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DOE OSTI2021

Subacute safety assessment of recombinant Lactococcus lactis on the gut microbiota of male Sprague–Dawley rats

Abstract BACKGROUND Lactococcus lactis strain pGSMT/MG1363 is a genetically modified microorganism (GMM) that constitutively expresses human metallothionein‐I fusion protein to combine with intracellular lead. Unlike traditional probiotics, pGSMT/MG1363 lacks a history of safe use in food. Administration of microorganism could influence the gut microbial community and consequently confer health benefits or cause disadvantages to the host. To date, little has been done to assess the influence of recombinant strain pGSMT/MG1363 on the stability of gut microbiota. RESULTS Liver, testis and kidney sections of male Sprague–Dawley rats orally administered pGSMT/MG1363 for 6 weeks showed normal structure and no pathological damage. There were no adverse effects on the analyzed serum biochemical parameters between the pGSMT/MG1363 group and the MG1363 group. Principal coordinate analysis showed that, compared with the MG1363 group, the 6‐week‐old fecal gut microbiota of rats fed with pGSMT/MG1363 was not significantly different (Adonis, P = 0.802). pGSMT/MG1363 treatment for 6 weeks did not significantly change the relative abundance of gut microbiota at the phylum and genus levels in comparison with MG1363 treatment. CONCLUSION Compared to the non‐GM strain MG1363 group, administration of the recombinant strain pGSMT/MG1363 for 6 weeks showed no adverse effects on the analyzed physiological parameters and gut microbial compositions of male Sprague–Dawley rats. The results suggested that, in terms of gut microbiota stability, pGSMT/MG1363 could be considered as safe as MG1363, at least for short‐term intake. Further toxicological evaluations still need to be considered before drawing a definite conclusion concerning the safe use of pGSMT/MG1363. © 2021 Society of Chemical Industry.

Chen, Guowei↗

DOE OSTI2024

Developing a pipeline to expand the genetic code of diverse bacteria for microbial engineering

Microbial biotechnologies are key to addressing grand challenges to promote human health, reverse carbon emissions, recycle mixed plastic waste, remediate contaminated soils, and achieve sustainable economies. Synthetic biology has enabled design of diverse microbes and their proteins for useful purposes, but the narrowness of the natural genetic code limits functional diversity (e.g., biosynthesis) of engineered microbes. The natural genetic code defines the fundamental rules of translating genetic information into proteins comprised of 22 ‘canonical’ amino acids. However, using a technique called genetic code expansion (GCE), the chemical properties and therefore functions of proteins can be transformed by incorporation of one or more of ~200 chemically diverse ‘non-canonical’ amino acids. The effective application of genetic code expansion in diverse microbes has the potential to revolutionize biotechnology. However, despite over 50 years of research and its transformative potential, the application of genetic code expansion has been limited to a handful of bacterial species. In this project, we will perform three tasks to both overcome the barriers that prevent wide spread adoption of GCE as molecular tool and demonstrate its potential for biotechnological applications. Specifically, we will (1) develop a genetic engineering methodology that will enable use of GCE in a broad range of bacterial hosts, (2) use high-throughput functional genomics methods to identify physiological responses to both genetic code expansion and exposure to non-canonical amino acids in three different bacteria, and (3) demonstrate an application of GCE by selectively incorporate non-canonical amino acids into surface displayed peptides such as those used for biomining.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2011

Automated Miniaturized Instrument for Space Biology Applications and the Monitoring of the Astronauts Health Onboard the ISS

