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At least 253 records · Page 14
All sourcesNASA NTRSDOE OSTI
NASA NTRS1972

Development of a tape transport bacterial detection system

The feasibility of a tape transport chemiluminescence system for bacterial monitoring of regenerated water was demonstrated using a manually operated laboratory breadboard. The principle of detection is based on measuring the increase in chemiluminescence produced by the catalytic action of bacterial porphyrins on a luminol-hydrogen peroxide mixture. Viable organisms are distinguished from nonviable by comparing the signals of incubated and unincubated water samples. Using optimized protocols, sensitivities were obtained with 400 ml suspensions of E. coli and Cl. sporogenes. The sensitivity of the unincubated cycle E. coli (aerobe) was found to be 30 to 35 cells/m1, and that of the Cl. sporogenes (anaerobe) was 1000 to 10,000 cells/m1. The lower sensitivity toward Cl. sporogenes is attributed to several factors, namely the lower cytochrome content, the tendency to sporulate, long lag periods and the lower growth rate of Clostridia in general. The operational procedures used for processing the incubated and unincubated samples involved the following sequence: (1) concentrating the sample by filtration through a membrane filter, (2) washing with Dextrose-Thioglycollate Broth (3) incubating (0 to 4 hrs as required), (4) washing with 4M Urea, and (5) reacting with reagent in front of a photomultiplier tube. The signal output was recorded on a strip chart recorder.

Witz, S.↗
NASA NTRS1972

Survival of bacterial isolates exposed to simulated Jovian trapped radiation belt electrons and solar wind protons

With missions to Jupiter, the spacecraft will be exposed for extended duration to solar wind radiation and the Jovian trapped radiation belt. This study is designed to determine the effect of these radiation environments on spacecraft bacterial isolates. The information can be used in the probability of contamination analysis for these missions. A bacterial subpopulation from Mariner Mars 1971 spacecraft (nine sporeforming and three nonsporeforming isolates) plus two comparative organisms, Staphylococcus epidermidis ATCC 17917 and a strain of Bacillus subtilis var. niger, were exposed to 2-, 12-, and 25-MeV electrons at different doses with simultaneous exposure to a vacuum of 0.0013 N/sqm at 20 and -20 C. The radioresistance of the subpopulation was dependent on the isolate, dose, and energy of electrons. Temperature affected the radioresistance of only the sporeforming isolates. Survival data indicated that spores were reduced approximately 1 log/1500 J/kg, while nonsporeforming isolates (micrococci) were reduced 1.5 to 2 logs/1500 J/kg with the exception of an apparent radioresistant isolate whose resistance approached that of the spores. The subpopulation was found to be less resistant to lower energy than to higher energy electrons.

Taylor, D. M.↗
NASA NTRS2003

Radiation-Dependent Limit for the Viability of Bacterial Spores in Halite Fluid Inclusions and on Mars

When claims for the long-term survival of viable organisms are made, either within terrestrial minerals or on Mars, considerations should be made of the limitations imposed by the naturally occurring radiation dose to which they have been exposed. We investigated the effect of ionizing radiation on different bacterial spores by measuring the inactivation constants for B. subtilis and s. marismortui spores in solution as well as for dry spores of B. subtilis and B. thuringiensis. S. marismortui is a halophilic spore that is genetically similar to the recently discovered 2-9-3 bacterium from a halite fluid inclusion, claimed to be 250 million years old, B. thuringiensis is a soil bacterium that is genetically similar to the human pathogens B. anthracis and B. cereus. To relate the inactivation constant to some realistic environments, we calculated the radiation regimen in a halite fluid inclusion and in the Martian subsurface over time. Our conclusion is that the ionizing dose of radiation in those environments limits the survival of viable bacterial spores over long periods. In the absence of an active repair mechanism in the dormant state, the long-term survival of spores is limited to less than 109 million years in halite fluid inclusions, to 100 to 160 million years in the Martian subsurface below 3 m, and to less than 600,000 years in the upper-most meter of Mars.

