Results for “Extraction buffer”

DOE OSTI2024

Exploring viral particle, soil, and extraction buffer physicochemical characteristics and their impacts on extractable viral communities

Viruses are expected to be pivotal members of soil ecosystems, and recent advances in viral size fraction metagenomic (viromic) approaches have improved our ability to interrogate soil viral ecology. However, viromics relies on extraction buffers to effectively desorb viral particles from the soil matrix for downstream analysis, and viral extraction efficiency could be affected by the interplay between viral particles, soils, and extraction buffer chemistry. Here, we investigated whether extraction buffer chemistry affected extractable viral community composition measured by viromics from different soil types, for both biological (samples collected 1 m apart) and technical (subsamples from the same soil homogenate) replicates. We investigated pH manipulation of protein-supplemented phosphate-buffered saline buffer (PPBS, pHs 4.5, 5.5, 6.5, and 7.5) on forest, grassland and wetland soils that exhibited different soil edaphic properties, and we tested different buffer chemistries (PPBS, Carbonated Buffer, Glycine, and Saline Magnesium) on just the wetland soil. Spatial distance (i.e., biological replicate) was the primary driver of extractable viral community composition across all buffers and soils tested. Differences in viral community composition according to extraction buffer properties were only observed in the grassland technical replicates at PPBS buffer pH 4.5, and in both the wetland technical and biological replicates treated with different buffer chemistries, but the effects of buffer chemistry were secondary to spatial distance in the biological replicates. The lack of buffering capacity in the grassland soil technical replicates likely increased sorption of viral particles at pH 4.5, but neither protein composition nor isoelectric point explained this phenomenon. In conclusion, given that most soil viral ecological studies to date include sample collection over distances much farther apart than the 1-m distances considered here, results suggest that extraction buffer chemistry is likely of much lower importance than ecological considerations, such as spatial distance, in the design of future soil viral ecological investigations.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1986

[3H]Indole-3-acetyl-myo-inositol hydrolysis by extracts of Zea mays L. vegetative tissue

[3H]Indole-3-acetyl-myo-inositol was hydrolyzed by buffered extracts of acetone powders prepared from 4 day shoots of dark grown Zea mays L. seedlings. The hydrolytic activity was proportional to the amount of extract added and was linear for up to 6 hours at 37 degrees C. Boiled or alcohol denatured extracts were inactive. Analysis of reaction mixtures by high performance liquid chromatography demonstrated that not all isomers of indole-3-acetyl-myo-inositol were hydrolyzed at the same rate. Buffered extracts of acetone powders were prepared from coleoptiles and mesocotyls. The rates of hydrolysis observed with coleoptile extracts were greater than those observed with mesocotyl extracts. Active extracts also catalyzed the hydrolysis of esterase substrates such as alpha-naphthyl acetate and the methyl esters of indoleacetic acid and naphthyleneacetic acid. Attempts to purify the indole-3-acetyl-myo-inositol hydrolyzing activity by chromatographic procedures resulted in only slight purification with large losses of activity. Chromatography over hydroxylapatite allowed separation of two enzymically active fractions, one of which catalyzed the hydrolysis of both indole-3-acetyl-myo-inositol and esterase substrates. With the other enzymic hydrolysis of esterase substrates was readily demonstrated, but no hydrolysis of indole-3-acetyl-myo-inositol was ever detected.

NASA Program Space Biology↗

DOE OSTI2020

Sensitive Top-Down Proteomics Analysis of a Low Number of Mammalian Cells Using a Nanodroplet Sample Processing Platform

