Planetary Protection Knowledge Gap Closure Enabling Crewed Missions to Mars.

As focus for exploration of Mars transitions from current robotic explorers to development of crewed missions, it remains important to protect the integrity of scientific investigations at Mars, as well as protect the Earth's biosphere from any potential harmful effects from returned martian material. This is the discipline of planetary protection, and the Committee on Space Research (COSPAR) maintains the consensus international policy and guidelines on how this is implemented. Based on National Aeronautics and Space Administration (NASA) and European Space Agency (ESA) studies that began in 2001, COSPAR adopted principles and guidelines for human missions to Mars in 2008. At that point, it was clear that to move from those qualitative provisions, a great deal of work and interaction with spacecraft designers would be necessary to generate meaningful quantitative recommendations that could embody the intent of the Outer Space Treaty (Article IX) in the design of such missions. Beginning in 2016, COSPAR then sponsored a multiyear interdisciplinary meeting series to address planetary protection "knowledge gaps" (KGs) with the intent of adapting and extending the current robotic mission-focused Planetary Protection Policy to support the design and implementation of crewed and hybrid exploration missions. This article describes the outcome of the interdisciplinary COSPAR meeting series, to describe and address these KGs, as well as identify potential paths to gap closure. It includes the background scientific basis for each topic area and knowledge updates since the meeting series ended. In particular, credible solutions for KG closure are described for the three topic areas of (1) microbial monitoring of spacecraft and crew health; (2) natural transport (and survival) of terrestrial microbial contamination at Mars, and (3) the technology and operation of spacecraft systems for contamination control. The article includes a KG data table on these topic areas, which is intended to be a point of departure for making future progress in developing an end-to-end planetary protection requirements implementation solution for a crewed mission to Mars. Overall, the workshop series has provided evidence of the feasibility of planetary protection implementation for a crewed Mars mission, given (1) the establishment of needed zoning, emission, transport, and survival parameters for terrestrial biological contamination and (2) the creation of an accepted risk-based compliance approach for adoption by spacefaring actors including national space agencies and commercial/nongovernment organizations.

Shock-Impacts and Vibrational g-Forces Can Dislodge Bacillus spp. Spores from Spacecraft Surfaces.

Mars spacecraft encounter numerous g-loads that occur along the launch or landing vectors (called axial vectors) or along lateral off-axes vectors. The goal of this research was to determine if there was a threshold for dislodging spores under brute-force dynamic shock compressional impacts (i.e., henceforth called shock-impacts) or long-term vibrationally induced g-loads that might simulate spacecraft launches or landings profiles. Results indicated that spores of Bacillus subtilis 168 and B. atrophaeus ATCC 9372 were dislodged from ChemFilm-coated aluminum coupons during shock impact events of 60 g's or higher. In contrast, the threshold for dislodging B. pumilus SAFR-032 spores was approx. 80 g's. Vibrational g-loading was conducted at approx. 12-15 g's (z-axis) and 77 Hz. All three Bacillus spp. exhibited very modest spore dislodgement at 1, 4, or 8 min of induced vibrational g-loads. However, the numbers of spores released depended on the Earth's g-vector relative to the bacterial monolayers. When the experimental hardware was placed in an 'Up' orientation (defined as the spores sat on the upper surface of the coupons and the coupons pointed up and away from Earth's g-vector), zero to only a few spores were dislodged. When the experimental hardware was inverted and the coupon surfaces were in a 'Down' orientation, the number of spores released increased by 20-30 times. Overall, the results of both assays suggest that spores on spacecraft surfaces will not likely be dislodged during nominal launch and landing scenarios, with the exception of jettisoned hardware (e.g., heat shields or backshells) during landing that might hit the Martian terrain at high g's. However, off-nominal landings hitting the Martian surface at >60 g's are likely to release low numbers of spores into the atmosphere and regolith.

Advancing the automation of plant nucleic acid extraction for rapid diagnosis of plant diseases in space.

