BENEFICIAL INTERACTIONS BETWEEN MICROBES AND THEIR ENVIRONMENT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1025685
• Project Start Date: Jan 14, 2021
• Project End Date: Jan 13, 2026
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
Microbiology and Cell Science

Non Technical Summary
Microbes are ubiquitous in the environment and play a vital role in maintaining the habitability of our planet. In this project, we examine the interactions that occur between microbes and their surrounding environment, whether that is an animal host, the ocean, or man-made structures, such as the International Space Station. Specifically, we examine how beneficial microbes change and regulate their biochemical and molecular responses to an ever-changing environment. This project will help build a stronger foundation to understand the molecular processes underlying the diverse roles that beneficial microbes play in maintaining and shaping their environment.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	3999	1040	50%
306	4010	1100	50%

Knowledge Area
305 - Animal Physiological Processes; 306 - Environmental Stress in Animals;

Subject Of Investigation
3999 - Animal research, general; 4010 - Bacteria;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology;

Keywords

microbes

spaceflight

symbiosis

microbial dark matter

Goals / Objectives
Microbes are ubiquitous in the environment and play a vital role in maintaining the habitability of our planet. In this project, we examine the interactions that occur between microbes and their surrounding environment, whether that is an animal host, the ocean, or man-made structures, such as the International Space Station. Specifically, we examine how beneficial microbes change and regulate their biochemical and molecular responses to an ever-changing environment. This project will help build a stronger foundation to understand the molecular processes underlying the diverse roles that beneficial microbes play in maintaining and shaping their environment. The specific research objectives of this project are to: 1)reconstruct the genetic content of hubs of unknown microbial taxa within microbialite ecosystems; 2)isolate and enrich the hubs of unknown taxa using functionalized magnetic-quantum dots in living microbialites; 3)elucidate changes in activation and mediation of the extrinsic and intrinsic apoptosis pathway(s) associated with bacteria-induced apoptosis in animal hosts under modeled microgravity conditions; and 4) characterize the changes in the transcriptome of a host animal in the presence and absence of beneficial bacteria under the stress of spaceflight conditions.

