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At least 415 records · Page 23
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Binding of Campylobacter jejuni FliW Adjacent to the CsrA RNA-Binding Pockets Modulates CsrA Regulatory Activity

Campylobacter jejuni CsrA is an mRNA-binding, post-transcriptional regulator that controls many metabolic- and virulence-related characteristics of this important pathogen. In contrast to E. coli CsrA, whose activity is modulated by binding to small non-coding RNAs (sRNAs), C. jejuni CsrA activity is controlled by binding to the CsrA antagonist FliW. In this study, we identified the FliW binding site on CsrA. Deletion of the C-terminus of C. jejuni CsrA, which is extended relative to sRNA-binding CsrA proteins, abrogated FliW binding. Bacterial two-hybrid experiments were used to assess the interaction of FliW with wild-type CsrA and mutants thereof, in which every amino acid was individually mutated. Two CsrA mutations (V51A and N55A) resulted in a significant decrease in FliW binding. The V51A and N55A mutants also showed a decrease in CsrA-FliW complex formation, as assessed by size-exclusion chromatography and surface plasmon resonance. These residues were highly conserved in bacterial species containing CsrA orthologs whose activities are predicted to be regulated by FliW. The location of FliW binding was immediately adjacent to the two RNA-binding sites of the CsrA homodimer, suggesting the model that FliW binding to CsrA modulates its ability to bind to its mRNA targets either by steric hindrance, electrostatic repulsion, or by altering the overall structure of the RNA-binding sites.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Construction of chicken infectious anemia virus infectious clone and study on its pathogenicity

Chicken infectious anemia virus (CIAV) can be transmitted through contaminated live poultry vaccine. However, the pathogenicity of contaminated CIAV strains is rarely reported. Previously, the chickens showed the typical symptoms of anemia after using the attenuated live fowl pox virus (FPV) vaccine. Therefore, exogenous CIAV contamination was suspected. We detected anti-CIAV antibodies in SPF chicks vaccinated with the FPV vaccine. CIAV contamination was confirmed in the FPV vaccine, and the CIAV strain was named JS2020-FPV. This study aims to rescue JS2020-FPV by reverse genetic assays and investigate its pathogenicity. Firstly, double-copies infectious clone of JS2020-FPV was constructed. For the pathogenicity study, infectious clone of JS2020-FPV was used to inoculate 1-day-old SPF chicks. The typical symptoms of anemia were observed in the JS2020-PFV group 14 days post inoculation. The hematocrit and body weight of chicks in the JS2020-PFV group were significantly lower than those in the mock group. Notably, the thymus development index and antibody levels of NDV were lower in chicks in the JS2020-PFV group than those in the mock group. Different degrees of apoptosis of MSB1 and DF-1 were observed after inoculated with the JS2020-FPV VP3 recombinant fusion protein expressed by E. coli system, indicating that VP3 induced apoptosis in the transformed cells. Overall, the pathogenicity of the CIAV detected in the contaminated vaccine was confirmed by inoculating SPF chicks with the double-copies infectious DNA clone in this study. Our findings indicate that the dangers of vaccine contamination cannot be ignored.

Wang, Jinjin↗
DOE OSTI2025

High-Throughput Microfluidic Electroporation (HTME): A Scalable, 384-Well Platform for Multiplexed Cell Engineering

Electroporation-mediated gene delivery is a cornerstone of synthetic biology, offering several advantages over other methods: higher efficiencies, broader applicability, and simpler sample preparation. Yet, electroporation protocols are often challenging to integrate into highly multiplexed workflows, owing to limitations in their scalability and tunability. These challenges ultimately increase the time and cost per transformation. As a result, rapidly screening genetic libraries, exploring combinatorial designs, or optimizing electroporation parameters requires extensive iterations, consuming large quantities of expensive custom-made DNA and cell lines or primary cells. To address these limitations, we have developed a High-Throughput Microfluidic Electroporation (HTME) platform that includes a 384-well electroporation plate (E-Plate) and control electronics capable of rapidly electroporating all wells in under a minute with individual control of each well. Fabricated using scalable and cost-effective printed-circuit-board (PCB) technology, the E-Plate significantly reduces consumable costs and reagent consumption by operating on nano to microliter volumes. Furthermore, individually addressable wells facilitate rapid exploration of large sets of experimental conditions to optimize electroporation for different cell types and plasmid concentrations/types. Use of the standard 384-well footprint makes the platform easily integrable into automated workflows, thereby enabling end-to-end automation. We demonstrate transformation of E. coli with pUC19 to validate the HTME's core functionality, achieving at least a single colony forming unit in more than 99% of wells and confirming the platform's ability to rapidly perform hundreds of electroporations with customizable conditions. This work highlights the HTME's potential to significantly accelerate synthetic biology Design-Build-Test-Learn (DBTL) cycles by mitigating the transformation/transfection bottleneck.