Human space exploration missions will continue the development of sustainable plant cultivation in what are obviously novel habitat settings. Effective pathology mitigation strategies are needed to cope with plant disease outbreaks in any space-based plant growth system. However, few technologies currently exist for space-based diagnosis of plant pathogens. Therefore, we developed a method of extracting plant nucleic acid that will facilitate the rapid diagnosis of plant diseases for future spaceflight applications. The microHomogenizer™ from Claremont BioSolutions, originally designed for bacterial and animal tissue samples, was evaluated for plant-microbial nucleic acid extractions. The microHomogenizer™ is an appealing device in that it provides automation and containment capabilities that would be required in spaceflight applications. Three different plant pathosystems were used to assess the versatility of the extraction process. Tomato, lettuce, and pepper plants were respectively inoculated with a fungal plant pathogen, an oomycete pathogen, and a plant viral pathogen. The microHomogenizer™, along with the developed protocols, proved to be an effective mechanism for producing DNA from all three pathosystems, in that PCR and sequencing of the resulting samples demonstrated clear DNA-based diagnoses. Thus, this investigation advances the efforts to automate nucleic acid extraction for future plant disease diagnosis in space.

Microbial Protocols for Spacecraft: 3. Spore Monolayer Preparation Methods for Ultraviolet Irradiation Exposures.

Developing robust microbial survival models for interplanetary and planetary spacecraft requires precise inactivation kinetics for vehicle bioburdens. To generate such data, reliable protocols are required for preparing, testing, and assaying microbial cells or spores on simulated spacecraft materials. New data are presented on the utility of the liquid droplet protocol for applying Bacillus subtilis spores to aluminum coupons. Results indicate that low-density spore monolayers should be created between 2 and 5 × 106 spores per cm2 on individual coupons to prevent the formation of aggregates or multilayers of spores. Such aggregation or multilayers will interfere with the precision of characterizing the effects of UV irradiation on spore survival. Optimum spore monolayers are defined as spore monolayers without overlapping or clustered cells and in which all spores will receive UV photons during assays. The best spore monolayers were created with sterile deionized water (SDIW) on uncoated aluminum coupons, or with SDIW + Triton X-100 (at 0.5 × of the critical micellar concentration) on either uncoated Al-coupons or on Chemfilm Class 1A-coated coupons. The Triton X-100 surfactant improved the uniformity of the monolayers without affecting the sensitivity of the spores to UV irradiation. Furthermore, spore layers created at either 2 × 107 or 2 × 108 spores/cm2 created multi-stacking effects that clearly reduced the precision of the UV irradiation assays. A set of standardized protocols is suggested for spacecraft processing and planetary protection communities to permit directly comparing results from divergent labs.

Microbial Protocols for Spacecraft: 1. Effects of Surface Texture, Low Pressure, and UV Irradiation on Recovery of Microorganisms from Surfaces.

Modeling risks for the forward contamination of planetary surfaces from endemic bioburdens on landed spacecraft requires precise data on the biocidal effects of space factors on microbial survival. Numerous studies have been published over the preceding 60 years on the survival of diverse microorganisms exposed to solar heating, solar ultraviolet (UV) irradiation, vacuum, ionizing radiation, desiccation, and many other planetary surface conditions. These data were generated with diverse protocols that can impair the interpretations of the results due to dynamic experimental errors inherent in all lab protocols. The current study (1) presents data on how metal surfaces can affect spore adhesion, (2) proposes doping and extraction protocols that can achieve very high recovery rates (close to 100%) from aluminum coupons with four Bacillus spp., (3) establishes a timeline in which dried spores on aluminum coupons should be used to minimize aging effects of spore monolayers, (4) confirms that vacuum alone does not dislodge spores dried on aluminum coupons, and (5) establishes that multiple UV irradiation sources yield similar results if properly cross-calibrated. The protocols are given to advance discussions in the planetary protection community on how to standardize lab protocols to align results from diverse labs into a coherent interpretation of how space conditions will degrade microbial survival over time.

Diagnosing an Opportunistic Fungal Pathogen on Spaceflight-Grown Plants Using the MinION Sequencing Platform.

Sustainable agriculture in microgravity is integral to future long-term human space exploration. To ensure the efficient and sustainable cultivation of plants in space, a contingency plan to monitor plant health and mitigate plant diseases is necessary. Yet, neither methods nor tools currently exist to evaluate the plant microbial interactions or to diagnose potential plant diseases in space-based bioregenerative life support systems. In this study, we show how the MinION sequencing platform can be used to diagnose the opportunistic pathogen Fusarium oxysporum sensu lato, a fungal infection on Zinnia hybrida (zinnia) plants that were grown on the International Space Station (ISS) in 2015-2016. Genomic DNA from the infected plant material (root and leaf tissues) retrieved from the ISS were extracted and sequenced. In addition, pure cultures of Burkholderia contaminans, F. oxysporum sensu lato, and Fusarium sporotrichioides were used as controls to test the specificity of the bioinformatics pipeline developed. The results show that the MinION platform can be used to accurately differentiate between fusaria species and strengthens the case for using the platform as a rapid plant disease diagnostic tool in space.