Top-down proteomics is a powerful tool for characterizing genetic variations and post-translational modifications at intact protein level. That being said, one significant technical gap of top-down proteomics is the inability to analyze low amount of biological samples, which limits its access to isolated rare cells, fine-needle aspiration biopsies, and tissue substructures. Herein, we developed an ultrasensitive top-down platform by incorporating a microfluidic sample preparation system, termed nanoPOTS (Nanodroplet Processing in One pot for Trace Samples), into a top-down proteomics workflow. A unique combination of a nonionic detergent dodecyl-ß-D-maltopyranoside (DDM) with urea as protein extraction buffer significantly improved both protein extraction efficiency and sample recovery. We hypothesize that the DDM detergent improves protein recovery by efficiently reducing non-specific adsorption of intact proteins on container surfaces, while urea serves as a strong denaturant to disrupt noncovalent complexes and release intact proteins for downstream analysis. The nanoPOTS-based top-down platform reproducibly and quantitatively identified ~170 to ~620 proteoforms from ~70 to ~770 HeLa cells containing ~10 to ~115 ng of total protein. A variety of post-translational modifications including acetylation, myristoylation, and iron binding were identified using only less than 800 cells. We anticipate the nanoPOTS top-down proteomics platform will be broadly applicable in biomedical researches, particularly where clinical specimens are not available in amounts amenable to standard workflows.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2025

Development of a high-throughput method for processing sponge-stick samples to detect viable Bacillus anthracis spores

Since the national validation of the sponge-stick based method for detection of Bacillus anthracis spores in environmental samples, there have not been focused efforts to address the low throughput nature of the method, which processes only one sample at one time. Sample processing remains a serious bottleneck for rapidly analyzing large numbers of samples expected from a biological warfare attack. Therefore, we developed a high-throughput method to simultaneously process multiple sponge-stick samples to be better prepared for rapid response and recovery after wide area anthrax incidents. In this method, sponges are placed in 50 mL tubes containing 25 mL extraction buffer and shaken to release spores, after which the suspension is recovered for analysis. Here, we determined that an additional extraction step, conducted in the same tubes with 10 mL buffer, further increased spore recovery from sponge-stick by approximately 10 %. We determined that orbital shaking and multi-tube vortexing were both more effective than reciprocating shaking for recovering spores. We conducted simultaneous processing of up to 12 sponge-stick samples and demonstrated comparable spore recovery efficiencies to the traditional low-throughput stomacher-based method (approximately 60 % recovery at 10 2 -spore level and 75 % recovery at 10 4 -spore level for both methods in three replicate experiments, P > 0.05 for two-tailed t-tests for each experiment and spore level). We also demonstrated that our high-throughput method could be integrated with Rapid Viability-Polymerase Chain Reaction (RV-PCR) analysis and could detect levels as low as 40 spores per sponge even when challenged by a PCR particulate contaminant.

Anthrax↗

DOE OSTI2022

Evaluation of Protein Extraction Methods for Metaproteomic Analyses of Root-Associated Microbes

Metaproteomics is a powerful tool for the characterization of metabolism, physiology, and functional interactions in microbial communities, including plant-associated microbiota. However, the metaproteomic methods that have been used to study plant-associated microbiota are very laborious and require large amounts of plant tissue, hindering wider application of these methods. We optimized and evaluated different protein extraction methods for metaproteomics of plant-associated microbiota in two different plant species (Arabidopsis and maize). Our main goal was to identify a method that would work with low amounts of input material (40 to 70 mg) and that would maximize the number of identified microbial proteins. We tested eight protocols, each comprising a different combination of physical lysis method, extraction buffer, and cell-enrichment method on roots from plants grown with synthetic microbial communities. We assessed the performance of the extraction protocols by liquid chromatography-tandem mass spectrometry–based metaproteomics and found that the optimal extraction method differed between the two species. For Arabidopsis roots, protein extraction by beating whole roots with small beads provided the greatest number of identified microbial proteins and improved the identification of proteins from gram-positive bacteria. For maize, vortexing root pieces in the presence of large glass beads yielded the greatest number of microbial proteins identified. Based on these data, we recommend the use of these two methods for metaproteomics with Arabidopsis and maize. Furthermore, detailed descriptions of the eight tested protocols will enable future optimization of protein extraction for metaproteomics in other dicot and monocot plants.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Assessing the Extractability of Rare Earth Elements from Coal Preparation Fines Refuse Using an Organic Acid Lixiviant

