Bacterial genome sequences of uncharacterized Chitinophaga species isolated from the International Space Station.

We report four Chitinophaga sp. strains isolated from wastewater collected onboard the International Space Station. Here, we present three finished and one draft genome. Taxonomic ranks established by genome-based analysis indicate that these Chitinophaga sp. strains represent candidates for a new species.

Genome Sequences of Bacteria Isolated from the International Space Station Water Systems.

We report draft genomes of five bacteria recovered from the U.S. and Russian water systems onboard the International Space Station. The five genera include Ralstonia, Burkholderia, Cupriavidus, Methylobacterium, and Pseudomonas. These sequences will help further the understanding of water reclamation and environmental control and life support systems in space.

Microbial isolation and characterization from two flex lines from the urine processor assembly onboard the international space station.

Urine, humidity condensate, and other sources of non-potable water are processed onboard the International Space Station (ISS) by the Water Recovery System (WRS) yielding potable water. While some means of microbial control are in place, including a phosphoric acid/hexavalent chromium urine pretreatment solution, many areas within the WRS are not available for routine microbial monitoring. Due to refurbishment needs, two flex lines from the Urine Processor Assembly (UPA) within the WRS were removed and returned to Earth. The water from within these lines, as well as flush water, was microbially evaluated. Culture and culture-independent analysis revealed the presence of Burkholderia, Paraburkholderia, and Leifsonia. Fungal culture also identified Fusarium and Lecythophora. Hybrid de novo genome analysis of the five distinct Burkholderia isolates identified them as B. contaminans, while the two Paraburkholderia isolates were identified as P. fungorum. Chromate-resistance gene clusters were identified through pangenomic analysis that differentiated these genomes from previously studied isolates recovered from the point-of-use potable water dispenser and/or current NCBI references, indicating that unique populations exist within distinct niches in the WRS. Beyond genomic analysis, fixed samples directly from the lines were imaged by environmental scanning electron microscopy, which detailed networks of fungal-bacterial biofilms. This is the first evidence of biofilm formation within flex lines from the UPA onboard the ISS. For all bacteria isolated, biofilm potential was further characterized, with the B. contaminans isolates demonstrating the most considerable biofilm formation. Moreover, the genomes of the B. contaminans revealed secondary metabolite gene clusters associated with quorum sensing, biofilm formation, antifungal compounds, and hemolysins. The potential production of these gene cluster metabolites was phenotypically evaluated through biofilm, bacterial-fungal interaction, and hemolytic assays. Collectively, these data identify the UPA flex lines as a unique ecological niche and novel area of biofilm growth within the WRS. Further investigation of these organisms and their resistance profiles will enable engineering controls directed toward biofilm prevention in future space station water systems.

Cyclic electron flow (CEF) and ascorbate pathway activity provide constitutive photoprotection for the photopsychrophile, Chlamydomonas sp. UWO 241 (renamed Chlamydomonas priscuii).

Under environmental stress, plants and algae employ a variety of strategies to protect the photosynthetic apparatus and maintain photostasis. To date, most studies on stress acclimation have focused on model organisms which possess limited to no tolerance to stressful extremes. We studied the ability of the Antarctic alga Chlamydomonas sp. UWO 241 (UWO 241) to acclimate to low temperature, high salinity or high light. UWO 241 maintained robust growth and photosynthetic activity at levels of temperature (2 °C) and salinity (700 mM NaCl) which were nonpermissive for a mesophilic sister species, Chlamydomonas raudensis SAG 49.72 (SAG 49.72). Acclimation in the mesophile involved classic mechanisms, including downregulation of light harvesting and shifts in excitation energy between photosystem I and II. In contrast, UWO 241 exhibited high rates of PSI-driven cyclic electron flow (CEF) and a larger capacity for nonphotochemical quenching (NPQ). Furthermore, UWO 241 exhibited constitutively high activity of two key ascorbate cycle enzymes, ascorbate peroxidase and glutathione reductase and maintained a large ascorbate pool. These results matched the ability of the psychrophile to maintain low ROS under short-term photoinhibition conditions. We conclude that tight control over photostasis and ROS levels are essential for photosynthetic life to flourish in a native habitat of permanent photooxidative stress. We propose to rename this organism Chlamydomonas priscuii.

A CRISPR-based assay for the study of eukaryotic DNA repair onboard the International Space Station.

As we explore beyond Earth, astronauts may be at risk for harmful DNA damage caused by ionizing radiation. Double-strand breaks are a type of DNA damage that can be repaired by two major cellular pathways: non-homologous end joining, during which insertions or deletions may be added at the break site, and homologous recombination, in which the DNA sequence often remains unchanged. Previous work suggests that space conditions may impact the choice of DNA repair pathway, potentially compounding the risks of increased radiation exposure during space travel. However, our understanding of this problem has been limited by technical and safety concerns, which have prevented integral study of the DNA repair process in space. The CRISPR/Cas9 gene editing system offers a model for the safe and targeted generation of double-strand breaks in eukaryotes. Here we describe a CRISPR-based assay for DNA break induction and assessment of double-strand break repair pathway choice entirely in space. As necessary steps in this process, we describe the first successful genetic transformation and CRISPR/Cas9 genome editing in space. These milestones represent a significant expansion of the molecular biology toolkit onboard the International Space Station.