Project Methods
Objective 1: Reconstruct the genetic content of hubs of unknown microbial taxa within target ecosystem.Approach 1: Generate metagenome-assembled genomes (MAGs) of key hubs of microbial 'dark matter' derived from microbialites. We propose to generate deeply sequenced metagenomes to the microbialite-forming communities of Highborne Cay, The Bahamas. These new datasets will be used to reconstruct metagenome-assembled genomes (MAGs) of unknown hubs using the following approaches. Hi-C libraries will be prepared and sequenced using an Illumina HiSeq platform. The resulting reads will be assembled to species-level genome bins using existing algorithms and annotated. These bins will be subsequently reviewed and refined via manual curation and comparison to reference genomes by Mash Screen and Fast-ANI algorithms. Single copy gene analysis methods, like CheckM, will be applied to evaluate MAG completeness. The ModelSEEDapproach will be used to construct genome-scale metabolic models for all MAGs estimated to be at least 75% complete.Approach 2. Metabolic modeling to predict active pathway and identify knowledge gaps. We will use Blast2GO to predict genes, and annotate them functionally. Operon structures will be identified and we will identify those operons resembling functional units where unknown gene functions are present. These new genes will be further analyzed by phylogenetic analysis to assess their conservation across newly identified species. Additionally, the MAG-based models generated in Approach 1 will be combined into a single compartmentalized community metabolic model using KBase.Objective 2: Enrich and isolate the hubs of unknown taxa using magnetic-carbon-quantum dots conjugated to DNA probes. Once hubs of microbial 'dark matter' have been bioinformatically identified and prioritized, we will then begin to isolate and enrich the organisms for further characterization using magnetic quantum dots (MQDs). MQDs are nanoparticles with wide spread engineering and biological applications (e.g. drug delivery, live in vivo imaging, microbial diagnostics)). We propose to synthesize MQDs using carbon quantum dots as the core structure as they are small (<10 nm), stable, water-soluble, and can have their optical properties "tuned" with various ions to create a colorful range of fluorescence emissions enabling the cells to be visualized under epifluorescent and confocal microscopy. The carbon dots will be hybridized with iron oxide (Fe2O3 ) and functionalized with amines (e.g. phenylenediamine). The newly formed MQDs will be concentrated using an available Magnetic Particle Concentrator. The iron content in the hybridized dots will be confirmed using standard colorimetric kits (e.g. BioAssay Systems).Objective 3. Elucidate changes in activation and mediation of bacteria-induced apoptosis pathway(s) associated animal hosts under modeled microgravity conditions. Using the symbiotic interaction between the bioluminescent marine Vibrio fischeri and the bobtail squid Euprymna scolopes as a model system, we propose to examine how environmental stress, such as microgravity, impacts normal bacteria-induced animal development. Specifically, we will examine bacteria-induced apoptosis through the extrinsic (e.g. initiated by bacterial molecules) and intrinsic (e.g. environmental stress) pathways. This work will be conducted under both simulated modeled microgravity using high-aspect ratio bioreactors and natural spaceflight aboard the International Space Station (ISS) . We will assess differential gene expression of extrinsic and intrinsic apoptosis genes in aposymbiotic (i.e., without bacteria) and symbiotic squid under modeled microgravity conditions using NanoString. Both aposymbiotic and symbiotic squid will be harvested every 2 h in triplicate and flash frozen in liquid nitrogen and stored at -80°C until RNA extraction as previously optimized. The translation of the genes transcripts will be confirmed with Western blotting and localized within the host tissues with ICC using commercially available polyclonal antibodies. Additionally, the onset of apoptosis will be co-monitored in a cohort of unfrozen animals using both acridine orange (pycnotic nuclei) staining and fluorometric TUNEL kit (chromosomal cleavage) as previously optimized. We expect that the extrinsic apoptosis pathways will be closely tied to the host innate immune system as apoptosis enables the clearance of damaged cells without inflammation.Objective 4. Characterize the changes in the transcriptome of a host animal in the presence and absence of beneficial bacteria under the stress of spaceflight conditions. Approach 1. RNA-Seq of host light organs exposed to microgravity. We propose to generate transcriptomes to host E. scolopes light organs in the presence and absence of the mutualistic V. fischeri immediately at hatching (0 h) and at three key developmental time points (2, 6, 12 h) post-colonization. There will be a total of 10 biological replicates collected for each flight treatment, we anticipate using six of the biological replicates (i.e., two light organs will be pooled for each library replicate) for the RNA-Seq transcriptome preparation, as described below. To examine the impact of microgravity on the host squid in the presence and absence of V. fischeri the transcriptome of the host squid will be partially sequenced using RNA-Seq, or direct transcriptome sequencing with the high throughput Illumina NextSeq500 sequencing platform. Prior to the flight, hatchling controls (0 h) will be preserved in RNAlater and frozen at -80°C. After the flight and ground control experiments, the light organ of all RNAlater-fixed animals will be dissected and total RNA will be extracted from each light organ separately using an RNAeasy kit (Qiagen) as previously optimized. The quality of the RNA will be assessed using a Bioanalyzer with a RNA6000 Nano kit (Agilent). High-quality RNA (e.g. RIN >8) will undergo polyA selection and cDNA library synthesis using a NEBNext Ultra RNA library prep kit for Illumina sequencing.