Use of NanoSIMS to Identify the Lower Limits of Metabolic Activity and Growth by Serratia liquefaciens Exposed to Sub-Zero Temperatures.

Serratia liquefaciens is a cold-adapted facultative anaerobic astrobiology model organism with the ability to grow at a Martian atmospheric pressure of 7 hPa. Currently there is a lack of data on its limits of growth and metabolic activity at sub-zero temperatures found in potential habitable regions on Mars. Growth curves and nano-scale secondary ion mass spectrometry (NanoSIMS) were used to characterize the growth and metabolic threshold for S. liquefaciens ATCC 27,592 grown at and below 0 °C. Cells were incubated in Spizizen medium containing three stable isotopes substituting their unlabeled counterparts; i.e., 13C-glucose, (15NH4)2SO4, and H218O; at 0, -1.5, -3, -5, -10, or -15 °C. The isotopic ratios of 13C/12C, 15N/14N, and 18O/16O and their corresponding fractions were determined for 240 cells. NanoSIMS results revealed that with decreasing temperature the cellular amounts of labeled ions decreased indicating slower metabolic rates for isotope uptake and incorporation. Metabolism was significantly reduced at -1.5 and -3 °C, almost halted at -5 °C, and shut-down completely at or below -10 °C. While growth was observed at 0 °C after 5 days, samples incubated at -1.5 and -3 °C exhibited significantly slower growth rates until growth was detected at 70 days. In contrast, cell densities decreased by at least half an order of magnitude over 70 days in cultures incubated at ≤ -5 °C. Results suggest that S. liquefaciens, if transported to Mars, might be able to metabolize and grow in shallow sub-surface niches at temperatures above -5 °C and might survive-but not grow-at temperatures below -5 °C.

Transcriptional response to prolonged perchlorate exposure in the methanogen Methanosarcina barkeri and implications for Martian habitability.

Observations of trace methane (CH4) in the Martian atmosphere are significant to the astrobiology community given the overwhelming contribution of biological methanogenesis to atmospheric CH4 on Earth. Previous studies have shown that methanogenic Archaea can generate CH4 when incubated with perchlorates, highly oxidizing chaotropic salts which have been found across the Martian surface. However, the regulatory mechanisms behind this remain completely unexplored. In this study we performed comparative transcriptomics on the methanogen Methanosarcina barkeri, which was incubated at 30˚C and 0˚C with 10-20 mM calcium-, magnesium-, or sodium perchlorate. Consistent with prior studies, we observed decreased CH4 production and apparent perchlorate reduction, with the latter process proceeding by heretofore essentially unknown mechanisms. Transcriptomic responses of M. barkeri to perchlorates include up-regulation of osmoprotectant transporters and selection against redox-sensitive amino acids. Increased expression of methylamine methanogenesis genes suggest competition for H2 with perchlorate reduction, which we propose is catalyzed by up-regulated molybdenum-containing enzymes and maintained by siphoning diffused H2 from energy-conserving hydrogenases. Methanogenesis regulatory patterns suggest Mars' freezing temperatures alone pose greater constraints to CH4 production than perchlorates. These findings increase our understanding of methanogen survival in extreme environments and confers continued consideration of a potential biological contribution to Martian CH4.

Fusarium oxysporum as an Opportunistic Fungal Pathogen on Zinnia hybrida Plants Grown on board the International Space Station.