We report that this work is a first-order study to assess the suitability of an organic acid lixiviant to extract rare earth elements (REE) from coal preparation fines refuse sourced from a Pennsylvania mine with a total REE of ~ 300 ppm. The extraction of REE using an organic acid, in this case 0.1 M citric acid and 0.5 M trisodium citrate solution, is compared against 0.5 M (NH 4 ) 2 SO 4 , 1 M HCl, 1.2 M H 2 SO 4 , and 0.5 M ethylenediaminetetraacetic acid (EDTA). Ashing the coal waste material prior to leaching tests with the citrate solution nominally improved REE extraction. Buffered citrate solution recovered 7% of the total REE in the as-received Isabella Fines as compared to 11% in ashed samples, whereas (NH 4 ) 2 SO 4 extracted 5–6%, respectively. EDTA recovered up to 33% of the total REE, most likely due to the higher coordination chelate bond. Mineral acids, however, outperformed the organic acids on ashed material (16–52% REE recovery), suggesting that organic acids may not be a suitable competitive option for REE extraction from these types of feedstocks.

01 COAL, LIGNITE, AND PEAT↗

NASA NTRS2020

Improving Quantitative Laboratory Analysis of Phycobiliproteins to Provide High Quality Validation Data for Ocean Color Remote Sensing Algorithm

Identification and characterization of phytoplankton communities and their physiology is a primary aim of NASA's PACE satellite mission. The concentration and composition of phytoplankton pigments modulate the spectral distribution of light emanating from the ocean, which is measured by ocean color satellites, and thus provide critical information on phytoplankton community composition and physiological parameters. One diagnostic class of pigments not routinely well-characterized is the phycobiliproteins (PBPs), and NASA has a requirement to collect and distribute high quality in situ data in support of data product validation activities for ocean color missions. Phycobiliproteins are light-harvesting proteins that are the predominant photosynthetic pigments in some classes of phytoplankton including cyanobacteria, such as Synechococcus, Trichodesmium, and Microcystis. With the advance of hyperspectral ocean color sensors such as on PACE (expected to launch in late 2022), it is essential that we implement routine analysis of PBPs that satisfies several considerations: reproducible, high extraction efficiency for a variety of environments, and Suitable for large scale analysis. Published techniques for PBP analysis vary in recommendations for: collection, extraction, disruption mode, and analysis; evidence suggests the variation in results may depend at least in part on the species and even strain(s) of interest. Experiments that tested variations in these parameters have drawn very different conclusions regarding extraction efficiency and reproducibility. Cyanobacteria are more difficult to extract than other PBP-containing algae such as cryptophytes, but can be important primary producers. We used a cryptophyte (Rhodomonas salina) and cyanobacterium (Synechococcus sp.) to compare extraction efficiencies of water samples concentrated via centrifugation to filtered samples using two different extraction buffers (phosphate and asolectin-CHAPS). Samples were analyzed on a fluorometer configured for PC and PE detection. The results have important implications for collection and storage of samples for routine analysis; some previous studies (although not all) have suggested that filtered samples have a much lower extraction efficiency than whole water samples.

Kenemer, Christopher↗

DOE OSTI2023

Spatially Resolved Top-Down Proteomics of Tissue Sections Based on a Microfluidic Nanodroplet Sample Preparation Platform

Conventional proteomics measures the averaged signal from mixed cell populations or bulk tissues, leading to the dilution of significant changes in subpopulations of cells that might serve as important biomarkers. Recent developments in bottom-up proteomics have enabled spatial mapping of cellular heterogeneity in tissue microenvironments. However, bottom-up proteomics cannot precisely infer the abundance changes of intact proteins, which are presented as proteoforms. Herein, we described a spatially resolved top-down proteomics (TDP) platform for proteoform identification and quantification directly on thin tissue sections. The spatial TDP platform consisted of a nanoPOTS (nanodroplet Processing in One pot for Trace Samples)-based sample preparation system and an LCM (laser capture microdissection)-based cell isolation system. We improved the nanoPOTS sample preparation by adding benzonase in the extraction buffer to enhance the coverage of nucleus proteins. Using ~200 cultured cells as model samples, the improved approach increased proteoform identifications from 493 to 700; newly identified proteoforms primarily corresponded to nuclear proteins. To demonstrate the spatial TDP platform in tissue samples, we analyzed LCM-isolated tissue voxels from rat brain cortex and hypothalamus regions. We quantified 426 proteoforms by combining identifications from TopPIC and TDPortal with the quantitation from ProMex. Several proteoforms corresponding to the same gene exhibited mixed abundance profiles between two tissue regions, suggesting potential PTM-specific spatial distributions. The spatial TDP workflow has prospects for biomarker discovery at proteoform level from small tissue sections.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