Progress 01/14/21 to 09/30/21

Outputs
Target Audience:The audience for this report are those researchers working in the field of symbiosis, space biology and environmental microbiology. Changes/Problems:There was only one significant change. Due to travel restrictions field work was not completed in 2021 and has been defered to summer of 2022. This delay has impacted the first objective of this project plan. What opportunities for training and professional development has the project provided?This project has enabled the training of one graduate student and two postdoctoral fellows in areas of space microbiology and biology research. Each of the individuals have worked one-on-one with the PI to build their professional development skills in communicating science through peer-review publications and presentations at scientific conferences. How have the results been disseminated to communities of interest?The work has been disseminated through four peer-reviewed publications and three presentations at scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?1)Reconstruct the genetic content of hubs of unknown microbial taxa within microbialite ecosystems. After field work in the summer of 2022, we anticipate being able to sequence the genomes of the prioritized hubs of unknown organisms and begin to ascertain the metabolic function of key unknown taxa within the microbialite communities 2)isolate and enrich the hubs of unknown taxa using functionalized magnetic-quantum dots in living microbialites; We anticipate using live samples to be able to optimize the mag-dots to try and isolate individual cells that represent key unknown organisms. We may have to resort to fixing and permeabilizing the cells but we will attempt to try and isolate the targeted cells. 3)Elucidate changes in activation and mediation of the extrinsic and intrinsic apoptosis pathway(s) associated with bacteria-induced apoptosis in animal hosts under modeled microgravity conditions We will begin to search the UMAMI spaceflight dataset for apoptosis genes to examine how these genes changed under actual microgravity conditions and then compare those results to the ground results generated in 2021. 4) Characterize the changes in the transcriptome of a host animal in the presence and absence of beneficial bacteria under the stress of spaceflight conditions. We will finalize the RNASeq analysis and identify, which genes (if any) were differentially expressed under spaceflight, and ascertain whether beneficial microbes altered the expression of these genes.

Impacts
What was accomplished under these goals? 1. Reconstruct the genetic content of hubs of unknown microbial taxa in microbialites.In the first year, we assembled, organized, and integrated the many different types of sequencing datasets my lab has generated over the past decade. It took several attempts to optimize the different assemblers and software selection but by the end of the year we had an optimized pipeline so that it could work using any type of data (e.g., amplicon, metagenomic or metatranscriptomic data). Through these analyses we have been able to prioritize the most highly interconnected unknown taxa for further analysis. For example, in the table below, we have determined that of the top 10 taxa with the highest hub scores (i.e., most interconnected taxa within the community) four are unable to be classified at the kingdom level. All but two can be classified to the species level. Due to the global pandemic, no international field work in The Bahamas was possible in 2021. This aspect of the project has been moved to Spring May 2022. We have booked travel to the island of Highborne Cay to collect the samples needed to deeply sequencing microbialite metagenome to enable the closure of genomes of hub taxa. We have, however, worked with currently available RNA-later preserved samples of the Highborne Cay thrombolites (a type of microbialite) and have ensured that high quality of DNA could be generated if needed from these previously collected samples (see mitigation strategies) should there be any additional delays in travel. 2. Isolate and enrich the hubs of unknown taxa using functionalized magnetic-quantum dots in living microbialites.Magnetic nanoparticles with fluorescent cores have been synthesized and efforts to conjugate them to 16S rRNA oligonucleotides is underway. Due to the inability to travel to collect fresh living samples from Highborne Cay, Bahamas, all optimization work is currently being done in the marine bacterium Vibrio fischeri ES114 to improve the labeling and isolation conditions. We have been able to optimize the FISH methodology using the magnetic particles conjugated to V. fischeri ES114 cells and are working to now remove these targeted species from mixed populations. In collaboration with Dr. David Arnold of the University of Florida, we have built "magnetic tweezers" that can move and manipulate magnet nanoparticle-tagged cells on a standard upright microscope. Work is underway to optimize this approach to work in live microbialite cultures and target the prioritized unknown taxa. 3. Elucidate changes in activation and mediation of the extrinsic and intrinsic apoptosis pathway(s) associated with bacteria-induced apoptosis in animal hosts under modeled microgravity conditions. We have identified genes associated with both extrinsic and intrinsic apoptosis. See pathway map that we have generated below. Most of the genes (55%) discovered have a positive effect on apoptosis (i.e., induce apoptosis) whereas 34% of the recovered genes had a negative effect (i.e., suppress apoptosis). Eleven percent of the genes had multiple or undetermined function. Interestingly, the majority of the genes differently expressed during modeled microgravity were associated with the intrinsic apoptosis pathway (40%) suggesting that internal stressors may be triggering the apoptosis pathway in the host animals, whereas only 16% of the observed genes were associated with extrinsic, or external, activation of apoptosis. One of the major outcomes this goal was the discovery of numerous caspase-encoding genes for enzymes that are involved in the apoptotic cell death process. We exampled the differential expression of these genes and found that that several of the genes (cas9, cas3, cas8, cas2 and cas10) are all differentially upregulated under modeled microgravity conditions when compare to gravity controls. We are now in the process of evaluating the enzyme activity to ensure that the protein is active during similar time points. The delay due to COVID-19 slowed our progress in this area, but we have now optimized the enzyme detection assays and will be examining caspase activity for additional time points and genes. We will be testing the activity of Caspase 3/7, 8 and 9 enzymes over the first 20 hours post-infection with the bacteria. Lastly, we are trying to assess whether we can mitigate the increase in apoptotic cell death and further elucidate the different mechanisms of caspase activation. We have started to ascertain and optimize the use of inhibitors to prevent the onset of apoptosis. Preliminary data has identified that the inhibition of Caspase-8 seems to have a more pronounced effect on the levels of apoptosis compared to other Caspase enzymes suggesting this caspase may play a more prominent role in the bacteria-induced cell death of the host animal. We will also ascertain whether this inhibitor can also have a similar effect under modeled microgravity. 4. Characterize the changes in the transcriptome of a host animal in the presence and absence of beneficial bacteria under the stress of spaceflight conditions. The Understanding of Microgravity on Animal-Microbe Interactions (UMAMI) spaceflight experiment launched aboard Space-X22 in June 2021. This mission was designed to examine the impact microgravity and spaceflight on beneficial interactions between animals and symbiotic microbes. The experiment was completed aboard the International Space Station and the samples were returned in July 2021. RNASeq libraries were created to the various treatments (+/- symbiotic microbes) across a timeline. Additionally, samples were given to the UF SECIM core facility generate a metabolomics and lipidomic profiles. These additional datasets are still being processed. Analysis is currently underway on the RNASeq datasets. The data are of high quality and are being statistically analyzed.