Gaillard, William R↗
DOE OSTI2021

AlphaFold Protein Structure Database for Sequence-Independent Molecular Replacement

Crystallographic phasing recovers the phase information that is lost during a diffraction experiment. Molecular replacement is a commonly used phasing method for crystal structures in the protein data bank. In one form it uses a protein sequence to search a structure database to find suitable templates for phasing. However, sequence information is not always available, such as when proteins are crystallized with unknown binding partner proteins or when the crystal is of a contaminant. The recent development of AlphaFold published the predicted protein structures for every protein from twenty distinct species. In this work, we tested whether AlphaFold-predicted E. coli protein structures were accurate enough to enable sequence-independent phasing of diffraction data from two crystallization contaminants of unknown sequence. Using each of more than 4000 predicted structures as a search model, robust molecular replacement solutions were obtained, which allowed the identification and structure determination of YncE and YadF. Our results demonstrate the general utility of the AlphaFold-predicted structure database with respect to sequence-independent crystallographic phasing.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Magnetically Recoverable and Reusable Titanium Dioxide Nanocomposite for Water Disinfection

A bifunctional magnetic Fe3O4@SiO2@TiO2 or MS-TiO2 antimicrobial nanocomposite was prepared based on simple sol-gel methods with common equipment and chemicals. Reaction pH was found to influence the TiO2 upload in the nanocomposite. The alkaline condition produced the greatest TiO2 upload, while the acidic condition the least. Annealing at 300 °C turned the as-synthesized amorphous TiO2 into one with high content of anatase, the most photoactive form of TiO2. Irradiated by 365 nm UV light, a sample of 30 mg/mL of annealed nanocomposite containing 12.6 wt.% Ti was shown to be able to completely eradicate 104 CFU/mL of the laboratory-grown E. coli within 25 min, 25 min faster than the control when the 365 nm UV light was employed alone. The nanocomposite demonstrated consistent antimicrobial performance over repeated uses and was easily recoverable magnetically due to its high magnetization value (33 emu/g). Additionally, it was shown to reduce the bacterial count in a real surface water sample containing 500–5000 CFU/mL of different microbes by 62 ± 3% within 30 min. The irradiating 365 nm UV light alone was found to have generated little biocidal effect on this surface water sample. The nanocomposite is promising to serve as an effective, safe, and eco-friendly antimicrobial agent, especially for surface water disinfection.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2022

Viral Capsid Change upon Encapsulation of Double-Stranded DNA into an Infectious Hypodermal and Hematopoietic Necrosis Virus-like Particle

In this study, we aimed to encapsulate the sizable double-stranded DNA (dsDNA, 3.9 kbp) into a small-sized infectious hypodermal and hematopoietic necrosis virus-like particle (IHHNV-VLP; T = 1) and compared the changes in capsid structure between dsDNA-filled VLP and empty VLP. Based on our encapsulation protocol, IHHNV-VLP was able to load dsDNA at an efficiency of 30–40% (w/w) into its cavity. Structural analysis revealed two subclasses of IHHNV-VLP, so-called empty and dsDNA-filled VLPs. The three-dimensional (3D) structure of the empty VLP produced in E. coli was similar to that of the empty IHHNV-VLP produced in Sf9 insect cells. The size of the dsDNA-filled VLP was slightly bigger (50 Å) than its empty VLP counterpart; however, the capsid structure was drastically altered. The capsid was about 1.5-fold thicker due to the thickening of the capsid interior, presumably from DNA–capsid interaction evident from capsid protrusions or nodules on the interior surface. In addition, the morphological changes of the capsid exterior were particularly observed in the vicinity of the five-fold axes, where the counter-clockwise twisting of the “tripod” structure at the vertex of the five-fold channel was evident, resulting in a widening of the channel’s opening. Whether these capsid changes are similar to virion capsid maturation in the host cells remains to be investigated. Nevertheless, the ability of IHHNV-VLP to encapsulate the sizable dsDNA has opened up the opportunity to package a dsDNA vector that can insert exogenous genes and target susceptible shrimp cells in order to halt viral infection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell's survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. In this work, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13 C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