High Throughput Genome Releaser

In this study, we present the development of a High Throughput Genome Releaser, an innovative device addressing common challenges in screening PCR. This genome DNA releaser is designed for rapid, cost-effective, and efficient DNA extraction, optimized for subsequent PCR reactions. Our experimentation with various synthetic materials led us to select a particular type of plastic that mirrors the properties of glass cover slides, providing a smooth surface and effective compression capabilities. We engineered a 96-well device equipped with a 96-well plate and a top rod, operable both manually and automatically, which is compatible with widely used liquid-handling robot decks. This compatibility enhances ease of use in high-throughput PCR setups. Additionally, we developed software to support its automatic functions. The genome releaser facilitates the extraction of PCR-amplifiable genomic DNA from 96 samples within minutes, eliminates the need for extraction buffers, and is adaptable to a wide range of microorganisms and cells. This versatility could significantly advance biomanufacturing processes.

42 ENGINEERING↗

DOE OSTI2025

An Innovative High Throughput Genome Releaser for Rapid and Efficient PCR Screening

High-throughput PCR screening is vital in synthetic biology and metabolic engineering as it allows researchers to rapidly analyze and detect numerous targeted genetic mutation in the genome. Current challenges for high-throughput PCR screening in synthetic biology include efficiently preparing genomic DNA, optimizing protocols for diverse sample types, managing contamination risks, and effectively analyzing the large volumes of data generated while ensuring consistent and accurate results. In this study, we present the development of a High Throughput Genome Releaser (HTGR), an innovative device addressing common challenges in screening PCR. This genome DNA releaser is designed based on a squash method for rapid, cost-effective, and efficient DNA release, optimized for subsequent PCR reactions. After experimenting with various synthetic materials, we selected a plastic that closely replicates the smooth surface and compression properties of microscope slides, ensuring reliable performance. We engineered a device featuring a 96-Well Plate and a shear applicator, operable both manually and automatically, and compatible with standard liquid-handling robot platform. This compatibility enhances ease of use in high-throughput PCR workflows. Additionally, we developed software to support its automatic functions. Our results demonstrated that the specially engineered 96-Well Plate and HTGR can effectively squash fungal spores , which release enough genome DNA for PCR screening. The genome releaser facilitates the preparation of PCR-amplifiable genomic DNA substrate from 96 samples within minutes, eliminates the need for extraction buffers, and is adaptable to a wide range of microorganisms and cells, which could significantly advance biomanufacturing processes.

Yuan, Guoliang [BATTELLE (PACIFIC NW LAB)]↗

NASA NTRS1984

The atmosphere of Mars - Resources for the exploration and settlement of Mars

This paper describes methods of processing the Mars atmosphere to supply water, oxygen and buffer gas for a Mars base. Existing life support system technology is combined with innovative methods of water extraction, and buffer gas processing. The design may also be extended to incorporate an integrated greenhouse to supply food, oxygen and water recycling. It is found that the work required to supply one kilogram of an argon/nitrogen buffer gas is 9.4 kW-hr. To extract water from the dry Martian atmosphere can require up to 102.8 kW-hr per kilogram of water depending on the relative humidity of the air.

Meyer, T. R.↗

DOE OSTI2022

Project 18-15263 X-ray Studies of Interfacial Molecular Complexes in ALSEP Back-Extraction (Research Performance Final Report)

