Aspergillus niger as a cell factory for the production of pyomelanin, a molecule with UV-C radiation shielding activity.

Melanins are complex pigments with various biological functions and potential applications in space exploration and biomedicine due to their radioprotective properties. Aspergillus niger, a fungus known for its high radiation resistance, is widely used in biotechnology and a candidate for melanin production. In this study, we investigated the production of fungal pyomelanin (PyoFun) in A. niger by inducing overproduction of the pigment using L-tyrosine in a recombinant ΔhmgA mutant strain (OS4.3). The PyoFun pigment was characterized using three spectroscopic methods, and its antioxidant properties were assessed using a DPPH-assay. Additionally, we evaluated the protective effect of PyoFun against non-ionizing radiation (monochromatic UV-C) and compared its efficacy to a synthetically produced control pyomelanin (PyoSyn). The results confirmed successful production of PyoFun in A. niger through inducible overproduction. Characterization using spectroscopic methods confirmed the presence of PyoFun, and the DPPH-assay demonstrated its strong antioxidant properties. Moreover, PyoFun exhibited a highly protective effect against radiation-induced stress, surpassing the protection provided by PyoSyn. The findings of this study suggest that PyoFun has significant potential as a biological shield against harmful radiation. Notably, PyoFun is synthesized extracellularly, differing it from other fungal melanins (such as L-DOPA- or DHN-melanin) that require cell lysis for pigment purification. This characteristic makes PyoFun a valuable resource for biotechnology, biomedicine, and the space industry. However, further research is needed to evaluate its protective effect in a dried form and against ionizing radiation.

Colony growth and biofilm formation of Aspergillus niger under simulated microgravity.

The biotechnology- and medicine-relevant fungus Aspergillus niger is a common colonizer of indoor habitats such as the International Space Station (ISS). Being able to colonize and biodegrade a wide range of surfaces, A. niger can ultimately impact human health and habitat safety. Surface contamination relies on two key-features of the fungal colony: the fungal spores, and the vegetative mycelium, also known as biofilm. Aboard the ISS, microorganisms and astronauts are shielded from extreme temperatures and radiation, but are inevitably affected by spaceflight microgravity. Knowing how microgravity affects A. niger colony growth, in particular regarding the vegetative mycelium (biofilm) and spore production, will help prevent and control fungal contaminations in indoor habitats on Earth and in space. Because fungal colonies grown on agar can be considered analogs for surface contamination, we investigated A. niger colony growth on agar in normal gravity (Ground) and simulated microgravity (SMG) conditions by fast-clinorotation. Three strains were included: a wild-type strain, a pigmentation mutant (ΔfwnA), and a hyperbranching mutant (ΔracA). Our study presents never before seen scanning electron microscopy (SEM) images of A. niger colonies that reveal a complex ultrastructure and biofilm architecture, and provide insights into fungal colony development, both on ground and in simulated microgravity. Results show that simulated microgravity affects colony growth in a strain-dependent manner, leading to thicker biofilms (vegetative mycelium) and increased spore production. We suggest that the Rho GTPase RacA might play a role in A. niger's adaptation to simulated microgravity, as deletion of ΔracA leads to changes in biofilm thickness, spore production and total biomass. We also propose that FwnA-mediated melanin production plays a role in A. niger's microgravity response, as ΔfwnA mutant colonies grown under SMG conditions showed increased colony area and spore production. Taken together, our study shows that simulated microgravity does not inhibit A. niger growth, but rather indicates a potential increase in surface-colonization. Further studies addressing fungal growth and surface contaminations in spaceflight should be conducted, not only to reduce the risk of negatively impacting human health and spacecraft material safety, but also to positively utilize fungal-based biotechnology to acquire needed resources in situ.

Spaceflight Virology: What Do We Know about Viral Threats in the Spaceflight Environment?

Viruses constitute a significant part of the human microbiome, so wherever humans go, viruses are brought with them, even on space missions. In this mini review, we focus on the International Space Station (ISS) as the only current human habitat in space that has a diverse range of viral genera that infect microorganisms from bacteria to eukaryotes. Thus, we have reviewed the literature on the physical conditions of space habitats that have an impact on both virus transmissibility and interaction with their host, which include UV radiation, ionizing radiation, humidity, and microgravity. Also, we briefly comment on the practices used on space missions that reduce virus spread, that is, use of antimicrobial surfaces, spacecraft sterilization practices, and air filtration. Finally, we turn our attention to the health threats that viruses pose to space travel. Overall, even though efforts are taken to ensure safe conditions during human space travel, for example, preflight quarantines of astronauts, we reflect on the potential risks humans might be exposed to and how those risks might be aggravated in extraterrestrial habitats.

