Bridging Place-Based Astrobiology Education with Genomics, Including Descriptions of Three Novel Bacterial Species Isolated from Mars Analog Sites of Cultural Relevance.

Democratizing genomic data science, including bioinformatics, can diversify the STEM workforce and may, in turn, bring new perspectives into the space sciences. In this respect, the development of education and research programs that bridge genome science with "place" and world-views specific to a given region are valuable for Indigenous students and educators. Through a multi-institutional collaboration, we developed an ongoing education program and model that includes Illumina and Oxford Nanopore sequencing, free bioinformatic platforms, and teacher training workshops to address our research and education goals through a place-based science education lens. High school students and researchers cultivated, sequenced, assembled, and annotated the genomes of 13 bacteria from Mars analog sites with cultural relevance, 10 of which were novel species. Students, teachers, and community members assisted with the discovery of new, potentially chemolithotrophic bacteria relevant to astrobiology. This joint education-research program also led to the discovery of species from Mars analog sites capable of producing N-acyl homoserine lactones, which are quorum-sensing molecules used in bacterial communication. Whole genome sequencing was completed in high school classrooms, and connected students to funded space research, increased research output, and provided culturally relevant, place-based science education, with participants naming three novel species described here. Students at St. Andrew's School (Honolulu, Hawai'i) proposed the name Bradyrhizobium prioritasuperba for the type strain, BL16AT, of the new species (DSM 112479T = NCTC 14602T). The nonprofit organization Kauluakalana proposed the name Brenneria ulupoensis for the type strain, K61T, of the new species (DSM 116657T = LMG = 33184T), and Hawai'i Baptist Academy students proposed the name Paraflavitalea speifideiaquila for the type strain, BL16ET, of the new species (DSM 112478T = NCTC 14603T).

Toward sustainable space exploration: a roadmap for harnessing the power of microorganisms.

Finding sustainable approaches to achieve independence from terrestrial resources is of pivotal importance for the future of space exploration. This is relevant not only to establish viable space exploration beyond low Earth-orbit, but also for ethical considerations associated with the generation of space waste and the preservation of extra-terrestrial environments. Here we propose and highlight a series of microbial biotechnologies uniquely suited to establish sustainable processes for in situ resource utilization and loop-closure. Microbial biotechnologies research and development for space sustainability will be translatable to Earth applications, tackling terrestrial environmental issues, thereby supporting the United Nations Sustainable Development Goals.

The smallest space miners: principles of space biomining.

As we aim to expand human presence in space, we need to find viable approaches to achieve independence from terrestrial resources. Space biomining of the Moon, Mars and asteroids has been indicated as one of the promising approaches to achieve in-situ resource utilization by the main space agencies. Structural and expensive metals, essential mineral nutrients, water, oxygen and volatiles could be potentially extracted from extraterrestrial regolith and rocks using microbial-based biotechnologies. The use of bioleaching microorganisms could also be applied to space bioremediation, recycling of waste and to reinforce regenerative life support systems. However, the science around space biomining is still young. Relevant differences between terrestrial and extraterrestrial conditions exist, including the rock types and ores available for mining, and a direct application of established terrestrial biomining techniques may not be a possibility. It is, therefore, necessary to invest in terrestrial and space-based research of specific methods for space applications to learn the effects of space conditions on biomining and bioremediation, expand our knowledge on organotrophic and community-based bioleaching mechanisms, as well as on anaerobic biomining, and investigate the use of synthetic biology to overcome limitations posed by the space environments.

Microbially-Enhanced Vanadium Mining and Bioremediation Under Micro- and Mars Gravity on the International Space Station.

As humans explore and settle in space, they will need to mine elements to support industries such as manufacturing and construction. In preparation for the establishment of permanent human settlements across the Solar System, we conducted the ESA BioRock experiment on board the International Space Station to investigate whether biological mining could be accomplished under extraterrestrial gravity conditions. We tested the hypothesis that the gravity (g) level influenced the efficacy with which biomining could be achieved from basalt, an abundant material on the Moon and Mars, by quantifying bioleaching by three different microorganisms under microgravity, simulated Mars and Earth gravitational conditions. One element of interest in mining is vanadium (V), which is added to steel to fabricate high strength, corrosion-resistant structural materials for buildings, transportation, tools and other applications. The results showed that Sphingomonas desiccabilis and Bacillus subtilis enhanced the leaching of vanadium under the three gravity conditions compared to sterile controls by 184.92 to 283.22%, respectively. Gravity did not have a significant effect on mean leaching, thus showing the potential for biomining on Solar System objects with diverse gravitational conditions. Our results demonstrate the potential to use microorganisms to conduct elemental mining and other bioindustrial processes in space locations with non-1 × g gravity. These same principles apply to extraterrestrial bioremediation and elemental recycling beyond Earth.