Human space travelers experience a unique environment that affects homeostasis and physiologic adaptation. The spacecraft environment subjects the traveler to noise, chemical and microbiological contaminants, increased radiation, and variable gravity forces. As humans prepare for long-duration missions to the International Space Station (ISS) and beyond, effective measures must be developed, verified and implemented to ensure mission success. Limited biomedical quantitative capabilities are currently available onboard the ISS. Therefore, the development of versatile instruments to perform space biological analysis and to monitor astronauts' health is needed. We are developing a fully automated, miniaturized system for measuring gene expression on small spacecraft in order to better understand the influence of the space environment on biological systems. This low-cost, low-power, multi-purpose instrument represents a major scientific and technological advancement by providing data on cellular metabolism and regulation. The current system will support growth of microorganisms, extract and purify the RNA, hybridize it to the array, read the expression levels of a large number of genes by microarray analysis, and transmit the measurements to Earth. The system will help discover how bacteria develop resistance to antibiotics and how pathogenic bacteria sometimes increase their virulence in space, facilitating the development of adequate countermeasures to decrease risks associated with human spaceflight. The current stand-alone technology could be used as an integrated platform onboard the ISS to perform similar genetic analyses on any biological systems from the tree of life. Additionally, with some modification the system could be implemented to perform real-time in-situ microbial monitoring of the ISS environment (air, surface and water samples) and the astronaut's microbiome using 16SrRNA microarray technology. Furthermore, the current system can be enhanced substantially by combining it with other technologies for automated, miniaturized, high-throughput biological measurements, such as fast sequencing, protein identification (proteomics) and metabolite profiling (metabolomics). Thus, the system can be integrated with other biomedical instruments in order to support and enhance telemedicine capability onboard ISS. NASA's mission includes sustained investment in critical research leading to effective countermeasures to minimize the risks associated with human spaceflight, and the use of appropriate technology to sustain space exploration at reasonable cost. Our integrated microarray technology is expected to fulfill these two critical requirements and to enable the scientific community to better understand and monitor the effects of the space environment on microorganisms and on the astronaut, in the process leveraging current capabilities and overcoming present limitations.

Human space travelers↗

DOE OSTI2022

Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA). Final report

The goal of the Stahl lab is to use a combination of isotopic, genomic, and activity-based analyses in the field, of selected field isolates, and defined synthetic communities to identify sources and sinks of different nitrogen species in the field, focusing on the FRC at the ORNL. The most general research objective is to characterize variants in the pathway for denitrification among field isolates and synthetic microbial assemblies as a foundation for predicting the biotic and abiotic controls of nitrogen and carbon transformations in the field. Specifically, we have focused on characterizing environmental controls of nitrous oxide emissions, an important greenhouse gas and ozone depleting molecule. Studies have mapped the flux of nitrous oxide and controlling genetic elements within the FRC subsurface, indicating the importance of different enzyme variants in nitrous oxide production and consumption in controlling surface emissions. We have developed methods to quantify and catalog the isotopic signatures imparted on nitrous oxide by different nitrous oxide reductase variants in isolated organisms and model synthetic communities. We have developed methods to quantify and catalog the nitrous oxide affinity of field relevant isolates and their biomass yield when respiring nitrous oxide on different carbon substrates. We continue to expand the collection of field-relevant microbial isolates within ENIGMA by focused enrichment and isolation from field material. These objectives have been facilitated by the development of new technologies, including: 1) the development of a multiplexed automated optical density measurement system for quantifying growth kinetics of microorganisms dependent on gaseous substrates in monoculture or gas exchange in synthetic communities and 2) the development of a microcalorimetric analysis format to quantify the impact of environmental stress on microbial maintenance energy. Since field relevant processes are mediated by assemblies of organisms interacting with each other and their local environment, we have also collaborated with other ENIGMA investigators on the assembly and characterization of synthetic communities. These studies have evaluated the role of organisms lacking a complete pathway for denitrification in the control of field processes, measuring process rates of assemblies of organisms that individually lack the full denitrification pathway but mediate the complete denitrification when growing together. These efforts have so far resulted in the publication of 18 peer reviewed papers ranging from ecological and physiological drivers of pathway optimization to field-scale process characterization.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRS2010