The objective of the project was to use synchrotron X-rays to measure the molecular-scale organization of extractants, complexants, buffers, and metal ions at the organic-aqueous (liquid-liquid) interface during solvent back-extraction under conditions relevant to ALSEP (Actinide-Lanthanide Separation Process). The capability of X-ray fluorescence near total reflection (XFNTR) was extended to explore the distribution of Eu(III) between the liquid-liquid interface and bulk organic and aqueous phases which contained extractants HDEHP (bis(2-ethylhexyl) phosphoric acid) and HEHEHP (2-ethylhexyl phosphonic acid mono-2-ethylhexyl ester), the complexant DTPA (diethylene triamine pentaacetic acid), and citric and nitric acids. Although an Am-compatible sample cell was developed, extending x-ray measurements of flat liquid-liquid interfaces to the actinide Am(III) did not prove to be possible within constraints imposed by the Advanced Photon Source. However, preliminary measurements with a drop cell may eventually provide a path forward to investigate Am(III) at a liquid interface. It was also demonstrated that competitive binding that occurs during back-extraction of lanthanide ions to either a phosphoric acid extractant (DHDP, di-hexadecyl phosphate) at the liquid interface or to complexant DTPA in the bulk aqueous phase could be reversibly tuned by X-ray exposure. Separately, X-ray measurements from the liquid-vapor interface explored the role of the aqueous solubility of HDEHP and HEHEHP and their interactions with a range of lanthanides and transition metal ions. Finally, the capability of X-ray absorption spectroscopy (EXAFS) was extended to probe metal ion coordination in lanthanide ion-extractant complexes at the liquid-vapor interface. Complementary to the specific interfacial systems studied during this project, the development of x-ray techniques expands our ability to understand ion ordering at liquid interfaces.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗

DOE OSTI2021

Rapid extraction of short-lived isotopes from a buffer gas cell for use in gas-phase chemistry experiments, Part II: On-line studies with short-lived accelerator-produced radionuclides

We report a novel combination of advanced gas-chromatography and detection systems coupled to a buffer-gas cell was characterized on-line to allow gas-phase chemical studies of accelerator-produced short-lived α -decaying mercury, francium, and astatine isotopes. These were produced in 40 Ar- and 48 Ca-induced nuclear fusion–evaporation reactions, subsequently isolated in the recoil separators MARS at Texas A&M University, USA, and TASCA at GSI Darmstadt, Germany, before being thermalized in a buffer-gas-stopping cell. From the latter, the nuclear reaction products were extracted into gas-phase chromatographic systems, suitable for registering α-decaying short-lived radionuclides, such as isotopes of superheavy elements. Efficiencies of 21(3)% for 204-209 Fr were reached for the extraction into the optimized miniCOMPACT gas-chromatography setup, indicating that this technique enables the identification of isotopes of volatile as well as non-volatile elements. These studies guide the path towards chemical investigations of superheavy elements beyond flerovium, which are out of reach with currently used setups.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

DOE OSTI2020

Development of a High-Rate Front-End ASIC for X-Ray Spectroscopy and Diffraction Applications

We developed a new front-end application-specific integrated circuit (ASIC) to upgrade the Maia X-ray microprobe. The ASIC instruments 32 configurable channels that perform either positive or negative charge amplification, pulse shaping, peak amplitude, and time extraction along with buffered analog storage. At a gain of 3.6 V/fC, 1-μs peaking time, and a temperature of 248 K, an electronic resolution of 13 and 10 e- rms was measured with and without a silicon drift detector (SDD) sensor, respectively. A spectral resolution of 170-eV full-width at half-maximum (FWHM) at 5.9 keV was obtained with an 55Fe source. The channel linearity was better than ± 1% with rate capabilities up to 40 kcps. The ASIC was fabricated in a commercial 250-nm process with a footprint of 6.3 mm × 3.9 mm and dissipates 167 mW of static power.

Vernon, Emerson↗

DOE OSTI2026

Separation of radium and actinium in acetic acid/acetate solutions using TK101 resin

Here, the uptake and elution behavior of Ra and Ac was studied on TK101 resin using acetic acid and acetate buffer (acetic acid/lithium acetate) solutions. There is high extraction of Ra (>10 3 ) over a wide range of acetic acid concentrations (0.02 to 4 M) with slightly lower extraction (>10 2 ) in the acetate buffer solutions (pH 3.9 to 5.6). The Ac extraction is considerably lower with a maximum D w of ∼100, and negligible extraction at high acetic acid concentrations (> 1 M) and in the acetate buffer solutions. The difference in D w can be leveraged for highly efficient separations of Ac from Ra, with no detectable Ra in the Ac fractions and high Ac yields (∼100%). These separations may be useful for radiopharmaceutical applications as the Ac purity is extremely high and the elution conditions can be optimized to be biologically safe and appropriate for chelation to molecules relevant to targeted alpha therapy.

