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At least 379 records · Page 21
All sourcesNASA NTRSDOE OSTI
NASA NTRS1999

Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp. nov

The genus Shewanella has been studied since 1931 with regard to a variety of topics of relevance to both applied and environmental microbiology. Recent years have seen the introduction of a large number of new Shewanella-like isolates, necessitating a coordinated review of the genus. In this work, the phylogenetic relationships among known shewanellae were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations. This polyphasic taxonomy takes into account all available phenotypic and genotypic data and integrates them into a consensus classification. Based on information generated from this study and obtained from the literature, a scheme for the identification of Shewanella species has been compiled. Key phenotypic characteristics were sulfur reduction and halophilicity. Fatty acid and quinone profiling were used to impart an additional layer of information. Molecular characterizations employing small-subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in some cases. As a result, DNA-DNA hybridization and sequence analyses of a more rapidly evolving molecule (gyrB gene) were performed. Species-specific PCR probes were designed for the gyrB gene and used for the rapid screening of closely related strains. With this polyphasic approach, in addition to the ten described Shewanella species, two new species, Shewanella oneidensis and 'Shewanella pealeana', were recognized; Shewanella oneidensis sp. nov. is described here for the first time.

NASA Discipline Exobiology↗
DOE OSTI2021

Data from PNA-Assisted DNAzymes to Cleave Double-Stranded DNA for Genetic Engineering with High Sequence Fidelity

DNAzymes have been widely used in many sensing and imaging applications but have rarely been used for genetic engineering since their discovery in 1994, because their substrate scope is mostly limited to single-stranded DNA or RNA, whereas genetic information is stored mostly in double-stranded DNA (dsDNA). To overcome this major limitation, we herein report peptide nucleic acid (PNA)-assisted double-stranded DNA nicking by DNAzymes (PANDA) as the first example to expand DNAzyme activity toward dsDNA. We show that PANDA is programmable in efficiently nicking or causing double strand breaks on target dsDNA, which mimics protein nucleases and can act as restriction enzymes in molecular cloning. In addition to being much smaller than protein enzymes, PANDA has a higher sequence fidelity compared with CRISPR/Cas under the condition we tested, demonstrating its potential as a novel alternative tool for genetic engineering and other biochemical applications.

Conversion↗
DOE OSTI2021

PNA-Assisted DNAzymes to Cleave Double-Stranded DNA for Genetic Engineering with High Sequence Fidelity

DNAzymes have been widely used in many sensing and imaging applications but have rarely been used for genetic 5 engineering since their discovery in 1994, because their substrate scope is mostly limited to single-stranded DNA or RNA, whereas 6 genetic information is stored mostly in double-stranded DNA (dsDNA). To overcome this major limitation, in this paper we report 7 peptide nucleic acid (PNA)-assisted double-stranded DNA nicking by DNAzymes (PANDA) as the first example to expand 8 DNAzyme activity toward dsDNA. We show that PANDA is programmable in efficiently nicking or causing double strand breaks on 9 target dsDNA, which mimics protein nucleases and can act as restriction enzymes in molecular cloning. In addition to being much 10 smaller than protein enzymes, PANDA has a higher sequence fidelity compared with CRISPR/Cas under the condition we tested, 11 demonstrating its potential as a novel alternative tool for genetic engineering and other biochemical applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Optimizing Cell-based Antimicrobials through Pooled Genomic Libraries

DNA synthesis and assembly technologies ushered in through synthetic biology have great promise for biomanufacturing, bioremediation, and the development of living therapeutics. Unfortunately, predicting sequence to function relationships, including for biosynthetic pathways expressed in a new host organism, is difficult and often requires many iterative cycles of design, construction, and testing. We are working to develop data-driven approaches to identify the genetic determinants of growth defects and productivity for the expression of a cell-based antimicrobial. We assayed the growth, pigment production, and antimicrobial activity of a collection of over 10,000 genetic mutants of the violacein biosynthetic pathway and sequenced the genetic variation of these mutants. Through this project, we have developed an innovative codebase to automate the determination of pigmentation and antimicrobial clearing diameter for tens of thousands of genetic mutants cultivated on agar dishes. Further, we have written DNA sequence analysis code to demultiplex & provide consensus sequences from high-throughput PacBio long-read circular consensus sequencing (CCS) datasets. From this foundation, we plan to map DNA sequence to function to predict an optimal genetic design to maximize antimicrobial activity while minimizing deleterious growth effects. The workflows and algorithms developed through this project can be broadly applied to other engineered functions in microbes, uncovering sequence to function relationships for complex phenotypes where function impacts fitness.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Catalytic DNA Polymerization Can Be Expedited by Active Product Release**