Kminek, Gerhard↗
NASA NTRS1999

Effects of space flight and mixing on bacterial growth in low volume cultures

Previous investigations have shown that liquid suspension bacterial cultures grow to higher cell concentrations in spaceflight than on Earth. None of these studies included ground-control experiments designed to evaluate the fluid effects potentially responsible for the reported increases. Therefore, the emphasis of this research was to both confirm differences in final cell concentration between 1g and microgravity cultures, and to examine the effects of mixing as a partial explanation for this difference. Flight experiments were performed in the Fluid Processing Apparatus (FPA), aboard Space Shuttle Missions STS-63 and STS-69, with simultaneous 1g static and agitated controls. Additional static 1g, agitated, and clino-rotated controls were performed in 9-ml culture tubes. This research revealed that both E. coli and B. subtilis samples cultured in space flight grew to higher final cell densities (120-345% increase) than simultaneous static 1g controls. The final cell concentration of E. coli cells cultured under agitation was 43% higher than in static 1g cultures and was 102% higher with clino-rotation. However, for B. subtilis cultures grown while being agitated on a shaker or clino-rotated, the final cell concentrations were nearly identical to those of the simultaneous static 1g controls. Therefore, these data suggest that the unique fluid quiescence in the microgravity environment (lack of sedimentation, creating unique transfer of nutrients and waste products), was responsible for the enhanced bacterial proliferation reported in this and other studies.

STS-63 Shuttle Project↗
NASA NTRS2005

Intraspecific differences in bacterial responses to modelled reduced gravity

AIMS: Bacteria are important residents of water systems, including those of space stations which feature specific environmental conditions, such as lowered effects of gravity. The purpose of this study was to compare responses with modelled reduced gravity of space station, water system bacterial isolates with other isolates of the same species. METHODS AND RESULTS: Bacterial isolates, Stenotrophomonas paucimobilis and Acinetobacter radioresistens, originally recovered from the water supply aboard the International Space Station (ISS) were grown in nutrient broth under modelled reduced gravity. Their growth was compared with type strains S. paucimobilis ATCC 10829 and A. radioresistens ATCC 49000. Acinetobacter radioresistens ATCC 49000 and the two ISS isolates showed similar growth profiles under modelled reduced gravity compared with normal gravity, whereas S. paucimobilis ATCC 10829 was negatively affected by modelled reduced gravity. CONCLUSIONS: These results suggest that microgravity might have selected for bacteria that were able to thrive under this unusual condition. These responses, coupled with impacts of other features (such as radiation resistance and ability to persist under very oligotrophic conditions), may contribute to the success of these water system bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Water quality is a significant factor in many environments including the ISS. Efforts to remove microbial contaminants are likely to be complicated by the features of these bacteria which allow them to persist under the extreme conditions of the systems.

Non-NASA Center↗
NASA NTRS2010

Process to Selectively Distinguish Viable from Non-Viable Bacterial Cells

The combination of ethidium monoazide (EMA) and post-fragmentation, randomly primed DNA amplification technologies will enhance the analytical capability to discern viable from non-viable bacterial cells in spacecraft-related samples. Intercalating agents have been widely used since the inception of molecular biology to stain and visualize nucleic acids. Only recently, intercalating agents such as EMA have been exploited to selectively distinguish viable from dead bacterial cells. Intercalating dyes can only penetrate the membranes of dead cells. Once through the membrane and actually inside the cell, they intercalate DNA and, upon photolysis with visible light, produce stable DNA monoadducts. Once the DNA is crosslinked, it becomes insoluble and unable to be fragmented for post-fragmentation, randomly primed DNA library formation. Viable organisms DNA remains unaffected by the intercalating agents, allowing for amplification via post-fragmentation, randomly primed technologies. This results in the ability to carry out downstream nucleic acid-based analyses on viable microbes to the exclusion of all non-viable cells.