A plant production system called Veggie was launched to the International Space Station (ISS) in 2014. In late 2015, during the growth of Zinnia hybrida cv. 'Profusion' in the Veggie hardware, plants developed chlorosis, leaf curling, fungal growth that damaged leaves and stems, and eventually necrosis. The development of symptoms was correlated to reduced air flow leading to a significant buildup of water enveloping the leaves and stems in microgravity. Symptomatic tissues were returned to Earth on 18 May 2016 and were immediately processed to determine the primary causal agent of the disease. The presumptive pathogen was identified as Fusarium oxysporum by morphological features of microconidia and conidiophores on symptomatic tissues; that is, by epifluorescent microscopy (EFM), scanning electron microscopy (SEM), metabolic microarrays, and ITS sequencing. Both EFM and SEM imaging of infected tissues showed that germinating conidia were capable of stomatal penetration and thus acted as the primary method for infecting host tissues. A series of ground-based pathogenicity assays were conducted with healthy Z. hybrida plants that were exposed to reduced-airflow and high-water stress (i.e., encased in sealed bags) or were kept in an unstressed configuration. Koch's postulates were successfully completed with Z. hybrida plants in the lab, but symptoms only matched ISS-flown symptomatic tissues when the plants were stressed with high-water exposure. Unstressed plants grown under similar lab conditions failed to develop the symptoms observed with plants on board the ISS. The overall results of the pathogenicity tests imply that F. oxysporum acted as an opportunistic pathogen on severely high-water stressed plants. The source of the opportunistic pathogen is not known, but virulent strains of F. oxysporum were not recovered from unused materials in the Veggie plant pillow growth units assayed after the flight.

Salt Tolerance and UV Protection of Bacillus subtilis and Enterococcus faecalis under Simulated Martian Conditions.

Ultraviolet (UV) irradiation on the surface of Mars is an important factor that affects the survivability of microorganisms on Mars. The possibility of martian brines made from Fe2(SO4)3, MnSO4, and MgSO4 salts providing a habitable niche on Mars via attenuation of UV radiation was investigated on the bacteria Bacillus subtilis and Enterococcus faecalis. Results demonstrate that it is possible for brines containing Fe2(SO4)3 on Mars to provide protection from harmful UV irradiation, even at concentrations as low as 0.5%. Brines made from MnSO4 and MgSO4 did not provide significant UV protection, and most spores/cells died over the course of short-term experiments. However, Fe2(SO4)3 brines are strongly acidic and thus were lethal to E. faecalis, when cells were exposed for 7 days. In contrast, B. subtilis, a spore-forming bacterium resistant to pH extremes, was unaffected by the acidic conditions of the brines and did not experience any significant lethal effects in Fe2(SO4)3. Any extant microbial life in martian Fe2(SO4)3 brines (if present) would need to be capable of surviving acidic environments, if these brines are to be considered a possible habitable niche. The results from this work are important to the search for life on planets with atmospheres that do not significantly attenuate UV radiation (i.e., like Mars) and to planetary protection, since it is possible that terrestrial bacteria in the genus Bacillus are likely to survive in Fe-sulfate brines on Mars.

Addition of anaerobic electron acceptors to solid media did not enhance growth of 125 spacecraft bacteria under simulated low-pressure Martian conditions.

To protect Mars from microbial contamination, research on growth of microorganisms found in spacecraft assembly clean rooms under simulated Martian conditions is required. This study investigated the effects of low atmospheric pressure on the growth of chemoorganotrophic spacecraft bacteria and whether the addition of Mars relevant anaerobic electron acceptors might enhance growth. The 125 bacteria screened here were recovered from actual Mars spacecraft. Growth at 7 hPa, 0 °C, and a CO2-enriched anoxic atmosphere (called low-PTA conditions) was tested on five TSA-based media supplemented with anaerobic electron acceptors. None of the 125 spacecraft bacteria showed active growth under the tested low-PTA conditions and amended media. In contrast, a decrease in viability was observed in most cases. Growth curves of two hypopiezotolerant strains, Serratia liquefaciens and Trichococcus pasteurii, were performed to quantify the effects of the added anaerobic electron acceptors. Slight variations in growth rates were determined for both bacteria. However, the final cell densities were similar for all media tested, indicating no general preference for any specific anaerobic electron acceptor. By demonstrating that a broad diversity of chemoorganotrophic and culturable spacecraft bacteria do not grow under the tested conditions, we conclude that there may be low risk of growth of chemoorganotrophic bacteria typically recovered from Mars spacecraft during planetary protection bioburden screenings.

A Cruise-Phase Microbial Survival Model for Calculating Bioburden Reductions on Past or Future Spacecraft Throughout Their Missions with Application to Europa Clipper.