Space station biomining experiment demonstrates rare earth element extraction in microgravity and Mars gravity.

Microorganisms are employed to mine economically important elements from rocks, including the rare earth elements (REEs), used in electronic industries and alloy production. We carried out a mining experiment on the International Space Station to test hypotheses on the bioleaching of REEs from basaltic rock in microgravity and simulated Mars and Earth gravities using three microorganisms and a purposely designed biomining reactor. Sphingomonas desiccabilis enhanced mean leached concentrations of REEs compared to non-biological controls in all gravity conditions. No significant difference in final yields was observed between gravity conditions, showing the efficacy of the process under different gravity regimens. Bacillus subtilis exhibited a reduction in bioleaching efficacy and Cupriavidus metallidurans showed no difference compared to non-biological controls, showing the microbial specificity of the process, as on Earth. These data demonstrate the potential for space biomining and the principles of a reactor to advance human industry and mining beyond Earth.

No Effect of Microgravity and Simulated Mars Gravity on Final Bacterial Cell Concentrations on the International Space Station: Applications to Space Bioproduction.

Microorganisms perform countless tasks on Earth and they are expected to be essential for human space exploration. Despite the interest in the responses of bacteria to space conditions, the findings on the effects of microgravity have been contradictory, while the effects of Martian gravity are nearly unknown. We performed the ESA BioRock experiment on the International Space Station to study microbe-mineral interactions in microgravity, simulated Mars gravity and simulated Earth gravity, as well as in ground gravity controls, with three bacterial species: Sphingomonas desiccabilis, Bacillus subtilis, and Cupriavidus metallidurans. To our knowledge, this was the first experiment to study simulated Martian gravity on bacteria using a space platform. Here, we tested the hypothesis that different gravity regimens can influence the final cell concentrations achieved after a multi-week period in space. Despite the different sedimentation rates predicted, we found no significant differences in final cell counts and optical densities between the three gravity regimens on the ISS. This suggests that possible gravity-related effects on bacterial growth were overcome by the end of the experiment. The results indicate that microbial-supported bioproduction and life support systems can be effectively performed in space (e.g., Mars), as on Earth.

The hypoxic expression of the glucose transporter RAG1 reveals the role of the bHLH transcription factor Sck1 as a novel hypoxic modulator in Kluyveromyces lactis.

Glucose is the preferred nutrient for most living cells and is also a signaling molecule that modulates several cellular processes. Glucose regulates the expression of glucose permease genes in yeasts through signaling pathways dependent on plasma membrane glucose sensors. In the yeast Kluyveromyces lactis, sufficient levels of glucose induction of the low-affinity glucose transporter RAG1 gene also depends on a functional glycolysis, suggesting additional intracellular signaling. We have found that the expression of RAG1 gene is also induced by hypoxia in the presence of glucose, indicating that glucose and oxygen signaling pathways are interconnected. In this study we investigated the molecular mechanisms underlying this crosstalk. By analyzing RAG1 expression in various K. lactis mutants, we found that the bHLH transcriptional activator Sck1 is required for the hypoxic induction of RAG1 gene. The RAG1 promoter region essential for its hypoxic induction was identified by promoter deletion experiments. Taken together, these results show that the RAG1 glucose permease gene is synergistically induced by hypoxia and glucose and highlighted a novel role for the transcriptional activator Sck1 as a key mediator in this mechanism.

The hypoxic transcription factor KlMga2 mediates the response to oxidative stress and influences longevity in the yeast Kluyveromyces lactis.