LaDuc, Myron T.↗
NASA NTRS2023

Evolutionary Flexibility and Rigidity in the Bacterial Methylerythritol Phosphate (Mep) Pathway

Terpenoids are a diverse class of compounds with wide-ranging uses including as industrial solvents, pharmaceuticals, and fragrances. Efforts to produce terpenoids sustainably by engineering microbes for fermentation are ongoing, but industrial production still largely relies on nonrenewable sources. The methylerythritol phosphate (MEP) pathway generates terpenoid precursor molecules and includes the enzyme Dxs and two iron–sulfur cluster enzymes: IspG and IspH. IspG and IspH are rate limiting-enzymes of the MEP pathway but are challenging for metabolic engineering because they require iron–sulfur cluster biogenesis and an ongoing supply of reducing equivalents to function. Therefore, identifying novel alternatives to IspG and IspH has been an on-going effort to aid in metabolic engineering of terpenoid biosynthesis. We report here an analysis of the evolutionary diversity of terpenoid biosynthesis strategies as a resource for exploration of alternative terpenoid biosynthesis pathways. Using comparative genomics, we surveyed a database of 4,400 diverse bacterial species and found that some may have evolved alternatives to the first enzyme in the pathway, Dxs making it evolutionarily flexible. In contrast, we found that IspG and IspH are evolutionarily rigid because we could not identify any species that appear to have enzymatic routes that circumvent these enzymes. The ever-growing repository of sequenced bacterial genomes has great potential to provide metabolic engineers with alternative metabolic pathway solutions. With the current state of knowledge, we found that enzymes IspG and IspH are evolutionarily indispensable which informs both metabolic engineering efforts and our understanding of the evolution of terpenoid biosynthesis pathways.

Bailey Marshall↗
NASA NTRS2025

Influence of Microgravity on Bacterial Pathogen Virulence and Immune Cell Function—Relevance for Infectious Disease Risk During Spaceflight

Spaceflight has measurable impacts on astronaut immune profiles as well as the virulence patterns of bacterial pathogens. Data with respect to human immunity indicate diminished T and NK cell function, altered cytokine profiles, persistent inflammation, and latent herpesvirus reactivation. Furthermore, evaluation of International Space Station (ISS) crewmembers gives evidence of compromised immunity, including atypical allergy, infectious disease, and dermatitis. Data with respect to certain human bacterial pathogens suggest modified virulence that may be enhanced. It is therefore critical to examine this interaction of immune dysregulation and increased microbial virulence and whether it might synergistically increase the risk of infectious disease to crew members. The goal of this study is to use modeled microgravity to study the impact of medically significant ISS bacteria that may have altered virulence on the immune response of the host. This study consists of two primary aims to assess this relationship. First, immune cells will be collected from healthy test subjects and cultured in static or in modeled microgravity conditions together with either control pathogens or with microgravity conditioned pathogens that were grown in bioreactors. Second, immune cells will be collected from astronauts before, during, and after flight and co-cultured with the control or the microgravity conditioned bacteria. Three pathogens were selected for this investigation: Pseudomonas aeruginosa , Salmonella enterica serovar Enteritidis and Burkholderia cepacia . Previously, the optimal cell to bacteria ratios that produce the greatest immune cell responses have been derived for these three bacteria. Cellular activation, as determined by the induction of cell surface activation markers and cytokine profiles, will be measured. Interactions between cells and bacteria will be assessed using fluorescent and electron microscopy techniques. This study will provide critical information to help understand how microgravity alters microbial virulence and the associated infectious disease risk to crewmembers during spaceflight missions. Over the past year, the Immunology and Microbiology laboratories at NASA Johnson Space Center have collaborated to process the astronaut subject samples to complete the last year of the flight portion of this study. This included the completion of four astronaut subjects full mission sampling sets. Sample processing included innate and adaptive cell flow cytometry as well as analysis of cytokine concentrations in the supernatant. The ground control segment of the study will take place during FY25 which involves parallel infections run under static and clinostat conditions. The acquired data sets over this 3-year study are now being analyzed to provide a comprehensive set of results and conclusions that will contribute to a more effective risk assessment for astronauts during spaceflight regarding this host-pathogen context.

Immunology↗
DOE OSTI2023

Genetically modified bacterial cells and methods useful for producing indigoidine

The present invention provides for a genetically modified bacterial host cell capable of producing indigoidine, wherein the host cell comprises a non-ribosomal peptide synthetase (NRPS) that converts glutamine to indigoidine, and the bacterial host cell is reduced in its expression of one or more of the sixteen indicated enzymes.