and nuclear chemistry↗

DOE OSTI2024

KBase Narrative - Porphyromonadaceae sp. W3.11 genome

Narratives for The phenotype and genotype of fermentative prokaryotes This is the Narrative for Porphyromonadaceae sp. W3.11. A complementary Narrative for Lachnospiraceae sp. C1.1 is available here. This is the Narrative for Lachnospiraceae sp. C1.1. A complementary Narrative for Porphyromonadaceae sp. W3.11 is available here. Background and Isolation This Narrative and its complementary Narrative contain assembly and annotation of two bacterial isolates that were isolated by our laboratory from the rumen of a Holstein heifer. All procedures with animals have been approved by University of California Davis’s Institutional Animal Care and Use Committee. Rumen contents were collected through a rumen fistula and strained through two layers of cheesecloth into a bottle. The bottle was sealed to exclude air and maintained at 39°C. Contents were brought to the laboratory and bubbled under O2-free CO2 within 15 min. At the laboratory, serial dilutions were made with anaerobic dilution solution for Lachnospiraceae sp. C1.1 and propionibacterium diluent for Porphyromonadaceae sp. W3.11 (table S2). Aliquots (0.1 ml) of each dilution were injected into anaerobic bottle plates (1) containing 9 ml of LH medium (table S2). After incubation at 37°C for 7 days, isolated colonies were picked. Lachnospiraceae sp. C1.1 was picked from a bottle inoculated with a 104 dilution of rumen contents, and Porphyromonadaceae sp. W3.11 was picked from a bottle inoculated with a 103 dilution. After initial isolation, these organisms were purified by growing on anaerobic roll tubes (2) and picking isolated colonies. We performed de novo sequencing of Lachnospiraceae sp. C1.1 and Porphyromonadaceae sp. W3.11. Aliquots of liquid culture (9 and 1.5 ml, respectively) were collected by syringe and centrifuged (21,000g for 10 min at 4°C). Cell pellets were submitted to Molecular Research LP for DNA extraction, library preparation, and sequencing. After resuspending pellets in 180 µl of ATL buffer (Qiagen), DNA was extracted using the MagAttract HMW DNA Kit (Qiagen). DNA was eluted in 100 µl of AE buffer (Qiagen) and then cleaned using the DNEasy PowerClean Pro Cleanup Kit (Qiagen). DNA was then sheared using the Covaris g-TUBE (Covaris). Sequencing libraries were prepared using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences) and 1500 ng of the sheared and purified DNA. The SMRTbell libraries were size-selected (>6 Kb) using a BluePippin instrument (Sage Science) and 0.75% agarose gel. Libraries were then sequenced using the PacBio Sequel II (Pacific Biosciences) platform and a 30-hour movie time. Narrative Summary In these Narratives, we filtered low-quality reads using Trimmomatic (v0.36), assembled filtered reads with SPAdes (v3.15.3), and then checked completeness and contamination of the assembled genomes with CheckM (v1.0.18). Statistics for sequencing and assembly are in table S3. Using the assembled contigs (genomes), we called genes and annotated them. Protein-coding genes were called using Prodigal (v2.6.3) (3) locally or using KBase via RASTtk (v1.073), with identical results. Genes were annotated with KO IDs using KAAS (4). They were further annotated with pfam and TIGRFAM IDs using KBase and the Annotate Domains in a Genome app. We classified putative genes for hydrogenases using HydDB. Genes for 16S ribosomal RNA (rRNA) were called using RASTtk (v1.073) in KBase. The contigs (genomes) were analyzed to determine whether they belonged to new species. Taxonomy was assigned using GTDB-Tk (v1.7.0) in KBase. The identity of 16S rRNA genes to other organisms was found using EzBioCloud (5). Values of digital DNA-DNA hybridization (dDDH) were found with Type (Strain) Genome Server (6). These analyses suggest that Lachnospiraceae sp. C1.1 and Porphyromonadaceae sp. W3.11 represent novel species or genera. GTDB-Tk assigned Lachnospiracae sp. C1.1 to family Lachnospiraceae and genus NK4A144, which contains no type strains. It assigned Porphyromonadaceae sp. W3.11 to Porphyromonadaceae and genus Porphyromonas_A. Values of 16S rRNA identity and dDDH with respect to type strains were low (table S4). Although more phenotypic data are needed, available evidence supports assignment of genomes to new species or genera. Related publication Hackmann TJ, Zhang B. The phenotype and genotype of fermentative prokaryotes. Sci Adv. 2023 Sep 29;9(39):eadg8687. doi: 10.1126/sciadv.adg8687. Epub 2023 Sep 27. PMID: 37756392; PMCID: PMC10530074.

