Geomicrobiology of a seawater-influenced active sulfuric acid cave.

Fetida Cave is an active sulfuric acid cave influenced by seawater, showing abundant microbial communities that organize themselves under three main different morphologies: water filaments, vermiculations and moonmilk deposits. These biofilms/deposits have different cave distribution, pH, macro- and microelement and mineralogical composition, carbon and nitrogen content. In particular, water filaments and vermiculations had circumneutral and slightly acidic pH, respectively, both had abundant organic carbon and high microbial diversity. They were rich in macro- and microelements, deriving from mineral dissolution, and, in the case of water filaments, from seawater composition. Vermiculations had different color, partly associated with their mineralogy, and unusual minerals probably due to trapping capacities. Moonmilk was composed of gypsum, poor in organic matter, had an extremely low pH (0\u20131) and low microbial diversity. Based on 16S rRNA gene analysis, the microbial composition of the biofilms/deposits included autotrophic taxa associated with sulfur and nitrogen cycles and biomineralization processes. In particular, water filaments communities were characterized by bacterial taxa involved in sulfur oxidation and reduction in aquatic, aphotic, microaerophilic/anoxic environments (Campylobacterales, Thiotrichales, Arenicellales, Desulfobacterales, Desulforomonadales) and in chemolithotrophy in marine habitats (Oceanospirillales, Chromatiales). Their biodiversity was linked to the morphology of the water filaments and their collection site. Microbial communities within vermiculations were partly related to their color and showed high abundance of unclassified Betaproteobacteria and sulfur-oxidizing Hydrogenophilales (including Sulfuriferula), and Acidiferrobacterales (including Sulfurifustis), sulfur-reducing Desulfurellales, and ammonia-oxidizing Planctomycetes and Nitrospirae. The microbial community associated with gypsum moonmilk showed the strong dominance (>60%) of the archaeal genus Thermoplasma and lower abundance of chemolithotrophic Acidithiobacillus, metal-oxidizing Metallibacterium, Sulfobacillus, and Acidibacillus. This study describes the geomicrobiology of water filaments, vermiculations and gypsum moonmilk from Fetida Cave, providing insights into the microbial taxa that characterize each morphology and contribute to biogeochemical cycles and speleogenesis of this peculiar seawater-influenced sulfuric acid cave

Global Proteome Analysis of Leptospira interrogans

Comparative global proteome analyses were performed on Leptospira interrogans serovar Copenhageni grown under conventional in vitro conditions and those mimicking in vivo conditions (iron limitation and serum presence). Proteomic analyses were conducted using iTRAQ and LC-ESI-tandem mass spectrometry complemented with two-dimensional gel electrophoresis and MALDI-TOF mass spec-trometry. A total of 563 proteins were identified in this study. Altered expression of 65 proteins, including upregulation of the L. interrogans virulence factor Loa22 and 5 novel proteins with homology to virulence factors found in other pathogens, was observed between the comparative conditions. Immunoblot analyses confirmed upregulation of 5 of the known or putative virulence factors in L. interrogans exposed to the in vivo-like environmental conditions. Further, ELISA analyses using serum from patients with leptospirosis and immunofluorescence studies performed on liver sections derived from L. interrogans-infected hamsters verified expression of all but one of the identified proteins during infection. These studies, which represent the first documented comparative global proteome analysis of Leptospira, demonstrated proteome alterations under conditions that mimic in vivo infection and allowed for the identification of novel putative L. interrogans virulence factors

MEXEM – Mars Exposed Extremophile Mixture – a space experiment to investigate the capability of anaerobic organisms to survive on Mars