Abstract The sequence‐specific hybridization of DNA facilitates its use as a building block for designer nanoscale structures and reaction networks that perform computations. However, the strong binding energy of Watson–Crick base pairing that underlies this specificity also causes the DNA dehybridization rate to depend sensitively on sequence length and temperature. This strong dependency imposes stringent constraints on the design of multi‐step DNA reactions. Here we show how an ATP‐dependent helicase, Rep‐X, can drive specific dehybridization reactions at rates independent of sequence length, removing the constraints of equilibrium on DNA hybridization and dehybridization. To illustrate how this new capacity can speed up designed DNA reaction networks, we show that Rep‐X extends the range of conditions where the primer exchange reaction, which catalytically adds a domain provided by a hairpin template to a DNA substrate, proceeds rapidly.

Moerman, Pepijn G.↗
DOE OSTI2022

Catalytic DNA Polymerization Can Be Expedited by Active Product Release**

Abstract The sequence‐specific hybridization of DNA facilitates its use as a building block for designer nanoscale structures and reaction networks that perform computations. However, the strong binding energy of Watson–Crick base pairing that underlies this specificity also causes the DNA dehybridization rate to depend sensitively on sequence length and temperature. This strong dependency imposes stringent constraints on the design of multi‐step DNA reactions. Here we show how an ATP‐dependent helicase, Rep‐X, can drive specific dehybridization reactions at rates independent of sequence length, removing the constraints of equilibrium on DNA hybridization and dehybridization. To illustrate how this new capacity can speed up designed DNA reaction networks, we show that Rep‐X extends the range of conditions where the primer exchange reaction, which catalytically adds a domain provided by a hairpin template to a DNA substrate, proceeds rapidly.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates

The sequence specificity and programmability of DNA binding and cleavage have enabled widespread applications of CRISPR-Cas12a in genetic engineering. As an RNA-guided CRISPR endonuclease, Cas12a engages a 20-base pair (bp) DNA segment by forming a three-stranded R-loop structure in which the guide RNA hybridizes to the DNA target. Here we use single-molecule torque spectroscopy to investigate the dynamics and mechanics of R-loop formation of two widely used Cas12a orthologs at base-pair resolution. We directly observe kinetic intermediates corresponding to a ~5 bp initial RNA-DNA hybridization and a ~17 bp intermediate preceding R-loop completion, followed by transient DNA unwinding that extends beyond the 20 bp R-loop. The complex multistate landscape of R-loop formation is ortholog-dependent and shaped by target sequence, mismatches, and DNA supercoiling. A four-state kinetic model captures essential features of Cas12a R-loop dynamics and provides a biophysical framework for understanding Cas12a activity and specificity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Genomic reconstruction of fossil and living microorganisms in ancient Siberian permafrost

