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At least 1,189 records · Page 66

Conserved and reproducible bacterial communities associate with extraradical hyphae of arbuscular mycorrhizal fungi

Abstract Extraradical hyphae (ERH) of arbuscular mycorrhizal fungi (AMF) extend from plant roots into the soil environment and interact with soil microbial communities. Evidence of positive and negative interactions between AMF and soil bacteria point to functionally important ERH-associated communities. To characterize communities associated with ERH and test controls on their establishment and composition, we utilized an in-growth core system containing a live soil–sand mixture that allowed manual extraction of ERH for 16S rRNA gene amplicon profiling. Across experiments and soils, consistent enrichment of members of the Betaproteobacteriales, Myxococcales, Fibrobacterales, Cytophagales, Chloroflexales, and Cellvibrionales was observed on ERH samples, while variation among samples from different soils was observed primarily at lower taxonomic ranks. The ERH-associated community was conserved between two fungal species assayed, Glomus versiforme and Rhizophagus irregularis, though R. irregularis exerted a stronger selection and showed greater enrichment for taxa in the Alphaproteobacteria and Gammaproteobacteria. A distinct community established within 14 days of hyphal access to the soil, while temporal patterns of establishment and turnover varied between taxonomic groups. Identification of a conserved ERH-associated community is consistent with the concept of an AMF microbiome and can aid the characterization of facilitative and antagonistic interactions influencing the plant-fungal symbiosis.

59 BASIC BIOLOGICAL SCIENCES↗

Bulk and Spatially Resolved Extracellular Metabolome of Free-Living Nitrogen Fixation

Free-living nitrogen-fixing bacteria contribute significantly to terrestrial nitrogen availability; however, the forms of nitrogen contributed by this process are poorly understood. This is in part because of inherent challenges to studying soil microorganisms in situ , such as vast differences in scale between microorganism and ecosystem and complexities of the soil system (e.g., opacity, chemical complexity).

59 BASIC BIOLOGICAL SCIENCES↗

Space Station Environmental Health System water quality monitoring

One of the unique aspects of the Space Station is that it will be a totally encapsulated environment and the air and water supplies will be reclaimed for reuse. The Environmental Health System, a subsystem of CHeCS (Crew Health Care System), must monitor the air and water on board the Space Station Freedom to verify that the quality is adequate for crew safety. Specifically, the Water Quality Subsystem will analyze the potable and hygiene water supplies regularly for organic, inorganic, particulate, and microbial contamination. The equipment selected to perform these analyses will be commercially available instruments which will be converted for use on board the Space Station Freedom. Therefore, the commercial hardware will be analyzed to identify the gravity dependent functions and modified to eliminate them. The selection, analysis, and conversion of the off-the-shelf equipment for monitoring the Space Station reclaimed water creates a challenging project for the Water Quality engineers and scientists.

Vincze, Johanna E.↗

Symbiosis and the origin of eukaryotic motility

Ongoing work to test the hypothesis of the origin of eukaryotic cell organelles by microbial symbioses is discussed. Because of the widespread acceptance of the serial endosymbiotic theory (SET) of the origin of plastids and mitochondria, the idea of the symbiotic origin of the centrioles and axonemes for spirochete bacteria motility symbiosis was tested. Intracellular microtubular systems are purported to derive from symbiotic associations between ancestral eukaryotic cells and motile bacteria. Four lines of approach to this problem are being pursued: (1) cloning the gene of a tubulin-like protein discovered in Spirocheata bajacaliforniesis; (2) seeking axoneme proteins in spirochets by antibody cross-reaction; (3) attempting to cultivate larger, free-living spirochetes; and (4) studying in detail spirochetes (e.g., Cristispira) symbiotic with marine animals. Other aspects of the investigation are presented.

Margulis, L.↗

Astrobiology Extends Biology into Deep Time and Space

To understand our own origins and to search for biospheres beyond Earth, we need a more robust concept of life itself. We must learn how to discriminate between attributes that are fundamental to all living systems versus those that represent principally local outcomes of long-term survival on Earth. We should identify the most basic environmental needs of life, chart the distribution of other habitable worlds, and understand the factors that created their distribution. Studies of microbial communities and the geologic record will be summarized that offer clues about the early evolution of our own biosphere as well as the signatures of life that we might find in the heavens.