Assessing the habitability of Mars and detecting life, if it ever existed there, depends on knowledge of whether the combined environmental stresses experienced on Mars are compatible with life as we know it and whether a record of that life could ever be detected. So far, only few investigations were performed to understand the combined effect of different environmental stresses on survival and growth of anaerobic and extremophilic organisms. In the space experiment MEXEM (formerly known as MASE-in-SPACE) the hypothesis will be tested that selected terrestrial organisms, enrichment cultures and original samples from extreme Mars-analogue environments on Earth are able to withstand the Martian environ- mental stress factors due to their highly effective cellular and molecular adaptation and repair mechanisms. In addition, artificially fossilized and aged isolates from Mars-analogue environments on Earth will be examined and assessed with respect to their suitability for biosigna- ture identification. MEXEM samples will be (i) oxygen-depleted natural sediment samples, (ii) natural sediments spiked with selected, defined strains representative for the respective analogue site, (iii) individual (facultative) anaerobic / micro-aerophilic species including ciliates and viruses, (iv) defined mixtures of these biological entities, (v) isolated strains from samples collected inside the ISS and (vi) artificially fossilized isolates from the natural environments. Most of these samples and isolates were obtained from Mars-analogue envi- ronments on Earth in the frame of the EC funded project MASE (Grant Agreement 607297) and from the space experiment EXTREMOPHILES (PI C. Moissl-Eichinger). MEXEM will be flown outside on the ISS in the new exobiology facility ESA is building now. It offers the possibility to simulate of the martian environment, in particular the martian UV cl e, which cannot be done in the lab, but also martian atmosphere and pressure in LEO

Limits of Life and the Habitability of Mars: The ESA Space Experiment BIOMEX on the ISS

BIOMEX (BIOlogy and Mars EXperiment) is an ESA/Roscosmos space exposure experiment housed within the exposure facility EXPOSE-R2 outside the Zvezda module on the International Space Station (ISS). The design of the multiuser facility supports—among others—the BIOMEX investigations into the stability and level of degradation of space-exposed biosignatures such as pigments, secondary metabolites, and cell surfaces in contact with a terrestrial and Mars analog mineral environment. In parallel, analysis on the viability of the investigated organisms has provided relevant data for evaluation of the habitability of Mars, for the limits of life, and for the likelihood of an interplanetary transfer of life (theory of lithopanspermia). In this project, lichens, archaea, bacteria, cyanobacteria, snow/permafrost algae, meristematic black fungi, and bryophytes from alpine and polar habitats were embedded, grown, and cultured on a mixture of martian and lunar regolith analogs or other terrestrial minerals. The organisms and regolith analogs and terrestrial mineral mixtures were then exposed to space and to simulated Mars-like conditions by way of the EXPOSE-R2 facility. In this special issue, we present the first set of data obtained in reference to our investigation into the habitability of Mars and limits of life. This project was initiated and implemented by the BIOMEX group, an international and interdisciplinary consortium of 30 institutes in 12 countries on 3 continents. Preflight tests for sample selection, results from ground-based simulation experiments, and the space experiments themselves are presented and include a complete overview of the scientific processes required for this space experiment and postflight analysis. The presented BIOMEX concept could be scaled up to future exposure experiments on the Moon and will serve as a pretest in low Earth orbit

Comparative study of fungal cell disruption—scope and limitations of the methods

Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems

ANALOGUE SAMPLES IN AN EUROPEAN SAMPLE CURATION FACILITY - THE EURO-CARES PROJECT.