MARSBOx: Fungal and Bacterial Endurance From a Balloon-Flown Analog Mission in the Stratosphere.

Whether terrestrial life can withstand the martian environment is of paramount interest for planetary protection measures and space exploration. To understand microbial survival potential in Mars-like conditions, several fungal and bacterial samples were launched in September 2019 on a large NASA scientific balloon flight to the middle stratosphere (∼38 km altitude) where radiation levels resembled values at the equatorial Mars surface. Fungal spores of Aspergillus niger and bacterial cells of Salinisphaera shabanensis, Staphylococcus capitis subsp. capitis, and Buttiauxella sp. MASE-IM-9 were launched inside the MARSBOx (Microbes in Atmosphere for Radiation, Survival, and Biological Outcomes Experiment) payload filled with an artificial martian atmosphere and pressure throughout the mission profile. The dried microorganisms were either exposed to full UV-VIS radiation (UV dose = 1148 kJ m-2) or were shielded from radiation. After the 5-h stratospheric exposure, samples were assayed for survival and metabolic changes. Spores from the fungus A. niger and cells from the Gram-(-) bacterium S. shabanensis were the most resistant with a 2- and 4-log reduction, respectively. Exposed Buttiauxella sp. MASE-IM-9 was completely inactivated (both with and without UV exposure) and S. capitis subsp. capitis only survived the UV shielded experimental condition (3-log reduction). Our results underscore a wide variation in survival phenotypes of spacecraft associated microorganisms and support the hypothesis that pigmented fungi may be resistant to the martian surface if inadvertently delivered by spacecraft missions.

Aspergillus niger Spores Are Highly Resistant to Space Radiation.

The filamentous fungus Aspergillus niger is one of the main contaminants of the International Space Station (ISS). It forms highly pigmented, airborne spores that have thick cell walls and low metabolic activity, enabling them to withstand harsh conditions and colonize spacecraft surfaces. Whether A. niger spores are resistant to space radiation, and to what extent, is not yet known. In this study, spore suspensions of a wild-type and three mutant strains (with defects in pigmentation, DNA repair, and polar growth control) were exposed to X-rays, cosmic radiation (helium- and iron-ions) and UV-C (254 nm). To assess the level of resistance and survival limits of fungal spores in a long-term interplanetary mission scenario, we tested radiation doses up to 1000 Gy and 4000 J/m2. For comparison, a 360-day round-trip to Mars yields a dose of 0.66 ± 0.12 Gy. Overall, wild-type spores of A. niger were able to withstand high doses of X-ray (LD90 = 360 Gy) and cosmic radiation (helium-ion LD90 = 500 Gy; and iron-ion LD90 = 100 Gy). Drying the spores before irradiation made them more susceptible toward X-ray radiation. Notably, A. niger spores are highly resistant to UV-C radiation (LD90 = 1038 J/m2), which is significantly higher than that of other radiation-resistant microorganisms (e.g., Deinococcus radiodurans). In all strains, UV-C treated spores (1000 J/m2) were shown to have decreased biofilm formation (81% reduction in wild-type spores). This study suggests that A. niger spores might not be easily inactivated by exposure to space radiation alone and that current planetary protection guidelines should be revisited, considering the high resistance of fungal spores.

Novel Genes Involved in Resistance to Both Ultraviolet Radiation and Perchlorate From the Metagenomes of Hypersaline Environments.