Hypoxia is defined as the decline of oxygen availability, depending on environmental supply and cellular consumption rate. The decrease in O2 results in reduction of available energy in facultative aerobes. The response and/or adaptation to hypoxia and other changing environmental conditions can influence the properties and functions of membranes by modifying lipid composition. In the yeast Kluyveromyces lactis, the KlMga2 gene is a hypoxic regulatory factor for lipid biosynthesis-fatty acids and sterols-and is also involved in glucose signaling, glucose catabolism and is generally important for cellular fitness. In this work we show that, in addition to the above defects, the absence of the KlMGA2 gene caused increased resistance to oxidative stress and extended lifespan of the yeast, associated with increased expression levels of catalase and SOD genes. We propose that KlMga2 might also act as a mediator of the oxidative stress response/adaptation, thus revealing connections among hypoxia, glucose signaling, fatty acid biosynthesis and ROS metabolism in K. lactis.

Three, two, one yeast fatty acid desaturases: regulation and function.

Fatty acid composition of biological membranes functionally adapts to environmental conditions by changing its composition through the activity of lipid biosynthetic enzymes, including the fatty acid desaturases. Three major desaturases are present in yeasts, responsible for the generation of double bonds in position C9-C10, C12-C13 and C15-C16 of the carbon backbone. In this review, we will report data addressed to define the functional role of basidiomycete and ascomycete yeast desaturase enzymes in response to various external signals and the regulation of the expression of their corresponding genes. Many yeast species have the complete set of three desaturases; however, only the Δ9 desaturase seems to be necessary and sufficient to ensure yeast viability. The evolutionary issue of this observation will be discussed.

Functional roles of the fatty acid desaturases encoded by KlOLE1, FAD2 and FAD3 in the yeast Kluyveromyces lactis.

Functional properties of cell membranes depend on their composition, particularly on the relative amount of saturated, unsaturated and polyunsaturated fatty acids present in the phospholipids. The aim of this study was to investigate the effect of cell membrane composition on cell fitness, adaptation and stress response in Kluyveromyces lactis. To this purpose, we have deleted the genes FAD2 and FAD3 encoding Δ12 and ω3 desaturases in Kluyveromyces lactis, thus generating mutant strains with altered fatty acid composition of membranes. These strains were viable and able to grow in stressing conditions like hypoxia and low temperature. Deletion of the Δ9 desaturase-encoding gene KlOLE1 resulted in lethality, suggesting that this enzyme has an essential role in this yeast. Transcription of the desaturase genes KlOLE1, FAD2 and FAD3 and cellular localization of the corresponding enzymes, have been studied under hypoxia and cold stress. Our findings indicate that expression of these desaturase genes and membrane composition were modulated by hypoxia and temperature stress, although the changes induced by these and other assayed conditions did not dramatically affect the general cellular fitness.

Unsaturated fatty acids-dependent linkage between respiration and fermentation revealed by deletion of hypoxic regulatory KlMGA2 gene in the facultative anaerobe-respiratory yeast Kluyveromyces lactis.

In the yeast Kluyveromyces lactis, the inactivation of structural or regulatory glycolytic and fermentative genes generates obligate respiratory mutants which can be characterized by sensitivity to the mitochondrial drug antimycin A on glucose medium (Rag(-) phenotype). Rag(-) mutations can occasionally be generated by the inactivation of genes not evidently related to glycolysis or fermentation. One such gene is the hypoxic regulatory gene KlMGA2. In this work, we report a study of the many defects, in addition to the Rag(-) phenotype, generated by KlMGA2 deletion. We analyzed the fermentative and respiratory metabolism, mitochondrial functioning and morphology in the Klmga2Δ strain. We also examined alterations in the regulation of the expression of lipid biosynthetic genes, in particular fatty acids, ergosterol and cardiolipin, under hypoxic and cold stress and the phenotypic suppression by unsaturated fatty acids of the deleted strain. Results indicate that, despite the fact that the deleted mutant strain had a typical glycolytic/fermentative phenotype and KlMGA2 is a hypoxic regulatory gene, the deletion of this gene generated defects linked to mitochondrial functions suggesting new roles of this protein in the general regulation and cellular fitness of K. lactis. Supplementation of unsaturated fatty acids suppressed or modified these defects suggesting that KlMga2 modulates membrane functioning or membrane-associated functions, both cytoplasmic and mitochondrial.