During transit between the Earth and planetary destinations, spacecraft encounter conditions that are deleterious to the survival of terrestrial microorganisms. To model the resulting bioburden reduction, a Cruise-Phase Microbial Survival (CPMS) model was prepared based upon the Lunar Microbial Survival model, which considers the effects of temperature, vacuum, ultraviolet (UV), and ionizing radiation found in the space environment. As an example, the CPMS was used to determine the expected bioburden reductions on the Europa Clipper spacecraft upon arrival at Jupiter under two different transit scenarios. Under a direct trajectory scenario, exterior surfaces are rapidly sterilized with tens of thousands of lethal doses (LDs) absorbed to the spacecraft exterior and at least one LD to all interior spaces of the spacecraft heated to at least 240 K. Under a Venus-Earth-Earth gravity assist (VEEGA) trajectory, we find substantially higher bioburden reductions resulting from the spacecraft spending much more time near the Sun and more time in transit overall. With VEEGA, the exterior absorbs hundreds of thousands of LDs and interior surfaces heated above 230 K would absorb at least one LD. From these simulations, we are able to generalize about bioburden reduction in transit on spacecraft in general, finding that all spacecraft surfaces would sustain at least one LD in ≤38.5 years even if completely unheated. Temperature and vacuum synergy dominates surface reductions out to at most 3.3 AU (for gold multilayer insulation), UV irradiation and temperature between 3.3 and 600 AU, and past 600 AU the effect of vacuum acting alone is the primary factor for all exterior and interior surfaces. Even under the most conservative estimates, if the average interior temperature of the Cassini spacecraft exceeded 218 K, or the Galileo spacecraft interior exceeded 222 K, neither spacecraft would have likely had any viable bioburdens onboard at disposal.

The Hypopiezotolerant Bacterium, Serratia liquefaciens, Failed to Grow in Mars Analog Soils under Simulated Martian Conditions at 7 hPa.

The search for life on Mars is predicated on the idea that Earth and Mars life (if present) should be both carbon- and water-based with similar forms of evolution. However, the astrobiology community can currently only investigate plausible Martian microbial ecosystems by using Terran life-forms as proxies. In order to examine how life might persist on Mars, we used a hypopiezotolerant bacterium (def., able to grow at 7-10 hPa)-Serratia liquefaciens-in growth assays with four Mars analog soils conducted under a subset of simulated Martian conditions including 7 hPa, 0 °C, and a CO2-enriched anoxic atmosphere (called low-PTA conditions). The four Mars analog soils included an Aeolian dust analog, the Mars JSC-1 analog, a Phoenix lander-site simulant, and a high-Salts analog. Serratia liquefaciens cells were able to grow at 30 °C in a liquid minimal basal medium (MBM) supplemented with 10- or 20-mM sucrose, Spizizen salts, and micronutrients. When the four analog soils were doped with both MBM and cells of S. liquefaciens, and subsequently incubated at 30 °C for 72 h, cell densities increased between 2-logs (Phoenix analog) and 4-logs (Aeolian and JSC-1 analogs); the Salts analog led to complete inactivation of S. liquefaciens within 24 h. In contrast, when the experiment was repeated, but incubated under low-PTA conditions, S. liquefaciens cells were either killed immediately by the Salts analog, or decreased by > 5 logs over 28 d by the Aeolian, JSC-1, and Phoenix analogs. The failure of S. liquefaciens to grow in the analog soils under low-PTA conditions was attributed to the synergistic interactions among six factors (i.e., low pressure, low temperature, anoxic atmosphere (i.e., the low-PTA conditions), low-pH in the Salts soil, dissolved salts in all analogs, and oligotrophic conditions) that increased the biocidal or inhibitory conditions within the analog soils. Results suggest that even if a hypopiezotolerant Terran microbe is displaced from a spacecraft surface on Mars, and lands in a hydrated and nutrient-rich niche, growth in the Martian regolith is not automatically assured.

Exploring Microbial Activity in Low-pressure Environments.

The importance of hypopiezophilic and hypopiezotolerant microorganisms (i.e., life that grows at low atmospheric pressures; see section 2) in the field of astrobiology cannot be overstated. The ability to reproduce and thrive at Martian atmospheric pressure (0.2 to 1.2 kPa) is of high importance to both modeling the forward contamination of its planetary surface and predicting the habitability of Mars. On Earth, microbial growth at low pressure also has implications for the dissemination of microorganisms within clouds or the bulk atmosphere. Yet our ability to understand the effect of low pressure on microbial metabolism, growth, cellular structure and integrity, and adaptation is still limited. We present current knowledge on hypopiezophilic and hypopiezotolerant microorganisms, methods for isolation and cultivation, justify why there should be more focus for future research, and discuss their importance for astrobiology.