Microorganisms that thrive in hypersaline environments on the surface of our planet are exposed to the harmful effects of ultraviolet radiation. Therefore, for their protection, they have sunscreen pigments and highly efficient DNA repair and protection systems. The present study aimed to identify new genes involved in UV radiation resistance from these microorganisms, many of which cannot be cultured in the laboratory. Thus, a functional metagenomic approach was used and for this, small-insert libraries were constructed with DNA isolated from microorganisms of high-altitude Andean hypersaline lakes in Argentina (Diamante and Ojo Seco lakes, 4,589 and 3,200 m, respectively) and from the Es Trenc solar saltern in Spain. The libraries were hosted in a UV radiation-sensitive strain of Escherichia coli (recA mutant) and they were exposed to UVB. The resistant colonies were analyzed and as a result, four clones were identified with environmental DNA fragments containing five genes that conferred resistance to UV radiation in E. coli. One gene encoded a RecA-like protein, complementing the mutation in recA that makes the E. coli host strain more sensitive to UV radiation. Two other genes from the same DNA fragment encoded a TATA-box binding protein and an unknown protein, both responsible for UV resistance. Interestingly, two other genes from different and remote environments, the Ojo Seco Andean lake and the Es Trenc saltern, encoded two hypothetical proteins that can be considered homologous based on their significant amino acid similarity (49%). All of these genes also conferred resistance to 4-nitroquinoline 1-oxide (4-NQO), a compound that mimics the effect of UV radiation on DNA, and also to perchlorate, a powerful oxidant that can induce DNA damage. Furthermore, the hypothetical protein from the Es Trenc salterns was localized as discrete foci possibly associated with damaged sites in the DNA in cells treated with 4-NQO, so it could be involved in the repair of damaged DNA. In summary, novel genes involved in resistance to UV radiation, 4-NQO and perchlorate have been identified in this work and two of them encoding hypothetical proteins that could be involved in DNA damage repair activities not previously described.

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.

Single-cell analysis reveals individual spore responses to simulated space vacuum.

Outer space is a challenging environment for all forms of life, and dormant spores of bacteria have been frequently used to study the survival of terrestrial life in a space journey. Previous work showed that outer space vacuum alone can kill bacterial spores. However, the responses and mechanisms of resistance of individual spores to space vacuum are unclear. Here, we examined spores' molecular changes under simulated space vacuum (~10-5 Pa) using micro-Raman spectroscopy and found that this vacuum did not cause significant denaturation of spore protein. Then, live-cell microscopy was developed to investigate the temporal events during germination, outgrowth, and growth of individual Bacillus spores. The results showed that after exposure to simulated space vacuum for 10 days, viability of spores of two Bacillus species was reduced up to 35%, but all spores retained their large Ca2+-dipicolinic acid depot. Some of the killed spores did not germinate, and the remaining germinated but did not proceed to vegetative growth. The vacuum treatment slowed spore germination, and changed average times of all major germination events. In addition, viable vacuum-treated spores exhibited much greater sensitivity than untreated spores to dry heat and hyperosmotic stress. Among spores' resistance mechanisms to high vacuum, DNA-protective α/ß-type small acid-soluble proteins, and non-homologous end joining and base excision repair of DNA played the most important roles, especially against multiple cycles of vacuum treatment. Overall, these results give new insight into individual spore's responses to space vacuum and provide new techniques for microorganism analysis at the single-cell level.

Design of a spaceflight biofilm experiment.

Biofilm growth has been observed in Soviet/Russian (Salyuts and Mir), American (Skylab), and International (ISS) Space Stations, sometimes jeopardizing key equipment like spacesuits, water recycling units, radiators, and navigation windows. Biofilm formation also increases the risk of human illnesses and therefore needs to be well understood to enable safe, long-duration, human space missions. Here, the design of a NASA-supported biofilm in space project is reported. This new project aims to characterize biofilm inside the International Space Station in a controlled fashion, assessing changes in mass, thickness, and morphology. The space-based experiment also aims at elucidating the biomechanical and transcriptomic mechanisms involved in the formation of a "column-and-canopy" biofilm architecture that has previously been observed in space. To search for potential solutions, different materials and surface topologies will be used as the substrata for microbial growth. The adhesion of bacteria to surfaces and therefore the initial biofilm formation is strongly governed by topographical surface features of about the bacterial scale. Thus, using Direct Laser-Interference Patterning, some material coupons will have surface patterns with periodicities equal, above or below the size of bacteria. Additionally, a novel lubricant-impregnated surface will be assessed for potential Earth and spaceflight anti-biofilm applications. This paper describes the current experiment design including microbial strains and substrata materials and nanotopographies being considered, constraints and limitations that arise from performing experiments in space, and the next steps needed to mature the design to be spaceflight-ready.