Eng, Thomas T.↗
DOE OSTI2022

Design and construction of 3D printed devices to investigate active and passive bacterial dispersal on hydrated surfaces

Background: To disperse in water-unsaturated environments, such as the soil, bacteria rely on the availability and structure of water films forming on biotic and abiotic surfaces, and, especially, along fungal mycelia. Dispersal along such “fungal highways” may be driven both by mycelial physical properties and by interactions between bacteria and fungi. However, we still do not have a way to disentangle the biotic and abiotic elements. Results: We designed and 3D printed two devices establishing stable liquid films that support bacteria dispersal in the absence of biotic interactions. The thickness of the liquid film determined the presence of hydraulic flow capable of transporting non-motile cells. In the absence of flow, only motile cells can disperse in the presence of an energy source. Non-motile cells could not disperse autonomously without flow but dispersed as “hitchhikers” when co-inoculated with motile cells. Conclusions: The 3D printed devices can be used as an abiotic control to study bacterial dispersal on hydrated surfaces, such as plant roots and fungal hyphae networks in the soil. By teasing apart the abiotic and biotic dimensions, these 3D printed devices will stimulate further research on microbial dispersal in soil and other water-unsaturated environments.

3D printing↗
NASA NTRS1997

Conserved gene clusters in bacterial genomes provide further support for the primacy of RNA

Five complete bacterial genome sequences have been released to the scientific community. These include four (eu)Bacteria, Haemophilus influenzae, Mycoplasma genitalium, M. pneumoniae, and Synechocystis PCC 6803, as well as one Archaeon, Methanococcus jannaschii. Features of organization shared by these genomes are likely to have arisen very early in the history of the bacteria and thus can be expected to provide further insight into the nature of early ancestors. Results of a genome comparison of these five organisms confirm earlier observations that gene order is remarkably unpreserved. There are, nevertheless, at least 16 clusters of two or more genes whose order remains the same among the four (eu)Bacteria and these are presumed to reflect conserved elements of coordinated gene expression that require gene proximity. Eight of these gene orders are essentially conserved in the Archaea as well. Many of these clusters are known to be regulated by RNA-level mechanisms in Escherichia coli, which supports the earlier suggestion that this type of regulation of gene expression may have arisen very early. We conclude that although the last common ancestor may have had a DNA genome, it likely was preceded by progenotes with an RNA genome.

Non-NASA Center↗
DOE OSTI2025

Bacterial microcompartment architectures as biomaterials for conversion of gaseous substrates

Bacterial microcompartments (BMCs) are protein shells encapsulating multiple enzymes of a metabolic pathway. Interpretations of early experiments on carboxysomes led to the narrative that transport of small gases (CO 2 , O 2 ) across the shell membrane is restricted. Since then, this notion has been largely contradicted by studies of engineered shells, although these shell constructs lack important proteins present in the native BMCs, altering the synthetic shells’ topology, surface and mechanical properties. Here, we discuss here an updated model of gas permeability that informs the design of engineered shells for catalysis on gas substrates and outline how nonshell suprastructures of BMC shell proteins could be used in formulating sustainable biomaterials for hydrogen generation via methane pyrolysis and for other greenhouse gas mitigations.

Bacterial microcompartment↗
DOE OSTI2021

Structure of the Extracellular Region of the Bacterial Type VIIb Secretion System Subunit EsaA

Gram-positive bacteria use type VII secretion systems (T7SSs) to export effector proteins that manipulate the physiology of nearby prokaryotic and eukaryotic cells. Several mycobacterial T7SSs have established roles in virulence. By contrast, the genetically distinct T7SSb pathway found in Firmicutes bacteria more often functions to mediate bacterial competition. A lack of structural information on the T7SSb has limited the understanding of effector export by this protein secretion apparatus. Here, we present the 2.4 angstrom crystal structure of the extracellular region of the T7SSb subunit EsaA from Streptococcus gallolyticus. Our structure reveals that homodimeric EsaA is an elongated, arrow-shaped protein with a surface-accessible "tip", which in some species of bacteria serves as a receptor for lytic bacteriophages. Because it is the only T7SSb subunit large enough to traverse the peptidoglycan layer of Firmicutes, we propose that EsaA plays a critical role in transporting effectors across the entirety of the Gram-positive cell envelope.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Classification of bacterial plasmid and chromosome derived sequences using machine learning