Abstract Background Total DNA (intracellular, iDNA and extracellular, eDNA) from ancient permafrost records the mixed genetic repository of the past and present microbial populations through geological time. Given the exceptional preservation of eDNA under perennial frozen conditions, typical metagenomic sequencing of total DNA precludes the discrimination between fossil and living microorganisms in ancient cryogenic environments. DNA repair protocols were combined with high throughput sequencing (HTS) of separate iDNA and eDNA fraction to reconstruct metagenome-assembled genomes (MAGs) from ancient microbial DNA entrapped in Siberian coastal permafrost. Results Despite the severe DNA damage in ancient permafrost, the coupling of DNA repair and HTS resulted in a total of 52 MAGs from sediments across a chronosequence (26–120 kyr). These MAGs were compared with those derived from the same samples but without utilizing DNA repair protocols. The MAGs from the youngest stratum showed minimal DNA damage and thus likely originated from viable, active microbial species. Many MAGs from the older and deeper sediment appear related to past aerobic microbial populations that had died upon freezing. MAGs from anaerobic lineages, including Asgard archaea, however exhibited minimal DNA damage and likely represent extant living microorganisms that have become adapted to the cryogenic and anoxic environments. The integration of aspartic acid racemization modeling and metaproteomics further constrained the metabolic status of the living microbial populations. Collectively, combining DNA repair protocols with HTS unveiled the adaptive strategies of microbes to long-term survivability in ancient permafrost. Conclusions Our results indicated that coupling of DNA repair protocols with simultaneous sequencing of iDNA and eDNA fractions enabled the assembly of MAGs from past and living microorganisms in ancient permafrost. The genomic reconstruction from the past and extant microbial populations expanded our understanding about the microbial successions and biogeochemical alterations from the past paleoenvironment to the present-day frozen state. Furthermore, we provided genomic insights into long-term survival mechanisms of microorganisms under cryogenic conditions through geological time. The combined strategies in this study can be extrapolated to examine other ancient non-permafrost environments and constrain the search for past and extant extraterrestrial life in permafrost and ice deposits on Mars.

54 ENVIRONMENTAL SCIENCES↗
NASA NTRS2000

A comparison of RNA with DNA in template-directed synthesis

Nonenzymatic template-directed copying of RNA sequences rich in cytidylic acid using nucleoside 5'-(2-methylimidazol-1-yl phosphates) as substrates is substantially more efficient than the copying of corresponding DNA sequences. However, many sequences cannot be copied, and the prospect of replication in this system is remote, even for RNA. Surprisingly, wobble-pairing leads to much more efficient incorporation of G opposite U on RNA templates than of G opposite T on DNA templates.

Non-NASA Center↗
NASA NTRS1997

Sequence information signal processor for local and global string comparisons

A sequence information signal processing integrated circuit chip designed to perform high speed calculation of a dynamic programming algorithm based upon the algorithm defined by Waterman and Smith. The signal processing chip of the present invention is designed to be a building block of a linear systolic array, the performance of which can be increased by connecting additional sequence information signal processing chips to the array. The chip provides a high speed, low cost linear array processor that can locate highly similar global sequences or segments thereof such as contiguous subsequences from two different DNA or protein sequences. The chip is implemented in a preferred embodiment using CMOS VLSI technology to provide the equivalent of about 400,000 transistors or 100,000 gates. Each chip provides 16 processing elements, and is designed to provide 16 bit, two's compliment operation for maximum score precision of between -32,768 and +32,767. It is designed to provide a comparison between sequences as long as 4,194,304 elements without external software and between sequences of unlimited numbers of elements with the aid of external software. Each sequence can be assigned different deletion and insertion weight functions. Each processor is provided with a similarity measure device which is independently variable. Thus, each processor can contribute to maximum value score calculation using a different similarity measure.

Peterson, John C.↗
NASA NTRS2004

Microbial Characterization During the Early Habitation of the International Space Station

An evaluation of the microbiota from air, water, and surface samples provided a baseline of microbial characterization onboard the International Space Station (ISS) to gain insight into bacterial and fungal contamination during the initial stages of construction and habitation. Using 16S genetic sequencing and rep-PCR, 63 bacterial strains were isolated for identification and fingerprinted for microbial tracking. Of the bacterial strains that were isolated and fingerprinted, 19 displayed similarity to each other. The use of these molecular tools allowed for the identification of bacteria not previously identified using automated biochemical analysis and provided a clear indication of the source of several ISS contaminants. Strains of Bradyrhizobium and Sphingomonas unable to be identified using sequencing were identified by comparison of rep-PCR DNA fingerprints. Distinct DNA fingerprints for several strains of Methylobacterium provided a clear indication of the source of an ISS water supply contaminant. Fungal and bacterial data acquired during monitoring do not suggest there is a current microbial hazard to the spacecraft, nor does any trend indicate a potential health risk. Previous spacecraft environmental analysis indicated that microbial contamination will increase with time and will require continued surveillance. Copyright 2004 Springer-Verlag.