Bacillus subtilis Spore Resistance to Simulated Mars Surface Conditions.

In a Mars exploration scenario, knowing if and how highly resistant Bacillus subtilis spores would survive on the Martian surface is crucial to design planetary protection measures and avoid false positives in life-detection experiments. Therefore, in this study a systematic screening was performed to determine whether B. subtilis spores could survive an average day on Mars. For that, spores from two comprehensive sets of isogenic B. subtilis mutant strains, defective in DNA protection or repair genes, were exposed to 24 h of simulated Martian atmospheric environment with or without 8 h of Martian UV radiation [M(+)UV and M(-)UV, respectively]. When exposed to M(+)UV, spore survival was dependent on: (1) core dehydration maintenance, (2) protection of DNA by α/ß-type small acid soluble proteins (SASP), and (3) removal and repair of the major UV photoproduct (SP) in spore DNA. In turn, when exposed to M(-)UV, spore survival was mainly dependent on protection by the multilayered spore coat, and DNA double-strand breaks represent the main lesion accumulated. Bacillus subtilis spores were able to survive for at least a limited time in a simulated Martian environment, both with or without solar UV radiation. Moreover, M(-)UV-treated spores exhibited survival rates significantly higher than the M(+)UV-treated spores. This suggests that on a real Martian surface, radiation shielding of spores (e.g., by dust, rocks, or spacecraft surface irregularities) might significantly extend survival rates. Mutagenesis were strongly dependent on the functionality of all structural components with small acid-soluble spore proteins, coat layers and dipicolinic acid as key protectants and efficiency DNA damage removal by AP endonucleases (ExoA and Nfo), non-homologous end joining (NHEJ), mismatch repair (MMR) and error-prone translesion synthesis (TLS). Thus, future efforts should focus on: (1) determining the DNA damage in wild-type spores exposed to M(+/-)UV and (2) assessing spore survival and viability with shielding of spores via Mars regolith and other relevant materials.

A Lunar Microbial Survival Model for Predicting the Forward Contamination of the Moon.

The surface conditions on the Moon are extremely harsh with high doses of ultraviolet (UV) irradiation (26.8 W · m-2 UVC/UVB), wide temperature extremes (-171°C to 140°C), low pressure (10-10 Pa), and high levels of ionizing radiation. External spacecraft surfaces on the Moon are generally >100°C during daylight hours and can reach as high as 140°C at local noon. A Lunar Microbial Survival (LMS) model was developed that estimated (1) the total viable bioburden of all spacecraft landed on the Moon as ∼4.57 × 1010 microbial cells/spores at contact, (2) the inactivation kinetics of Bacillus subtilis spores to vacuum as approaching -2 logs per 2107 days, (3) the inactivation of spores on external surfaces due to concomitant low-pressure and high-temperature conditions as -6 logs per 8 h for local noon conditions, and (4) the ionizing radiation by solar wind particles as approaching -3 logs per lunation on external surfaces only. When the biocidal factors of solar UV, vacuum, high-temperature, and ionizing radiation were combined into an integrated LMS model, a -231 log reduction in viable bioburden was predicted for external spacecraft surfaces per lunation at the equator. Results indicate that external surfaces of landed or crashed spacecraft are unlikely to harbor viable spores after only one lunation, that shallow internal surfaces will be sterilized due to the interactive effects of vacuum and thermal cycling from solar irradiation, and that deep internal surfaces would be affected only by vacuum with a degradation rate of -0.02 logs per lunation.

Transcriptomic responses of Serratia liquefaciens cells grown under simulated Martian conditions of low temperature, low pressure, and CO2-enriched anoxic atmosphere.

