at Gale Crater. Remarkably, we found a number of viable bacterial species in the soil profile at MES using a combination of molecular dependent and independent methods, unveiling the presence of life in the driest place on the Atacama Desert reported to date.

Biotechnological applications derived from microorganisms of the Atacama Desert.

The Atacama Desert in Chile is well known for being the driest and oldest desert on Earth. For these same reasons, it is also considered a good analog model of the planet Mars. Only a few decades ago, it was thought that this was a sterile place, but in the past years fascinating adaptations have been reported in the members of the three domains of life: low water availability, high UV radiation, high salinity, and other environmental stresses. However, the biotechnological applications derived from the basic understanding and characterization of these species, with the notable exception of copper bioleaching, are still in its infancy, thus offering an immense potential for future development.