Fungi/genetics↗
DOE OSTI2020

Reducing host DNA contamination in 16S rRNA gene surveys of anthozoan microbiomes using PNA clamps

Efforts to study the microbial communities associated with corals can be limited by inefficiencies in the sequencing process due to high levels of host amplification by universal bacterial 16S rRNA gene primers. Here, we develop an inexpensive peptide nucleic acid (PNA) clamp that binds to a target sequence of host DNA during PCR and blocks amplification. We then test the ability of this PNA clamp to mitigate host contamination and increase overall microbial sequence coverage on samples from three coral species: the gorgonians Eunicea flexuosa and Gorgonia ventalina, and the scleractinian Porites panamensis. The 20-bp PNA clamp was designed using DNA from E. flexuosa. Adding the PNA clamp during PCR increased the percentage of microbial reads in E. flexuosa samples more than 11-fold. Microbial community diversity was similar without- and with-PNA clamps, as were the relative frequencies of the ten most abundant ASVs (amplicon sequence variants), indicating that the clamps successfully blocked host DNA amplification while simultaneously increasing microbial DNA amplification proportionally across the most abundant taxa. The reduction of E. flexuosa DNA correlated with an increase in the abundance of rarer taxa. The clamp also increased the average percentage of microbial reads in another gorgonian, G. ventalina, by 8.6-fold without altering the microbial community beta diversity, and in a distantly related scleractinian coral, P. panamensis, by nearly double. The reduction of host contamination correlated with the number of nucleotide mismatches between the host amplicon and the PNA clamp. The PNA clamp costs as little as $0.48 per sample, making it an efficient and cost-effective solution to increase microbial sequence coverage for high-throughput sequencing of coral microbial communities.

16S rRNA gene sequencing↗
NASA NTRS2003

Spirochaeta americana sp. nov., a new haloalkaliphilic, obligately anaerobic spirochaete isolated from soda Mono Lake in California

A novel, obligately anaerobic, mesophilic, haloalkaliphilic spirochaete, strain ASpG1(T), was isolated from sediments of the alkaline, hypersaline Mono Lake in California, USA. Cells of the Gram-negative strain were motile and spirochaete-shaped with sizes of 0.2-0.22 x 8-18 microm. Growth of the strain was observed between 10 and 44 degrees C (optimum 37 degrees C), in 2-12% (w/v) NaCl (optimum 3% NaCl) and between pH 8 and 10.5 (optimum pH 9.5). The novel strain was strictly alkaliphilic, required high concentrations of carbonates in the medium and was capable of utilizing D-glucose, fructose, maltose, sucrose, starch and D-mannitol. End products of glucose fermentation were H2, acetate, ethanol and formate. Strain ASpG(T) was resistant to kanamycin and rifampicin, but sensitive to gentamicin, tetracycline and chloramphenicol. The G + C content of its DNA was 58.5 mol%. DNA-DNA hybridization analysis of strain ASpG1(T) with its most closely related species, Spirochaeta alkalica Z-7491(T), revealed a hybridization value of only 48.7%. On the basis of its physiological and molecular properties, strain ASpG1(T) appears to represent a novel species of the genus Spirochaeta, for which the name Spirochaeta americana is proposed (type strain ASpG1(T) =ATCC BAA-392(T) = DSM 14872(T)).

Sodium Chloride/metabolism↗
NASA NTRS1991

Phylogenetic diversity in the genus Bacillus as seen by 16S rRNA sequencing studies

Comparative sequence analysis of 16S ribosomal (r)RNAs or DNAs of Bacillus alvei, B. laterosporus, B. macerans, B. macquariensis, B. polymyxa and B. stearothermophilus revealed the phylogenetic diversity of the genus Bacillus. Based on the presently available data set of 16S rRNA sequences from bacilli and relatives at least four major "Bacillus clusters" can be defined: a "Bacillus subtilis cluster" including B. stearothermophilus, a "B. brevis cluster" including B. laterosporus, a "B. alvei cluster" including B. macerans, B. maquariensis and B. polymyxa and a "B. cycloheptanicus branch".