deeprob/ThioesteraseEnzymeSpecificity: EnZymClass-first-release

Characterizing the functional properties of plant acyl-ACP thioesterases (TEs), a key enzyme class used in the production of renewable oleochemicals in microbial hosts, experimentally, can be an expensive and time consuming process since it requires manual screening of thousands of candidates in a database. Using amino acid sequence to computationally predict an enzyme’s function might accelerate this process; however obtaining the necessary amount of information on previously characterized enzymes and their respective sequences required by standard Machine Learning (ML) based approaches to accurately infer sequence-function relationships can be prohibitive, especially with a low-throughput testing cycle. Experimental noise, unbalanced dataset where high sequence similarity does not always imply identical functional properties will further prevent robust prediction performance. Herein we present a ML method, Ensemble method for enZyme Classification (EnZymClass), that is specifically designed to address these issues. We used EnZymClass to classify TEs into short, long and mixed free fatty acid substrate specificity categories. While general guidelines for inferring substrate specificity have been proposed before, prediction of chain-length preference from primary sequence has remained elusive for plant acyl-ACP TEs. By applying EnZymClass to a subset of TEs in the ThYme database, we identified two medium chain TEs, ClFatB3 and CwFatB2, with previously uncharacterized activity in E. coli fatty acid production hosts.

Banerjee, Deepro↗
NASA NTRS1973

Tape Cassette Bacteria Detection System

The design, fabrication, and testing of an automatic bacteria detection system with a zero-g capability and based on the filter-capsule approach is described. This system is intended for monitoring the sterility of regenerated water in a spacecraft. The principle of detection is based on measuring the increase in chemiluminescence produced by the action of bacterial porphyrins (i.e., catalase, cytochromes, etc.) on a luminol-hydrogen peroxide mixture. Since viable as well as nonviable organisms initiate this luminescence, viable organisms are detected by comparing the signal of an incubated water sample with an unincubated control. Higher signals for the former indicate the presence of viable organisms. System features include disposable sealed sterile capsules, each containing a filter membrane, for processing discrete water samples and a tape transport for moving these capsules through a processing sequence which involves sample concentration, nutrient addition, incubation, a 4 Molar Urea wash and reaction with luminol-hydrogen peroxide in front of a photomultiplier tube. Liquids are introduced by means of a syringe needle which pierces a rubber septum contained in the wall of the capsule. Detection thresholds obtained with this unit towards E. coli and S. marcescens assuming a 400 ml water sample are indicated.

Source record↗
NASA NTRS1973

The prospect of life on Jupiter.

We have simulated electrical discharges in the Jovian atmosphere, using anhydrous methane-ammonia mixtures, and shown the formation of simple aliphatic nitriles, amino-nitriles, and their oligomers. Including hydrogen sulfide in the gas mixture, it appears that sulfur-containing amino-nitriles are not formed, since the hydrolysate of the products did not contain the corresponding amino-acids. There is a strong analogy between these reactions and the classical spark reactions simulating the primitive earth's atmosphere. We are attempting a closer simulation of Jupiter's atmosphere by using appropriate temperature and pressure conditions. It seems that prebiotic synthesis on Jupiter may have reached an advanced state. As an alternative approach we have tested the survival ability of common terrestrial microorganisms in aqueous media at 102 atmospheres pressure and at 20 C in a simulated Jovian atmosphere. E. coli, S. marcescens, A. aerogenes, and B. subtilis will all tolerate 24 hr under these conditions with little death.

Ponnamperuma, C.↗
NASA NTRS1974

Application of nitrogen metabolism in autotrophic bacteria to chemosynthetic bioregeneration in space missions, supplement

The chemolithotroph, Hydrogenomonas eutropha, was considered as a life support, bioregenerative system. This project focuses on several metabolic functions that are related to the proposed nitrogen cycle between man and this microbe. Specifically this organism has the capability to utilize as the sole nitrogen source such urine components as urea and fifteen individual amino acids, but not nine other amino acids. The effectiveness of utilization was high for many amino acids. Several specific growth inhibitions were also observed. The enzyme that catalyzes the incorporation of ammonia in the medium into amino acids was identified as a NADP-specific, L-glutamate dehydrogenase. This enzyme has a constitutive nature. This organism can synthesize all of its amino acids from carbon dioxide and ammonia. Therefore with the background literature of multiple pathways of individual amino acid biosyntheses, our evidence to date is consistent with the Hydrogeneomonas group having the same pathway of valine-isoleucine formation as the classical E. coli.