Publications

• Type: Other Status: Published Year Published: 2021 Citation: Everroad, R.C., Foster, J., Galazka, J.M., Jansson, J., Lee, J.A., Lera, M.P., Perera, I., Ricco, A., Szewczyk, N., Todd, P. and Zhang, Y., 2021. Space Biology Beyond LEO Instrumentation & Science Series-Science Working Group 2021 Annual Report. NASA
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Haveman, N.J., Khodadad, C.L., Dixit, A.R., Louyakis, A.S., Massa, G.D., Venkateswaran, K. and Foster, J.S., 2021. Evaluating the lettuce metatranscriptome with MinION sequencing for future spaceflight food production applications. npj Microgravity, 7(1), pp.1-11.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Vignale, F.A., Lencina, A.I., Stepanenko, T.M., Soria, M.N., Saona, L.A., Kurth, D., Guzm�n, D., Foster, J.S., Poir�, D.G., Villafa�e, P.G. and Albarrac�n, V.H., 2021. Lithifying and non-lithifying microbial ecosystems in the wetlands and salt flats of the central Andes. Microbial Ecology, pp.1-17.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Vroom, M.M., Rodriguez-Ocasio, Y., Lynch, J.B., Ruby, E.G. and Foster, J.S., 2021. Modeled microgravity alters lipopolysaccharide and outer membrane vesicle production of the beneficial symbiont Vibrio fischeri. npj Microgravity, 7(1), pp.1-10.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Zamkovaya, T., Foster, J.S., de Cr�cy-Lagard, V. and Conesa, A., 2021. A network approach to elucidate and prioritize microbial dark matter in microbial communities. The ISME journal, 15(1), pp.228-244.