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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

CRISPR–Cas9 bends and twists DNA to read its sequence

In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9–RNA–DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 ‘reads’ snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Bacterial diversity dynamics in microbial consortia selected for lignin utilization

Lignin is nature’s largest source of phenolic compounds. Its recalcitrance to enzymatic conversion is still a limiting step to increase the value of lignin. Although bacteria are able to degrade lignin in nature, most studies have focused on lignin degradation by fungi. To understand which bacteria are able to use lignin as the sole carbon source, natural selection over time was used to obtain enriched microbial consortia over a 12-week period. The source of microorganisms to establish these microbial consortia were commercial and backyard compost soils. Cultivation occurred at two different temperatures, 30°C and 37°C, in defined culture media containing either Kraft lignin or alkaline-extracted lignin as carbon source. iTag DNA sequencing of bacterial 16S rDNA gene was performed for each of the consortia at six timepoints (passages). The initial bacterial richness and diversity of backyard compost soil consortia was greater than that of commercial soil consortia, and both parameters decreased after the enrichment protocol, corroborating that selection was occurring. Bacterial consortia composition tended to stabilize from the fourth passage on. After the enrichment protocol, Firmicutes phylum bacteria were predominant when lignin extracted by alkaline method was used as a carbon source, whereas Proteobacteria were predominant when Kraft lignin was used. Bray-Curtis dissimilarity calculations at genus level, visualized using NMDS plots, showed that the type of lignin used as a carbon source contributed more to differentiate the bacterial consortia than the variable temperature. The main known bacterial genera selected to use lignin as a carbon source were Altererythrobacter , Aminobacter , Bacillus , Burkholderia , Lysinibacillus , Microvirga , Mycobacterium , Ochrobactrum , Paenibacillus , Pseudomonas , Pseudoxanthomonas , Rhizobiales and Sphingobium . These selected bacterial genera can be of particular interest for studying lignin degradation and utilization, as well as for lignin-related biotechnology applications.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1994

Inactivation, DNA double strand break induction and their rejoining in bacterial cells irradiated with heavy ions

Besides inactivation one of the major interests in our experiments is to study the primary damage in the DNA double strand breaks (DSB) after heavy ion irradiation. These damages lead not only to cell death but also under repair activities to mutations. In further experiments we have investigated the inactivation with two different strains of Deinococcus radiodurans (R1, Rec 30) and the induction of DSB as well as the rejoining of DSB in stationary cells of E. coli (strain B/r) irradiated with radiations of different quality. In the latter case irradiations were done so that the cell survival was roughly at the same level. We measured the DSB using the pulse field gelelectrophoresis which allows to separate between intact (circular) and damaged (linear) DNA. The irradiated cells were transferred to NB medium and incubated for different times to allow rejoining.

Schaefer, M.↗
DOE OSTI2026

Synthetic data-driven deep learning for label-free autonomous atomic force microscopy

Atomic force microscopy (AFM) is a widely used tool for nanoscale characterization across materials science, energy research, and biology. However, its adoption in high-throughput materials discovery and statistically driven studies remains limited by a strong dependence on expert operator input and by the scarcity of annotated experimental AFM datasets needed to enable data-driven automation. Here, we introduce SimuScan, a synthetic-data–driven framework that enables reliable AFM feature identification, segmentation, and targeted imaging without requiring large manually labeled experimental datasets. SimuScan generates tunable, high-fidelity synthetic AFM images of defined morphologies while incorporating realistic experimental artifacts, including tip–sample convolution, noise, flattening distortions, and surface debris. These datasets are shown to support scalable, label-free training of modern deep learning models for AFM analysis. When integrated into data-driven AFM workflows, SimuScan-trained models can locate and analyze nanoscale structures across large datasets and guide targeted follow-up imaging. We validate this approach on nanostructured surfaces, DNA assemblies, and bacterial cells, demonstrating robust generalization across diverse sample types with minimal operator intervention. More broadly, this work establishes a general strategy for generating explicitly conditioned, task-relevant synthetic data to improve the reliability of downstream models in autonomous microscopy.