Response of Staphylococcus Aureus to a Spaceflight Analogue

The decreased gravity of the spaceflight environment creates quiescent, low fluid shear conditions. This environment can impart considerable effects on the physiology of microorganisms as well as their interactions with potential hosts. Using the rotating wall vessel (RWV), as a spaceflight analogue, the consequence of low fluid shear culture on microbial pathogenesis has provided a better understanding of the risks to the astronaut crew from infectious microorganisms. While the outcome of low fluid shear culture has been investigated for several bacterial pathogens, little has been done to understand how this environmental factor affects Staphylococcus aureus. S. aureus is an opportunistic human pathogen which presents a high level of infection risk to the crew, as it has been isolated from both the space shuttle and International Space Station. Given that approximately forty percent of the population are carriers of the bacteria, eradication of this organism from in flight environments is impractical. These reasons have lead to us to assess the response of S. aureus to a reduced fluid shear environment. Culture in the RWV demonstrated that S. aureus grown under the low-shear condition had lower cell concentrations after 10 hours when compared to the control culture. Furthermore, the low-shear cultured bacteria displayed a reduction in carotenoid production, pigments responsible for their yellow/gold coloration. When exposed to various environmental stressors, post low-shear culture, a decrease in the ability to survive oxidative assault was observed compared to control cultures. The low fluid shear environment also resulted in a decrease in hemolysin secretion, a staphylococcal toxin responsible for red blood cell lysis. When challenged by the immune components present in human whole blood, low-shear cultured S. aureus demonstrated significantly reduced survival rates as compared to the control culture. Assays to determine the duration of these alterations demonstrated that the low-shear response could be lost in as few as 2.5 hours. These changes in phenotypic properties prompted investigation into variations occurring at the genetic level. Microarray analysis of low-shear cultured S. aureus revealed the differential regulation of genes involved in metabolism, stress response, and phosphate transfer. Additional genetic analysis with quantitative real-time PCR revealed alterations in the expression of Hfq, the conserved RNA chaperone protein involved in global gene regulation. Hfq has been connected to the regulation of a spaceflight microgravity response in S. typhimurium. These findings in S. aureus suggest an evolutionary conserved response to spaceflight conditions among structurally-diverse microorganisms. Furthermore, the reduction in pigmentation, hemolysin secretion, and survival against oxidative stress and immunologically active whole blood demonstrate an overall decrease in the virulence factors of S. aureus in response to spaceflight-like conditions.

Castro, S. L.↗

DOE OSTI2023

Physiological and genomic evidence of cysteine degradation and aerobic hydrogen sulfide production in freshwater bacteria

The sulfur-containing amino acid cysteine is abundant in the environment, including in freshwater lakes. Biological cysteine degradation can result in hydrogen sulfide (H 2 S), a toxic and ecologically relevant compound that is a central player in biogeochemical cycling in aquatic environments. Here, we investigated the ecological significance of cysteine in oxic freshwater, using isolated cultures, controlled experiments, and multiomics. We screened bacterial isolates enriched from natural lake water for their ability to produce H 2 S when provided cysteine. We identified 29 isolates (Bacteroidota, Proteobacteria, and Actinobacteria) that produced H 2 S. To understand the genomic and genetic basis for cysteine degradation and H 2 S production, we further characterized three isolates using whole-genome sequencing (using a combination of short-read and long-read sequencing) and tracked cysteine and H 2 S levels over their growth ranges: Stenotrophomonas maltophilia (Gammaproteobacteria), S. bentonitica (Gammaproteobacteria), and Chryseobacterium piscium (Bacteroidota). Cysteine decreased and H 2 S increased, and all three genomes had genes involved in cysteine degradation. Finally, to assess the presence of these organisms and genes in the environment, we surveyed a 5-year time series of metagenomic data from the same isolation source (Lake Mendota, Madison, WI, USA) and identified their presence throughout the time series. Overall, our study shows that diverse isolated bacterial strains can use cysteine and produce H 2 S under oxic conditions, and we show evidence using metagenomic data that this process may occur more broadly in natural freshwater lakes. Future considerations of sulfur cycling and biogeochemistry in oxic environments should account for H 2 S production from the degradation of organosulfur compounds.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Eukaryotic genomes from a global metagenomic data set illuminate trophic modes and biogeography of ocean plankton