Wixom, R. L.↗
NASA NTRS1973

Development of an iodine generator for reclaimed water purification in manned spacecraft applications

A successful 30-day test is described of a prototype Iodine Generating and Dispensing System (IGDS). The IGDS was sized to iodinate the drinking water nominally consumed by six men, 4.5 to 13.6 kg (10 to 30 lb) water per man-day with a + or - 10 to 20% variation with iodine (I2) levels of 0.5 to 20 parts per million (ppm). The I2 treats reclaimed water to prevent or eliminate microorganism contamination. Treatment is maintained with a residual of I2 within the manned spacecraft water supply. A simplified version of the chlorogen water disinfection concept, developed by life systems for on-site generation of chlorine (Cl2), was used as a basis for IGDS development. Potable water contaminated with abundant E. Coliform Group organisms was treated by electrolytically generated I2 at levels of 5 to 10 ppm. In all instances, the E. coli were eliminated.

Wynveen, R. A.↗
NASA NTRS1974

The protective effect of a constant magnetic field

The protective effect of a constant magnetic field sharply reduced spontaneous lysis of E. coli cells when subjected to ultraviolet radiation. A protective effect of a CMF was found in a study of tissue cultures of normally growing cells (kidney epithelium) and cancer cells (cells from a cancer of the larynx). The protective effect of a CMF is also seen in a combined exposure of tissue cultures to X-rays and CMF energy (strength of the CMF was 2000 oersteds with a gradient of 500 oersteds/cm). The data obtained are of interest to experimental oncology (development of new methods of treating malignant tumors).

Sosunov, A. V.↗
NASA NTRS1979

Accuracy of RGD approximation for computing light scattering properties of diffusing and motile bacteria

The study tests the accuracy of the Rayleigh-Gans-Debye (RGD) approximation against a rigorous scattering theory calculation for a simplified model of E. coli (about 1 micron in size) - a solid spheroid. A general procedure is formulated whereby the scattered field amplitude correlation function, for both polarized and depolarized contributions, can be computed for a collection of particles. An explicit formula is presented for the scattered intensity, both polarized and depolarized, for a collection of randomly diffusing or moving particles. Two specific cases for the intermediate scattering functions are considered: diffusing particles and freely moving particles with a Maxwellian speed distribution. The formalism is applied to microorganisms suspended in a liquid medium. Sensitivity studies revealed that for values of the relative index of refraction greater than 1.03, RGD could be in serious error in computing the intensity as well as correlation functions.

Kottarchyk, M.↗
NASA NTRS1982

The chemical structure of DNA sequence signals for RNA transcription

The proposed recognition sites for RNA transcription for E. coli NRA polymerase, bacteriophage T7 RNA polymerase, and eukaryotic RNA polymerase Pol II are evaluated in the light of the requirements for efficient recognition. It is shown that although there is good experimental evidence that specific nucleic acid sequence patterns are involved in transcriptional regulation in bacteria and bacterial viruses, among the sequences now available, only in the case of the promoters recognized by bacteriophage T7 polymerase does it seem likely that the pattern is sufficient. It is concluded that the eukaryotic pattern that is investigated is not restrictive enough to serve as a recognition site.

George, D. G.↗
NASA NTRS1988

The Antibio experiment

An experiment was flown on Spacelab to confirm the results of the Cytos 2 experiment on Salyut 7, which found an increase in minimal inhibitory concentration in in-flight cultures, i.e., an increase of antibiotic resistance. The 1 g centrifuge on Biorack was also used to differentiate the effects of cosmic rays and microgravity. The antibiotic sensitivity of bacteria cultivated in vitro during orbital flight was studied. The bacteria was E. coli, the antibiotic was Colistin. An increase of antibiotic resistance is observed. Three explanations are offered: stimulation of bacterial proliferation in space; a relationship between the transport of antibiotics into cells and modifications of cellular envelope permeability; and a combined effect of both phenomena.

Lapchine, L.↗