Millan-Solsona, Ruben [Oak Ridge National Laborato↗
NASA NTRS2004

A Survey of Staphylococcus sp and its Methicillin Resistance aboard the International Space Station

Background: Within the past few years, methicillin-resistant Staphylococcus aureus has emerged in environments with susceptible hosts in close proximity, such as hospitals and nursing homes. As the International Space Station (ISS) represents a semi-closed environment with a high level of crewmember interaction, an evaluation of isolates of clinical and environmental Staphylococcus aureus and coagulase negative Staphylococcus was performed to determine if this trend was also present in astronauts occupying ISS or on surfaces of the space station itself. Methods: Identification of isolates was completed using VITEK (GPI cards, BioMerieux), 16S ribosomal DNA analysis (MicroSeq 500, ABI), and Rep-PCR DNA fingerprinting (Divemilab, Bacterial Barcodes). Susceptibility tests were performed using VITEK (GPS-105 cards, BioMerieux) and resistance characteristics were evaluated by testing for the presence of the mecA gene (PBP2' MRSA test kit, Oxoid). Results: Rep-PCR analysis indicated the transfer of S. aureus between crewmembers and between crewmembers and ISS surfaces. While a variety of S. aureus were identified from both the crewmembers and environment, evaluations of the microbial population indicated minimal methicillin resistance. Results of this study indicated that within the semi-closed ISS environment, transfer of bacteria between crewmembers and their environment has been occurring, although there was no indication of a high concentration of methicillin resistant Staphylococcus species. Conclusions: While this study suggests that the spread of methicillin resistant S. aureus is not currently a concern aboard ISS, the increasing incidence of Earth-based antibiotic resistance indicates a need for continued clinical and environmental monitoring.

Bassinger, V. J.↗
NASA NTRS2013

Detection of Only Viable Bacterial Spores Using a Live/Dead Indicator in Mixed Populations

This method uses a photoaffinity label that recognizes DNA and can be used to distinguish populations of bacterial cells from bacterial spores without the use of heat shocking during conventional culture, and live from dead bacterial spores using molecular-based methods. Biological validation of commercial sterility using traditional and alternative technologies remains challenging. Recovery of viable spores is cumbersome, as the process requires substantial incubation time, and the extended time to results limits the ability to quickly evaluate the efficacy of existing technologies. Nucleic acid amplification approaches such as PCR (polymerase chain reaction) have shown promise for improving time to detection for a wide range of applications. Recent real-time PCR methods are particularly promising, as these methods can be made at least semi-quantitative by correspondence to a standard curve. Nonetheless, PCR-based methods are rarely used for process validation, largely because the DNA from dead bacterial cells is highly stable and hence, DNA-based amplification methods fail to discriminate between live and inactivated microorganisms. Currently, no published method has been shown to effectively distinguish between live and dead bacterial spores. This technology uses a DNA binding photoaffinity label that can be used to distinguish between live and dead bacterial spores with detection limits ranging from 109 to 102 spores/mL. An environmental sample suspected of containing a mixture of live and dead vegetative cells and bacterial endospores is treated with a photoaffinity label. This step will eliminate any vegetative cells (live or dead) and dead endospores present in the sample. To further determine the bacterial spore viability, DNA is extracted from the spores and total population is quantified by real-time PCR. The current NASA standard assay takes 72 hours for results. Part of this procedure requires a heat shock step at 80 degC for 15 minutes before the sample can be plated. Using a photoaffinity label would remove this step from the current assay as the label readily penetrates both live and dead bacterial cells. Secondly, the photoaffinity label can only penetrate dead bacterial spores, leaving behind the viable spore population. This would allow for rapid bacterial spore detection in a matter of hours compared to the several days that it takes for the NASA standard assay.

Behar, Alberto E.↗
DOE OSTI2021

A CRISPR-Cas9–integrase complex generates precise DNA fragments for genome integration

CRISPR-Cas9 is an RNA-guided DNA endonuclease involved in bacterial adaptive immunity and widely repurposed for genome editing in human cells, animals and plants. In bacteria, RNA molecules that guide Cas9 s activity derive from foreign DNA fragments that are captured and integrated into the host CRISPR genomic locus by the Cas1-Cas2 CRISPR integrase. How cells generate the specific lengths of DNA required for integrase capture is a central unanswered question of type II-A CRISPR-based adaptive immunity. Here, we show that an integrase supercomplex comprising guide RNA and the proteins Cas1, Cas2, Csn2 and Cas9 generates precisely trimmed 30-base pair DNA molecules required for genome integration. The HNH active site of Cas9 catalyzes exonucleolytic DNA trimming by a mechanism that is independent of the guide RNA sequence. These results show that Cas9 possesses a distinct catalytic capacity for generating immunological memory in prokaryotes.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2002

Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

Non-NASA Center↗
DOE OSTI2020

Peatland Microbial Community Composition Is Driven by a Natural Climate Gradient

Peatlands are important players in climate change–biosphere feedbacks via long-term net carbon (C) accumulation in soil organic matter and as potential net C sources including the potent greenhouse gas methane (CH 4 ). Interactions of climate, site-hydrology, plant community, and groundwater chemical factors influence peatland development and functioning, including C dioxide (CO 2 ) and CH 4 fluxes, but the role of microbial community composition is not well understood. To assess microbial functional and taxonomic dissimilarities, we used high throughput sequencing of the small subunit ribosomal DNA (SSU rDNA) to determine bacterial and archaeal community composition in soils from twenty North American peatlands. Targeted DNA metabarcoding showed that although Proteobacteria, Acidobacteria, and Actinobacteria were the dominant phyla on average, intermediate and rich fens hosted greater diversity and taxonomic richness, as well as an array of candidate phyla when compared with acidic and nutrient-poor poor fens and bogs. Moreover, pH was revealed to be the strongest predictor of microbial community structure across sites. Predictive metagenome content (PICRUSt) showed increases in specific genes, such as purine/pyrimidine and amino-acid metabolism in mid-latitude peatlands from 38 to 45° N, suggesting a shift toward utilization of microbial biomass over utilization of initial plant biomass in these microbial communities. Overall, there appears to be noticeable differences in community structure between peatland classes, as well as differences in microbial metabolic activity between latitudes. Finally, these findings are in line with a predicted increase in the decomposition and accelerated C turnover, and suggest that peatlands north of 37° latitude may be particularly vulnerable to climate change.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genome expansion by a CRISPR trimmer-integrase

CRISPR–Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1–Cas2 integrase is necessary but not sufficient. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation, but many CRISPR–Cas systems lack Cas4. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1–Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited exonucleases for faithful acquisition of new CRISPR immune sequences.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Multisubstrate DNA stable isotope probing reveals guild structure of bacteria that mediate soil carbon cycling

Significance Soil organic matter contains a large fraction of global C whose fate is largely governed by soil microbial communities. Global C-cycle models can be improved using information about microbial processes in soil, but the inner workings of soil communities remain largely hidden from view. We used stable isotopes to characterize the guild structure of soil bacteria based on how they assimilated C from different sources. Guild structure corresponded with growth dynamics and rrn copy number. We also show that this guild structure explains microbial biogeographical patterns at continental and global scales. These results suggest that microbial contributions to soil C cycling are largely governed by an anabolic process that governs energy allocation to growth, competition, and survival.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Genomic Metrics Applied to Rhizobiales ( Hyphomicrobiales ): Species Reclassification, Identification of Unauthentic Genomes and False Type Strains

Taxonomic decisions within the order Rhizobiales have relied heavily on the interpretations of highly conserved 16S rRNA sequences and DNA–DNA hybridizations (DDH). Currently, bacterial species are defined as including strains that present 95–96% of average nucleotide identity (ANI) and 70% of digital DDH (dDDH). Thus, ANI values from 520 genome sequences of type strains from species of Rhizobiales order were computed. From the resulting 270,400 comparisons, a ≥95% cut-off was used to extract high identity genome clusters through enumerating maximal cliques. Coupling this graph-based approach with dDDH from clusters of interest, it was found that: (i) there are synonymy between Aminobacter lissarensis and Aminobacter carboxidus, Aurantimonas manganoxydans and Aurantimonas coralicida, “Bartonella mastomydis,” and Bartonella elizabethae, Chelativorans oligotrophicus, and Chelativorans multitrophicus, Rhizobium azibense, and Rhizobium gallicum, Rhizobium fabae, and Rhizobium pisi, and Rhodoplanes piscinae and Rhodoplanes serenus; (ii) Chelatobacter heintzii is not a synonym of Aminobacter aminovorans; (iii) “Bartonella vinsonii” subsp. arupensis and “B. vinsonii” subsp. berkhoffii represent members of different species; (iv) the genome accessions GCF_003024615.1 (“Mesorhizobium loti LMG 6125T”), GCF_003024595.1 (“Mesorhizobium plurifarium LMG 11892T”), GCF_003096615.1 (“Methylobacterium organophilum DSM 760T”), and GCF_000373025.1 (“R. gallicum R-602 spT”) are not from the genuine type strains used for the respective species descriptions; and v) “Xanthobacter autotrophicus” Py2 and “Aminobacter aminovorans” KCTC 2477T represent cases of misuse of the term “type strain”. Aminobacter heintzii comb. nov. and the reclassification of Aminobacter ciceronei as A. heintzii is also proposed. To facilitate the downstream analysis of large ANI matrices, we introduce here ProKlust (“Prokaryotic Clusters”), an R package that uses a graph-based approach to obtain, filter, and visualize clusters on identity/similarity matrices, with settable cut-off points and the possibility of multiple matrices entries.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2009