The objective of the H2020-funded EURO-CARES project (grant agreement n° 640190) was to create a roadmap for the implementation of a European Extraterrestrial Sample Curation Facility (ESCF) that would be suitable for the curation of samples from all possible return missions likely over the next few decades, i.e. from the Moon, asteroids and Mars. The return of extraterrestrial samples brought to Earth will require specific storage conditions and handling procedures, in particular for those coming from Mars. For practical reasons and sterility concerns it might be necessary for such a facility to have its own collection of analogue samples permitting the testing of storage conditions, and to develop protocols for sample prepartion and analyses. Within the framework of the EURO-CARES project, we havecreated a list of the different types of samples that would be relevant for such a curation facility. The facility will be used for receiving and opening of the returned sample canisters, as well as for handling and preparation of the returned samples. Furthermore, it will provide some analysis of the returned samples, i.e. early sample characterisation, and is expected to provide longterm storage of the returned samples. Each of these basic functions requires special equipment. Equipment, handling protocols and long-term storage conditions will strongly depend on the characteristics of the materials, and on whether returned samples are from the Moon, Mars or an asteroidal body. Therefore the different types and aspects of analogue samples one need to be considered, i.e. the nature of the materials, which analogues are needed for what purpose, what mass is needed, and how should the analogue samples be stored within the facility. We distinguished five different types of anologue samples: analogue (s.s.), witness plate, voucher specimen, reference sample, and standard. Analogues are materials that have one or more physical or chemical properties similar to Earth-returned extraterrestrial samples. Reference samples are well-characterised materials with known physical and chemical properties used for testing. They may not necessarily be the same materials as the analogues defined above. Standards are internationally recognised, homogeneous materials with known physical and chemical properties that are used for calibration. They can also be used as reference samples in certain circumstances. They may be made of natural materials but are often produced artificially. A voucher specimen is a duplicate of materials used at any stage during sample acquisition, storage, transport, treatment etc., e.g. spacecraft materials (including solar panels), lubricants, glues, gloves, saws, drills, and others. In addition, Earth landing site samples (from the touch down site) would be necessary in case of doubtful analysis, even if normally this type of contamination is not expected. Finally, a witness plate is defined as material left in an area where work is being done to detect any biological, particulate, chemical, and/or organic contamination. It is a spatial and temporal document of what happens in the work area. Analogue materials could be solids (including ices), liquids or gases. These could contain biological (extant and/or exinct) and/or organic components. They could be natural materials, e.g. rocks or minerals, or could be manufactured, such as mixtures of different components, which may be biologically and/or organically doped. Analogues with appropriate sample size and nature will be well-suited for testing and training of sample handling procedures, and for transport protocols. The training of science and curation teams also requires reference samples and standards. Long-term storage needs special witness plates and voucher specimes. Developing and testing sample preparation protocols needs all sample types

The responses of an anaerobic microorganism, Yersinia intermedia MASE-LG-1 to individual and combined simulated Martian stresses

The limits of life of aerobic microorganisms are well understood, but the responses of anaerobic microorganisms to individual and combined extreme stressors are less well known. Motivated by an interest in understanding the survivability of anaerobic microorganisms under Martian conditions, we investigated the responses of a new isolate, Yersinia intermedia MASE-LG-1 to individual and combined stresses associated with the Martian surface. This organism belongs to an adaptable and persistent genus of anaerobic microorganisms found in many environments worldwide. The effects of desiccation, low pressure, ionizing radiation, varying temperature, osmotic pressure, and oxidizing chemical compounds were investigated. The strain showed a high tolerance to desiccation, with a decline of survivability by four orders of magnitude during a storage time of 85 days. Exposure to X-rays resulted in dose-dependent inactivation for exposure up to 600 Gy while applied doses above 750 Gy led to complete inactivation. The effects of the combination of desiccation and irradiation were additive and the survivability was influenced by the order in which they were imposed. Ionizing irradiation and subsequent desiccation was more deleterious than vice versa. By contrast, the presence of perchlorates was not found to significantly affect the survival of the Yersinia strain after ionizing radiation. These data show that the organism has the capacity to survive and grow in physical and chemical stresses, imposed individually or in combination that are associated with Martian environment. Eventually it lost its viability showing that many of the most adaptable anaerobic organisms on Earth would be killed on Mars today

The contamination impact of human exploration to a subterranean environment and the implications for further crewed space exploration

The quest of exploring and looking for life in new places is a human desire since centuries. Nowadays, we are not only looking on planet Earth any more, but our endeavours focus on nearby planets in our solar system. It is therefore of great importance to preserve the extra-terrestrial environment and not to contaminate it with terrestrial / human associated bacteria. At this point in time we are not able to send crewed missions to other planets; however, analysing the impact of human exploration on environments is of great planetary protection concern. This can be achieved by obtaining samples from a subterranean environment, where only expert speleologists have access and the human impact is considered very low. For this study, astronauts participating in the 2014 ESA CAVES (Cooperative Adventure for Valuing and Exercising human behaviour and performance Skills) training course, obtained samples from deep within a subterranean environment and returned them to the laboratory for molecular microbial analysis