Desmarais, David↗

Engineering of Methane Metabolism in Pichia Pastoris Through Methane Monooxygenase Expression

Exploration of the solar system is constrained by the cost of moving mass off Earth. Producing materials in situ will reduce the mass that must be delivered from earth. CO2 is abundant on Mars and manned spacecraft. On the ISS, NASA reacts excess CO2 with H2 to generate CH4 and H2O using the Sabatier System. The resulting water is recovered into the ISS, but the methane is vented to space. Thus, there is a capability need for systems that convert methane into valuable materials. Methanotrophic bacteria consume methane but these are poor synthetic biology platforms. Thus, there is a knowledge gap in utilizing methane in a robust and flexible synthetic biology platform. The yeast Pichia pastoris is a refined microbial factory that is used widely by industry because it efficiently secretes products. Pichia could produce a variety of useful products in space. Pichia does not consume methane but robustly consumes methanol, which is one enzymatic step removed from methane. Our goal is to engineer Pichia to consume methane thereby creating a powerful methane-consuming microbial factory.

Pichia↗

New Frontiers in Synthetic Biology for Spaceflight

Exploration of the solar system is constrained by the cost of moving mass off Earth. Producing materials in situ will reduce the mass that must be delivered from earth. CO2 is abundant on Mars and manned spacecraft. On the ISS, NASA reacts excess CO2 with H2 to generate CH4 and H2O using the Sabatier System. The resulting water is recovered into the ISS, but the methane is vented to space. Thus, there is a capability need for systems that convert methane into valuable materials. Methanotrophic bacteria consume methane but these are poor synthetic biology platforms. Thus, there is a knowledge gap in utilizing methane in a robust and flexible synthetic biology platform. The yeast Pichia pastoris is a refined microbial factory that is used widely by industry because it efficiently secretes products. Pichia could produce a variety of useful products in space. Pichia does not consume methane but robustly consumes methanol, which is one enzymatic step removed from methane. Our goal is to engineer Pichia to consume methane thereby creating a powerful methane-consuming microbial factory.

Galazka, Jonathan M.↗

Methane Metabolism by Yeast for Solar System Exploration

Exploration of the solar system is constrained by the cost of moving mass off Earth. Producing materials in situ will reduce the mass that must be delivered from earth. CO2 is abundant on Mars and manned spacecraft. On the ISS, NASA reacts excess CO2 with H2 to generate CH4 and H2O using the Sabatier System. The resulting water is recovered into the ISS, but the methane is vented to space. Thus, there is a capability need for systems that convert methane into valuable materials. Methanotrophic bacteria consume methane but these are poor synthetic biology platforms. Thus, there is a knowledge gap in utilizing methane in a robust and flexible synthetic biology platform. The yeast Pichia pastoris is a refined microbial factory that is used widely by industry because it efficiently secretes products. Pichia could produce a variety of useful products in space. Pichia does not consume methane but robustly consumes methanol, which is one enzymatic step removed from methane. Our goal is to engineer Pichia to consume methane thereby creating a powerful methane-consuming microbial factory.

Galazka, Jonathan M.↗

Engineering of Methane Metabolism in Pichia Pastoris Through Methane Monooxygenase Expression

Exploration of the solar system is constrained by the cost of moving mass off Earth. Producing materials in situ will reduce the mass that must be delivered from earth. CO2 is abundant on Mars and manned spacecraft. On the ISS, NASA reacts excess CO2 with H2 to generate CH4 and H2O using the Sabatier System. The resulting water is recovered into the ISS, but the methane is vented to space. Thus, there is a capability need for systems that convert methane into valuable materials. Methanotrophic bacteria consume methane but these are poor synthetic biology platforms. Thus, there is a knowledge gap in utilizing methane in a robust and flexible synthetic biology platform. The yeast Pichia pastoris is a refined microbial factory that is used widely by industry because it efficiently secretes products. Pichia could produce a variety of useful products in space. Pichia does not consume methane but robustly consumes methanol, which is one enzymatic step removed from methane. Our goal is to engineer Pichia to consume methane thereby creating a powerful methane-consuming microbial factory.

Methane↗

New Frontiers in Synthetic Biology for Spaceflight

Exploration of the solar system is constrained by the cost of moving mass off Earth. Producing materials in situ will reduce the mass that must be delivered from earth. CO2 is abundant on Mars and manned spacecraft. On the ISS, NASA reacts excess CO2 with H2 to generate CH4 and H2O using the Sabatier System. The resulting water is recovered into the ISS, but the methane is vented to space. Thus, there is a capability need for systems that convert methane into valuable materials. Methanotrophic bacteria consume methane but these are poor synthetic biology platforms. Thus, there is a knowledge gap in utilizing methane in a robust and flexible synthetic biology platform. The yeast Pichia pastoris is a refined microbial factory that is used widely by industry because it efficiently secretes products. Pichia could produce a variety of useful products in space. Pichia does not consume methane but robustly consumes methanol, which is one enzymatic step removed from methane. Our goal is to engineer Pichia to consume methane thereby creating a powerful methane-consuming microbial factory.