Results from previous experiments indicated that the Gram-negative α-proteobacterium Serratia liquefaciens strain ATCC 27592 was capable of growth under low temperature (0 °C), low pressure (0.7 kPa), and anoxic, CO2-dominated atmosphere-conditions intended to simulate the near-subsurface environment of Mars. To probe the response of its transcriptome to this extreme environment, S. liquefaciens ATCC 27592 was cultivated under 4 different environmental simulations: 0 °C, 0.7 kPa, CO2 atmosphere (Condition A); 0 °C, ~101.3 kPa, CO2 atmosphere (Condition B); 0 °C, ~101.3 kPa, ambient N2/O2 atmosphere (Condition C); and 30 °C, ~101.3 kPa, N2/O2 atmosphere (Condition D; ambient laboratory conditions). RNA-seq was performed on ribosomal RNA-depleted total RNA isolated from triplicate cultures grown under Conditions A-D and the datasets generated were subjected to transcriptome analyses. The data from Conditions A, B, or C were compared to laboratory Condition D. Significantly differentially expressed transcripts were identified belonging to a number of KEGG pathway categories. Up-regulated genes under all Conditions A, B, and C included those encoding transporters (ABC and PTS transporters); genes involved in translation (ribosomes and their biogenesis, biosynthesis of both tRNAs and aminoacyl-tRNAs); DNA repair and recombination; and non-coding RNAs. Genes down-regulated under all Conditions A, B, and C included: transporters (mostly ABC transporters); flagellar and motility proteins; genes involved in phenylalanine metabolism; transcription factors; and two-component systems. The results are discussed in the context of Mars astrobiology and planetary protection.

Metabolic fingerprints of Serratia liquefaciens under simulated Martian conditions using Biolog GN2 microarrays.

Microorganisms growing at atmospheric pressures of 0.7 kPa may have a significant impact on the search for life on Mars. Data on their nutrient requirements in a simulated Martian environment are required to ascertain both the potential risk of forward contamination and the potential of past or present habitability of Mars. Serratia liquefaciens can grow at concomitant conditions of low pressure, low temperature, and anoxic atmosphere. Changes in the metabolic fingerprint of S. liquefaciens grown under varying physical conditions including diverse atmospheric pressures (0.7 kPa to 101.3 kPa), temperatures (30 °C or 0 °C), and atmospheric gas compositions (Earth or CO2) were investigated using Biolog GN2 assays. Distinct patterns for each condition were observed. Above 10 kPa S. liquefaciens performed similar to Earth-normal pressure conditions (101.3 kPa) whereas below 10 kPa shifts in metabolic patterns were observed. The differences indicated a physiological alteration in which S. liquefaciens lost its ability to metabolize the majority of the provided carbon sources at 0.7 kPa with a significant decrease in the oxidation of amino acids. By measuring the physiological responses to different carbon sources we were able to identify nutritional constraints that support cellular replication under simulated shallow Mars subsurface conditions.

The Photochemistry of Unprotected DNA and DNA inside Bacillus subtilis Spores Exposed to Simulated Martian Surface Conditions of Atmospheric Composition, Temperature, Pressure, and Solar Radiation.

DNA is considered a potential biomarker for life-detection experiments destined for Mars. Experiments were conducted to examine the photochemistry of bacterial DNA, either unprotected or within Bacillus subtilis spores, in response to exposure to simulated martian surface conditions consisting of the following: temperature (-10°C), pressure (0.7 kPa), atmospheric composition [CO2 (95.54%), N2 (2.7%), Ar (1.6%), O2 (0.13%), and H2O (0.03%)], and UV-visible-near IR solar radiation spectrum (200-1100 nm) calibrated to 4 W/m2 of UVC (200-280 nm). While the majority (99.9%) of viable spores deposited in multiple layers on spacecraft-qualified aluminum coupons were inactivated within 5 min, a detectable fraction survived for up to the equivalent of ∼115 martian sols. Spore photoproduct (SP) was the major lesion detected in spore DNA, with minor amounts of cyclobutane pyrimidine dimers (CPD), in the order TT CPD > TC CPD >> CT CPD. In addition, the (6-4)TC, but not the (6-4)TT, photoproduct was detected in spore DNA. When unprotected DNA was exposed to simulated martian conditions, all photoproducts were detected. Surprisingly, the (6-4)TC photoproduct was the major photoproduct, followed by SP ∼ TT CPD > TC CPD > (6-4)TT > CT CPD > CC CPD. Differences in the photochemistry of unprotected DNA and spore DNA in response to simulated martian surface conditions versus laboratory conditions are reviewed and discussed. The results have implications for the planning of future life-detection experiments that use DNA as the target, and for the long-term persistence on Mars of forward contaminants or their DNA. Key Words: Bacillus subtilis-DNA-Mars-Photochemistry-Spore-Ultraviolet. Astrobiology 18, 393-402.