Discrimination of Spore-Forming Bacilli Using spoIVA

A method of discriminating between spore-forming and non-spore-forming bacteria is based on a combination of simultaneous sporulation-specific and non-sporulation-specific quantitative polymerase chain reactions (Q-PCRs). The method was invented partly in response to the observation that for the purposes of preventing or reducing biological contamination affecting many human endeavors, ultimately, only the spore-forming portions of bacterial populations are the ones that are problematic (or, at least, more problematic than are the non-spore-forming portions). In some environments, spore-forming bacteria constitute small fractions of the total bacterial populations. The use of sporulation-specific primers in Q-PCR affords the ability to assess the spore-forming fraction of a bacterial population present in an environment of interest. This assessment can provide a more thorough and accurate understanding of the bacterial contamination in the environment, thereby making it possible to focus contamination- testing, contamination-prevention, sterilization, and decontamination resources more economically and efficiently. The method includes the use of sporulation-specific primers in the form of designed, optimized deoxyribonucleic acid (DNA) oligonucleotides specific for the bacterial spoIVA gene (see table). [In "spoIVA," "IV" signifies Roman numeral four and the entire quoted name refers to gene A for the fourth stage of sporulation.] These primers are mixed into a PCR cocktail with a given sample of bacterial cells. A control PCR cocktail into which are mixed universal 16S rRNA primers is also prepared. ["16S rRNA" denotes a ribosomal ribonucleic acid (rRNA) sequence that is common to all organisms.] Following several cycles of heating and cooling according to the PCR protocol to amplify amounts of DNA molecules, the amplification products can be analyzed to determine the types of bacterial cells present within the samples. If the amplification product is strong, relative to the product of a control PCR sequence, then it is concluded that the bacterial population in the sample consists predominantly of spore-forming cells. If the amplification product is weak or nonexistent, then it is concluded that the bacterial population in the sample consists predominantly or entirely of non-spore-forming cells.

Venkateswaran, Kasthuri↗
DOE OSTI2020

Supplemental selenium source on gut health: insights on fecal microbiome and fermentation products of growing puppies

ABSTRACT Selenium is an essential trace element that can modulate the gut microbiome with an impact on host health. The present study aimed to evaluate the effects of organic (selenium-enriched yeast) vs inorganic (sodium selenite) selenium source on fecal end-fermentation products and gut microbiome of puppies from 20 to 52 weeks of age. Alpha and beta diversity of the gut bacterial community were affected by age but not by gender or selenium source. The relative abundance of taxa was differently affected by age, and the DNA concentration of all selected bacterial groups increased with age, although total volatile fatty acids (VFA), acetate, propionate, caproate and lactate concentrations decreased. Organic selenium was associated with a higher concentration of total VFA, propionate and butyrate, a higher number of DNA copies of Lactobacillus, and a trend to lower DNA copies of Escherichia coli. Effects on fecal microbiome during growth differed with selenium source. Females had higher fecal end-fermentation products related to protein degradation, whereas males had higher DNA concentration of Bifidobacterium. Organic selenium might be beneficial over inorganic for dog food supplementation due to the positive modulation of the gut microbiome observed in puppies.

Pereira, Ana Margarida↗
DOE OSTI2025

Interrelationships among methods of estimating microbial biomass across multiple soil orders and biomes

Understanding the role of soil microbes is critical to ecosystem processes, and more thorough comparisons of measurement proxies for soil microbial biomass could broaden the inclusion of explicit microbial parameterization in soil carbon cycling and earth system models. We measured physical, chemical, and biological data from eight soil orders representing 11 major biomes and four climate regions. Four prominent methods to measure microbial abundance—chloroform fumigation extraction (CFE), total DNA yield, gene copy number by quantitative polymerase chain reaction (GCN), and phospholipid fatty acids (PLFA)—were compared to assess their relationships with each other and with soil characteristics. Correlations were observed when comparing methods, with CFE correlating strongly with total DNA yield, GCN, and PLFA; CFE with bacterial GCN and bacterial PLFA; and to a lesser extent, total PLFA and total DNA yield. Correlations improved with the removal of organic soils (Histosols, Gelisols). Comparisons involving extracted DNA were improved by correcting for clay content, due to DNA extraction inefficiencies in clay-rich soils. Correlations involving fungi (PLFA or GCN) were always less significant. These methods could serve as reliable, inter-relatable proxies for the estimation of total soil microbial biomass while recognizing that the proxies are less effective at parsing differences between bacteria and fungi. Here, we provide specific equations to relate measures of soil microbial biomass by these four different methods to enable microbial models to utilize a greater diversity of observed data sources in parameterizations and simulations. Caveats for the equations and their values are also discussed.

59 BASIC BIOLOGICAL SCIENCES↗