methane↗

Enterococci in Space: Adaptation, Antibiotic Resistance, and Clinical Implications

Enterococci are gram-positive bacteria that originated when our ancient animal ancestors emerged from the oceans to live on land, and brought their gut flora with them. Enterococcus faecalis (EF) and Enterococcus faecium, are common human commensals and can harbor multidrug resistance. Both have been previously isolated from the International Space Station (ISS). Likely as a consequence of their evolutionary origins, enterococci show remarkable stress resistance within, but also outside, their human hosts. Their antibiotic resistance, coupled with tolerance to desiccation, starvation, and disinfection, make some EF strains potent pathogens in the built environment (e.g., hospitals), and a potential risk to crew health during space missions. Here we describe our planned flight studies, currently in development. Genomic Enumeration of Antibiotic Resistance in Space (GEARS) will characterize the frequency and genomic identity of antibiotic resistant organisms, including enterococci, on the ISS, and expand our future in-space sequencing-based diagnostic capabilities. Enterococcus Growth Advantage on ISS via Tn-seq (EnteroGAIT) will assess the evolutionary selective pressure of the space environment (microgravity, space radiation) using EF as a model system during a long-duration culture and persistence experiment. Adaptation & Evolution of Resilient Enterococcus in Space (AERES) will study existing and newly identified isolates to characterize the “natural” evolutionary history of EF on Earth and in space to reveal mechanisms of microbial adaption including, possibly, natural selection. Our work to date has identified that EF ISS isolates share virulence factors found in clinical strains and commensals, and revealed limitations of current pathogenicity assessment tools. While ISS isolates were, in some cases, multi-drug resistant, current data suggests these are largely commensal strains. Our work will further refine potential crew health risks and improve understanding of EF adaptation to the built environment, of great relevance on Earth where EF is the second leading cause of hospital acquired infection.

Jordan McKaig↗

Benchmarking Computational Tools for Calling SNPs and Indels in Complex Microbial Populations

The NASA BioNutrients missions seek to understand the suitability of microorganisms for bioproduction during space flight. One topic of interest is the stability of microbial genomes during long-term ambient storage and subsequent rehydration and growth. To address these questions, samples from 8 species were flown to ISS for 5 years of desiccated storage at ambient temperature (Stasis Packs) and 2 species were packaged along with powdered media inside a bioreactor system to allow hydration and growth in microgravity (Production Packs). For both systems, Whole Genome Sequencing (WGS) of the DNA extracted from the returned samples and paired ground controls will be conducted to identify changes in genome stability due to time, storage conditions and growth in space. Across the technical replicates, ground controls, 10 timepoints, and multiple experimental conditions, ~300 samples have been selected for initial analysis with WGS sequencing to 100x coverage. A flexible and resource efficient mutation calling pipeline is needed to process this large dataset and allow for comparisons between species. Many bioinformatics tools for calling Indels and Single Nucleotide Variants (SNVs) are designed for use with pure isolates, where true variations from the reference genome are expected to dominate the reads aligning to the location of mutation. In contrast, DNA from the Stasis Pack (SP) samples was collected directly after recovery from desiccated storage and the Production Pack (PP) samples were collected after fermentation. In this context, reads with mutations are expected to be less frequent than reads that align with the reference genome, as each sample will include multiple lines of cells. Thus, BioNutrients samples are expected to be similar to samples from cancer cell or “pooled” sequencing approaches. In preparation for the analysis of the BioNutrients samples, we have tested three mutation calling tools (GATK for Microbes, BreSeq and DiscoSNP) designed for complex samples. A challenge of validating mutation identification pipelines is a lack of “Ground Truth” datasets, especially for complex samples. To compare these three tools, we sought to identify mutations in pre-existing WGS data collected from populations of Chlamydomonas reinhardtii that were exposed to UV mutagenesis and growth in LEO as part of the Space Algae-1 mission. Here we present a summary of these tools against the analysis originally conducted using the CRISP tool. Critical metrics are compared such as runtime, the number of SNPs, the number and size of Indels, and patterns of transversion and transitions identified by each tool are reported. By sharing these benchmarking results collected in support of the BioNutrients mission, we aim to guide others seeking to identify SNVs in similarly complex microbial samples.