ABSTRACT Metagenomics is a powerful method for interpreting the ecological roles and physiological capabilities of mixed microbial communities. Yet, many tools for processing metagenomic data are neither designed to consider eukaryotes nor are they built for an increasing amount of sequence data. EukHeist is an automated pipeline to retrieve eukaryotic and prokaryotic metagenome-assembled genomes (MAGs) from large-scale metagenomic sequence data sets. We developed the EukHeist workflow to specifically process large amounts of both metagenomic and/or metatranscriptomic sequence data in an automated and reproducible fashion. Here, we applied EukHeist to the large-size fraction data (0.8–2,000 µm) from Tara Oceans to recover both eukaryotic and prokaryotic MAGs, which we refer to as TOPAZ (Tara Oceans Particle-Associated MAGs). The TOPAZ MAGs consisted of >900 environmentally relevant eukaryotic MAGs and >4,000 bacterial and archaeal MAGs. The bacterial and archaeal TOPAZ MAGs expand upon the phylogenetic diversity of likely particle- and host-associated taxa. We use these MAGs to demonstrate an approach to infer the putative trophic mode of the recovered eukaryotic MAGs. We also identify ecological cohorts of co-occurring MAGs, which are driven by specific environmental factors and putative host-microbe associations. These data together add to a number of growing resources of environmentally relevant eukaryotic genomic information. Complementary and expanded databases of MAGs, such as those provided through scalable pipelines like EukHeist, stand to advance our understanding of eukaryotic diversity through increased coverage of genomic representatives across the tree of life. IMPORTANCE Single-celled eukaryotes play ecologically significant roles in the marine environment, yet fundamental questions about their biodiversity, ecological function, and interactions remain. Environmental sequencing enables researchers to document naturally occurring protistan communities, without culturing bias, yet metagenomic and metatranscriptomic sequencing approaches cannot separate individual species from communities. To more completely capture the genomic content of mixed protistan populations, we can create bins of sequences that represent the same organism (metagenome-assembled genomes [MAGs]). We developed the EukHeist pipeline, which automates the binning of population-level eukaryotic and prokaryotic genomes from metagenomic reads. We show exciting insight into what protistan communities are present and their trophic roles in the ocean. Scalable computational tools, like EukHeist, may accelerate the identification of meaningful genetic signatures from large data sets and complement researchers’ efforts to leverage MAG databases for addressing ecological questions, resolving evolutionary relationships, and discovering potentially novel biodiversity.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Global Archaeal Diversity Revealed Through Massive Data Integration: Uncovering Just Tip of Iceberg

The domain of Archaea has gathered significant interest for its ecological and biotechnological potential and its role in helping us to understand the evolutionary history of Eukaryotes. In comparison to the bacterial domain, the number of adequately described members in Archaea is relatively low, with less than 1000 species described. It is not clear whether this is solely due to the cultivation difficulty of its members or, indeed, the domain is characterized by evolutionary constraints that keep the number of species relatively low. Based on molecular evidence that bypasses the difficulties of formal cultivation and characterization, several novel clades have been proposed, enabling insights into their metabolism and physiology. Given the extent of global sampling and sequencing efforts, it is now possible and meaningful to question the magnitude of global archaeal diversity based on molecular evidence. To do so, we extracted all sequences classified as Archaea from 500 thousand amplicon samples available in public repositories. After processing through our highly conservative pipeline, we named this comprehensive resource the ‘Global Archaea Diversity’ (GAD), which encompassed nearly 3 million molecular species clusters at 97% similarity, and organized it into over 500 thousand genera and nearly 100 thousand families. Saline environments have contributed the most to the novel taxa of this previously unseen diversity. The majority of those 16S rRNA gene sequence fragments were verified by matches in metagenomic datasets from IMG/M. These findings reveal a vast and previously overlooked diversity within the Archaea, offering insights into their ecological roles and evolutionary importance while establishing a foundation for the future study and characterization of this intriguing domain of life.

59 BASIC BIOLOGICAL SCIENCES↗