Plasmids are important genetic elements that facilitate horizonal gene transfer between bacteria and contribute to the spread of virulence and antimicrobial resistance. Most bacterial genome sequences in the public archives exist in draft form with many contigs, making it difficult to determine if a contig is of chromosomal or plasmid origin. Using a training set of contigs comprising 10,584 chromosomes and 10,654 plasmids from the PATRIC database, we evaluated several machine learning models including random forest, logistic regression, XGBoost, and a neural network for their ability to classify chromosomal and plasmid sequences using nucleotide k-mers as features. Based on the methods tested, a neural network model that used nucleotide 6-mers as features that was trained on randomly selected chromosomal and plasmid subsequences 5kb in length achieved the best performance, outperforming existing out-of-the-box methods, with an average accuracy of 89.38% ± 2.16% over a 10-fold cross validation. The model accuracy can be improved to 92.08% by using a voting strategy when classifying holdout sequences. In both plasmids and chromosomes, subsequences encoding functions involved in horizontal gene transfer—including hypothetical proteins, transporters, phage, mobile elements, and CRISPR elements—were most likely to be misclassified by the model. This study provides a straightforward approach for identifying plasmid-encoding sequences in short read assemblies without the need for sequence alignment-based tools.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Heterologous Assembly of Pleomorphic Bacterial Microcompartment Shell Architectures Spanning the Nano- to Microscale

Many bacteria use protein-based organelles known as bacterial microcompartments (BMCs) to organize and sequester sequential enzymatic reactions. Regardless of their specialized metabolic function, all BMCs are delimited by a shell made of multiple structurally redundant, yet functionally diverse, hexameric (BMC-H), pseudohexameric/trimeric (BMC-T), or pentameric (BMC-P) shell protein paralogs. When expressed without their native cargo, shell proteins have been shown to self-assemble into 2D sheets, open-ended nanotubes, and closed shells of ≈40 nm diameter that are being developed as scaffolds and nanocontainers for applications in biotechnology. Here, by leveraging a strategy for affinity-based purification, it is demonstrated that a wide range of empty synthetic shells, many differing in end-cap structures, can be derived from a glycyl radical enzyme-associated microcompartment. The range of pleomorphic shells observed, which span ≈2 orders of magnitude in size from ≈25 nm to ≈1.8 µm, reveal the remarkable plasticity of BMC-based biomaterials. In addition, new capped nanotube and nanocone morphologies are observed that are consistent with a multicomponent geometric model in which architectural principles are shared among asymmetric carbon, viral protein, and BMC-based structures.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Structural properties of optically clear bacterial cellulose produced by Komagataeibacter hansenii using arabitol

Here, bacterial cellulose (BC) exhibits beneficial properties for use in biomedical applications but is limited by its lack of tunable transparency capabilities. To overcome this deficiency, a novel method to synthesize transparent BC materials using an alternative carbon source, namely arabitol, was developed. Characterization of the BC pellicles was performed for yield, transparency, surface morphology, and molecular assembly. Transparent BC was produced using mixtures of glucose and arabitol. Zero percent arabitol pellicles exhibited 25% light transmittance, which increased with increasing arabitol concentration through to 75% light transmittance. While transparency increased, overall BC yield was maintained indicating that the altered transparency may be induced on a micro-scale rather than a macro-scale. Significant differences in fiber diameter and the presence of aromatic signatures were observed. Overall, this research outlines methods for producing BC with tunable optical transparency, while also bringing new insight to insoluble components of exopolymers produced by Komagataeibacter hansenii.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Engineered bacterial microcompartments: apps for programming metabolism

Bacterial Microcompartments (BMCs) are used by diverse bacteria to compartmentalize enzymatic reactions, functioning analogously to the organelles of eukaryotes. The bounding membrane and encapsulated components are composed entirely of protein, which makes them ideal targets for modification by genetic engineering. In contrast to viruses, in which generally only one protein forms the capsid, the shells of BMCs consist of a variety of shell proteins, each a potential unit of selection. Despite their differences in permeability, the shell proteins are surprisingly interchangeable. Recent developments have shown that they are also highly amenable to engineered modifications which poise them for a variety of biotechnological applications. Additionally, given their modular structure, with a module defined as a semi-autonomous functional unit, BMCs can be considered apps for programming metabolism that can be de-bugged by adaptive evolution.

59 BASIC BIOLOGICAL SCIENCES↗