Biology↗

The Need for Earth-Based Experiments to Inform Microbial Evolution on Planetary Surfaces

Introduction: Historically, the focus of planetary protection at NASA has been on unmanned, robotic missions. Such missions have paved the way for understanding how to implement planetary protection in a feasible and cost-sensitive way. However, with the introduction of crewed missions to Mars in the not-sodistant future, there is a need to better define and understand how to implement planetary protection under new circumstances, as well as understand the risk of contaminating Mars. One unavoidable fact is that microbes will go where humans go. Therefore, it is critical to understand how these microbes may (and will) impact our ability to conduct meaningful, reliable astrobiological science. Microorganisms have spent millions of years evolving to survive in extreme environments here on Earth. Already there are indications that microbes aboard the International Space Station evolve and adapt to life in low earth orbit. The microbes that are eventually taken to Mars with humans will also adapt, potentially causing harmful effects to crew and/or the planetary or astrobiological science conducted. Therefore, it is of critical interest that we evaluate and characterize the potential risks of microbial evolution on Mars. It is expected that microbes carried by humans will begin to evolve to new environments even before landing on Mars, during the several month cruise phase. Once landed, microbes will encounter different stressors within the crew habitats on Mars. During extravehicular activities, venting, or other release events, microbes will find their way out onto the Martian surface. The induced environments around crewed systems will create potentially-favorable conditions for microbes to continue evolving on Mars. Eventually, microbes may find their way beyond the close confines of the crewed area and continue evolving so as to fill new or distant niches on the Martian surface. It is challenging to replicate Martian environments here on Earth, making it nearly impossible to predict the evolutionary changes that microbes would undergo on Mars. But this work is critical. Serial passaging experiments performed by Richard Lenski on E. coli show the dramatic changes microbes can undergo even within a laboratory setting. Furthermore, experiments performed by Michael Baym also demonstrate the power of single mutations in microbial development of antibiotic resistance [3]. Long duration experiments should be performed on a suite of microbes exposed to environments likely to be experienced on the Martian surface. While simulating space environments can be challenging, facilities exist that can achieve individual and combinatorial environmental conditions to simulate space and planetary conditions. Such chambers should be employed for microbial studies. Currently, at the Marshall Space Flight Center, we have used various stressors like drying, vacuum, proton radiation, and ultraviolet light both separately and in combination, to evaluate the survival of cleanroom microbes. Shockingly, several non-spore forming isolates have demonstrated the ability to survive many extreme conditions (manuscript in preparation). These short duration exposures must be augmented with larger and more gradual studies to replicate what microbes might experience in the transition from cruise, to surface habitats, to induced surface environments, and finally true Martian environments. While no Earth-based experiment can perfectly replicate the Martian environment, nor could we test every possible microbe in simulation experimental regimes, efforts should be made to examine the evolutionary potential of the “usual suspects” seen on the ISS or in other crewed environments to begin to fill this important knowledge gap.

Chelsi D. Cassilly↗

Biological degradation and composition of inedible sweetpotato biomass

Many challenges are presented by biological degradation in a bioregenerative Controlled Ecological Life Support System (CELSS) as envisioned by the U.S. National Aeronautics and Space Administration (NASA). In the studies conducted with biodegradative microorganism indigenous to sweetpotato fields, it was determined that a particle size of 75 microns and incubation temperature of 30 degrees C were optimal for degradation. The composition of the inedible biomass and characterization of plant nutrient solution indicated the presence of potential energy sources to drive microbial transformations of plant waste. Selected indigenous soil isolates with ligno-cellulolytic or sulfate-reducing ability were utilized in biological studies and demonstrated diversity in ability to reduce sulfate in solution and to utilize alternative carbon sources: a lignin analog--4-hydroxy, 3-methoxy cinnamic acid, cellulose, arabinose, glucose, sucrose, mannitol, galactose, ascorbic acid.

Vegetables/chemistry/metabolism↗

Species- and site-specific genome editing in complex bacterial communities

Knowledge of microbial gene functions comes from manipulating the DNA of individual strains in isolation from their natural communities. While this approach to microbial genetics has been foundational, its requirement for culturable microorganisms has left the majority of microbes and their interactions genetically unexplored. Here, we describe a generalizable strategy for editing the genomes of specific organisms within microbial communities. We identified genetically tractable bacteria within a community using Environmental Transformation Sequencing (ET-Seq), an approach in which non-targeted transposon integrations are mapped and quantified following community delivery. We next developed and used DNA-editing All-in-one RNA-guided CRISPR-Cas Transposase (DART) systems for targeted DNA insertion into organisms identified as tractable by ET-Seq, enabling organism- and locus-specific genetic manipulation within the community context. To illustrate the utility of our approach, we selectively edited closely related strains, measured gene fitness, and enriched targeted members within soil and infant gut microbiota. These results establish a new paradigm for targeted community editing relevant to research and applications on medical, agricultural, and industrial microbiomes.