NASA Discipline Number 52-30↗
DOE OSTI2021

VirION2: a short- and long-read sequencing and informatics workflow to study the genomic diversity of viruses in nature

Microbes play fundamental roles in shaping natural ecosystem properties and functions, but do so under constraints imposed by their viral predators. However, studying viruses in nature can be challenging due to low biomass and the lack of universal gene markers. Though metagenomic short-read sequencing has greatly improved our virus ecology toolkit—and revealed many critical ecosystem roles for viruses—microdiverse populations and fine-scale genomic traits are missed. Some of these microdiverse populations are abundant and the missed regions may be of interest for identifying selection pressures that underpin evolutionary constraints associated with hosts and environments. Though long-read sequencing promises complete virus genomes on single reads, it currently suffers from high DNA requirements and sequencing errors that limit accurate gene prediction. Here we introduce VirION2, an integrated short- and long-read metagenomic wet-lab and informatics pipeline that updates our previous method (VirION) to further enhance the utility of long-read viral metagenomics. Using a viral mock community, we first optimized laboratory protocols (polymerase choice, DNA shearing size, PCR cycling) to enable 76% longer reads (now median length of 6,965 bp) from 100-fold less input DNA (now 1 nanogram). Using a virome from a natural seawater sample, we compared viromes generated with VirION2 against other library preparation options (unamplified, original VirION, and short-read), and optimized downstream informatics for improved long-read error correction and assembly. VirION2 assemblies combined with short-read based data (‘enhanced’ viromes), provided significant improvements over VirION libraries in the recovery of longer and more complete viral genomes, and our optimized error-correction strategy using long- and short-read data achieved 99.97% accuracy. In the seawater virome, VirION2 assemblies captured 5,161 viral populations (including all of the virus populations observed in the other assemblies), 30% of which were uniquely assembled through inclusion of long-reads, and 22% of the top 10% most abundant virus populations derived from assembly of long-reads. Viral populations unique to VirION2 assemblies had significantly higher microdiversity means, which may explain why short-read virome approaches failed to capture them. These findings suggest the VirION2 sample prep and workflow can help researchers better investigate the virosphere, even from challenging low-biomass samples. Our new protocols are available to the research community on protocols.io as a ‘living document’ to facilitate dissemination of updates to keep pace with the rapid evolution of long-read sequencing technology.

Long-reads↗
DOE OSTI2024

NovaDemux v39.07

This program is a sequence demultiplexer intended primarily for, but not limited to, Illumina sequencing machines. Typically, multiple experiments ("libraries") are pooled together and sequenced at once, with genetic molecules of these libraries tagged with a synthetic DNA "barcode". After sequencing, the data is demultiplexed into one file per library based on the barcode. However, errors in barcode reading cause misassignment and decrease yield. NovaDemux uses advanced statistical methods to maximize yield while minimizing misassignment compared to existing software.

Bushnell, Brian [Lawrence Berkeley National Labora↗
DOE OSTI2022

High-Resolution Structure of the Nuclease Domain of the Human Parvovirus B19 Main Replication Protein NS1

Two new structures of the N-terminal domain of the main replication protein, NS1, of human parvovirus B19 (B19V) are presented here. This domain (NS1-nuc) plays an important role in the “rolling hairpin” replication of the single-stranded B19V DNA genome, recognizing origin of replication sequences in double-stranded DNA, and cleaving (i.e., nicking) single-stranded DNA at a nearby site known as the terminal resolution site (trs). The three-dimensional structure of NS1-nuc is well conserved between the two forms, as well as with a previously solved structure of a sequence variant of the same domain; however, it is shown here at a significantly higher resolution (2.4 Å). Using structures of NS1-nuc homologues bound to single- and double-stranded DNA, models for DNA recognition and nicking by B19V NS1-nuc are presented that predict residues important for DNA cleavage and for sequence-specific recognition at the viral origin of replication.

59 BASIC BIOLOGICAL SCIENCES↗