Real-Time Culture-Independent Microbial Profiling Onboard the International Space Station Using Nanopore Sequencing.

For the past two decades, microbial monitoring of the International Space Station (ISS) has relied on culture-dependent methods that require return to Earth for analysis. This has a number of limitations, with the most significant being bias towards the detection of culturable organisms and the inherent delay between sample collection and ground-based analysis. In recent years, portable and easy-to-use molecular-based tools, such as Oxford Nanopore Technologies' MinION™ sequencer and miniPCR bio's miniPCR™ thermal cycler, have been validated onboard the ISS. Here, we report on the development, validation, and implementation of a swab-to-sequencer method that provides a culture-independent solution to real-time microbial profiling onboard the ISS. Method development focused on analysis of swabs collected in a low-biomass environment with limited facility resources and stringent controls on allowed processes and reagents. ISS-optimized procedures included enzymatic DNA extraction from a swab tip, bead-based purifications, altered buffers, and the use of miniPCR and the MinION. Validation was conducted through extensive ground-based assessments comparing current standard culture-dependent and newly developed culture-independent methods. Similar microbial distributions were observed between the two methods; however, as expected, the culture-independent data revealed microbial profiles with greater diversity. Protocol optimization and verification was established during NASA Extreme Environment Mission Operations (NEEMO) analog missions 21 and 22, respectively. Unique microbial profiles obtained from analog testing validated the swab-to-sequencer method in an extreme environment. Finally, four independent swab-to-sequencer experiments were conducted onboard the ISS by two crewmembers. Microorganisms identified from ISS swabs were consistent with historical culture-based data, and primarily consisted of commonly observed human-associated microbes. This simplified method has been streamlined for high ease-of-use for a non-trained crew to complete in an extreme environment, thereby enabling environmental and human health diagnostics in real-time as future missions take us beyond low-Earth orbit.

Prokaryotic and Fungal Characterization of the Facilities Used to Assemble, Test, and Launch the OSIRIS-REx Spacecraft.

To characterize the ATLO (Assembly, Test, and Launch Operations) environment of the OSIRIS-REx spacecraft, we analyzed 17 aluminum witness foils and two blanks for bacterial, archaeal, fungal, and arthropod DNA. Under NASA's Planetary Protection guidelines, OSIRIS-REx is a Category II outbound, Category V unrestricted sample return mission. As a result, it has no bioburden restrictions. However, the mission does have strict organic contamination requirements to achieve its primary objective of returning pristine carbonaceous asteroid regolith to Earth. Its target, near-Earth asteroid (101955) Bennu, is likely to contain organic compounds that are biologically available. Therefore, it is useful to understand what organisms were present during ATLO as part of the larger contamination knowledge effort-even though it is unlikely that any of the organisms will survive the multi-year deep space journey. Even though these samples of opportunity were not collected or preserved for DNA analysis, we successfully amplified bacterial and archaeal DNA (16S rRNA gene) from 16 of the 17 witness foils containing as few as 7 ± 3 cells per sample. Fungal DNA (ITS1) was detected in 12 of the 17 witness foils. Despite observing arthropods in some of the ATLO facilities, arthropod DNA (COI gene) was not detected. We observed 1,009 bacterial and archaeal sOTUs (sub-operational taxonomic units, 100% unique) and 167 fungal sOTUs across all of our samples (25-84 sOTUs per sample). The most abundant bacterial sOTU belonged to the genus Bacillus. This sOTU was present in blanks and may represent contamination during sample handling or storage. The sample collected from inside the fairing just prior to launch contained several unique bacterial and fungal sOTUs that describe previously uncharacterized potential for contamination during the final phase of ATLO. Additionally, fungal richness (number of sOTUs) negatively correlates with the number of carbon-bearing particles detected on samples. The total number of fungal sequences positively correlates with total amino acid concentration. These results demonstrate that it is possible to use samples of opportunity to characterize the microbiology of low-biomass environments while also revealing the limitations imposed by sample collection and preservation methods not specifically designed with biology in mind.

Glycerol Is an Osmoprotectant in Two Antarctic Chlamydomonas Species From an Ice-Covered Saline Lake and Is Synthesized by an Unusual Bidomain Enzyme.

Glycerol, a compatible solute, has previously been found to act as an osmoprotectant in some marine Chlamydomonas species and several species of Dunaliella from hypersaline ponds. Recently, Chlamydomonas reinhardtii and Dunaliella salina were shown to make glycerol with an unusual bidomain enzyme, which appears to be unique to algae, that contains a phosphoserine phosphatase and glycerol-3-phosphate dehydrogenase. Here we report that two psychrophilic species of Chlamydomonas (C. spp. UWO241 and ICE-MDV) from Lake Bonney, Antarctica also produce high levels of glycerol to survive in the lake's saline waters. Glycerol concentration increased linearly with salinity and at 1.3 M NaCl, exceeded 400 mM in C. sp. UWO241, the more salt-tolerant strain. We also show that both species expressed several isoforms of the bidomain enzyme. An analysis of one of the isoforms of C. sp. UWO241 showed that it was strongly upregulated by NaCl and is thus the likely source of glycerol. These results reveal another adaptation of the Lake Bonney Chlamydomonas species that allow them to survive in an extreme polar environment.