59 BASIC BIOLOGICAL SCIENCES↗

Veggie: Space Vegetables for the International Space Station and Beyond

The Veggie vegetable production system was launched to the International Space Station (ISS) in 2014. Veggie was designed by ORBITEC to be a compact, low mass, low power vegetable production system for astronaut crews. Veggie consists of a light cap containing red, blue, and green LEDs, an extensible transparent bellows, and a baseplate with a root mat reservoir. Seeds are planted in plant pillows, small growing bags that interface with the reservoir. The Veggie technology validation test, VEG-01, was initiated with the first test crop of 'Outredgeous' red romaine lettuce. Prior to flight, lettuce seeds were sanitized and planted in a substrate of arcillite (baked ceramic) mixed with controlled release fertilizer. Upon initiation, astronauts open the packaged plant pillows, install them in the Veggie hardware, and prime the system with water. Operations include plant thinning, watering, and photography. Plants were grown on the ISS for 33 days, harvested, and returned frozen to Earth for analysis. Ground controls were conducted at Kennedy Space Center in controlled environment chambers reproducing ISS conditions of temperature, relative humidity, and CO2. Returned plant samples were analyzed for microbial food safety and chemistry including elements, antioxidants, anthocyanins and phenolics. In addition the entire plant microbiome was sequenced, and returned plant pillows were analyzed via x-ray tomography. Food safety analyses allowed us to gain approvals for future consumption of lettuce by the flight surgeons and the payload safety office. A second crop of lettuce was grown in 2015, and the crew consumed half the produce, with the remainder frozen for later analysis. This growth test was followed by testing of a new crop in Veggie, zinnias. Zinnias were grown to test a longer duration flowering crop in preparation for tests of tomatoes and other fruiting crops in the future. Zinnias were harvested in February. Samples from the second harvest of lettuce and the zinnia harvest are frozen on the ISS and will return with the next cargo return flight. Some challenges occurred in all tests, especially in the area of watering, with plants receiving insufficient or excess water leading to stressed growth. Zinnia plants were also challenged with fungal growth. Initial tests with Veggie have given us great insight into future crop production scenarios as we work to develop regular supplemental salad crop production on ISS and larger food production systems for our journey to Mars. Funding for this research comes from NASA's Space Biology Program.

Food Safety↗

Research and Technology Development to Advance Environmental Monitoring, Food Systems, and Habitat Design for Exploration Beyond Low Earth Orbit

Exploration missions will carry crews far beyond the relatively safe environs of cis-lunar space. Such trips will have little or no opportunity for resupply or rapid aborts and will be of a duration that far exceeds our experience to date. The challenges this imposes on the requirements of systems that monitor the life support and provide food and shelter for the crew are the focus of much research within the Human Research Program. Making all of these technologies robust and reliable enough for multi-year missions with little or no ability to run for home calls for a thorough understanding of the risks and impacts of failure. The way we currently monitor for microbial contamination of water, air, and surfaces, by sampling and growing cultures on nutrient media, must be reconsidered for exploration missions which have limited capacity for consumables. Likewise, the shelf life of food must be increased so that the nutrients required to keep the crewmembers healthy do not degrade over the life of the mission. Improved formulations, preservation, packaging, and storage technologies are all being investigated for ways slow this process or replace stowed food with key food items grown fresh in situ. Ensuring that the mass and volume of a spacecraft are used to maximum efficiency calls for infusing human factors into the design from its inception to increase efficiency, improve performance, and retain robustness toward operational realities. Integrating the human system with the spacecraft systems is the focus of many lines of investigation.

Sullivan, Thomas A.↗

Plant-based engineering for production of high-valued natural products

Plants are a unique source of complex specialized metabolites, many of which play significant roles in human society. In many cases, however, the availability of these metabolites from naturally occurring sources fails to meet current demands. Thus, there is much interest in expanding the production capacity of target plant molecules. Traditionally, plant breeding, chemical synthesis, and microbial fermentation are considered the primary routes towards large scale production of natural products. Here, in this paper, we explore the advances, challenges, and future of plant engineering as a complementary path. Although plants are an integral part of our food and agricultural systems and sustain an extensive array of chemical constituents, their complex genetics and physiology have prevented the optimal exploitation of plants as a production chassis. We highlight emerging engineering tools and scientific advances developed in recent years that have improved the prospects of using plants as a sustainable and scalable production platform. We also discuss technological limitations and overall economic outlook of plant-based production of natural products.

59 BASIC BIOLOGICAL SCIENCES↗