Hackmann, Timothy↗

DOE OSTI2024

KBase Narrative - Lachnospiraceae sp. C1.1 genome

Narratives for The phenotype and genotype of fermentative prokaryotes This is the Narrative for Porphyromonadaceae sp. W3.11. A complementary Narrative for Lachnospiraceae sp. C1.1 is available here. This is the Narrative for Lachnospiraceae sp. C1.1. A complementary Narrative for Porphyromonadaceae sp. W3.11 is available here. Background and Isolation This Narrative and its complementary Narrative contain assembly and annotation of two bacterial isolates that were isolated by our laboratory from the rumen of a Holstein heifer. All procedures with animals have been approved by University of California Davis’s Institutional Animal Care and Use Committee. Rumen contents were collected through a rumen fistula and strained through two layers of cheesecloth into a bottle. The bottle was sealed to exclude air and maintained at 39°C. Contents were brought to the laboratory and bubbled under O2-free CO2 within 15 min. At the laboratory, serial dilutions were made with anaerobic dilution solution for Lachnospiraceae sp. C1.1 and propionibacterium diluent for Porphyromonadaceae sp. W3.11 (table S2). Aliquots (0.1 ml) of each dilution were injected into anaerobic bottle plates (1) containing 9 ml of LH medium (table S2). After incubation at 37°C for 7 days, isolated colonies were picked. Lachnospiraceae sp. C1.1 was picked from a bottle inoculated with a 104 dilution of rumen contents, and Porphyromonadaceae sp. W3.11 was picked from a bottle inoculated with a 103 dilution. After initial isolation, these organisms were purified by growing on anaerobic roll tubes (2) and picking isolated colonies. We performed de novo sequencing of Lachnospiraceae sp. C1.1 and Porphyromonadaceae sp. W3.11. Aliquots of liquid culture (9 and 1.5 ml, respectively) were collected by syringe and centrifuged (21,000g for 10 min at 4°C). Cell pellets were submitted to Molecular Research LP for DNA extraction, library preparation, and sequencing. After resuspending pellets in 180 µl of ATL buffer (Qiagen), DNA was extracted using the MagAttract HMW DNA Kit (Qiagen). DNA was eluted in 100 µl of AE buffer (Qiagen) and then cleaned using the DNEasy PowerClean Pro Cleanup Kit (Qiagen). DNA was then sheared using the Covaris g-TUBE (Covaris). Sequencing libraries were prepared using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences) and 1500 ng of the sheared and purified DNA. The SMRTbell libraries were size-selected (>6 Kb) using a BluePippin instrument (Sage Science) and 0.75% agarose gel. Libraries were then sequenced using the PacBio Sequel II (Pacific Biosciences) platform and a 30-hour movie time. Narrative Summary In these Narratives, we filtered low-quality reads using Trimmomatic (v0.36), assembled filtered reads with SPAdes (v3.15.3), and then checked completeness and contamination of the assembled genomes with CheckM (v1.0.18). Statistics for sequencing and assembly are in table S3. Using the assembled contigs (genomes), we called genes and annotated them. Protein-coding genes were called using Prodigal (v2.6.3) (3) locally or using KBase via RASTtk (v1.073), with identical results. Genes were annotated with KO IDs using KAAS (4). They were further annotated with pfam and TIGRFAM IDs using KBase and the Annotate Domains in a Genome app. We classified putative genes for hydrogenases using HydDB. Genes for 16S ribosomal RNA (rRNA) were called using RASTtk (v1.073) in KBase. The contigs (genomes) were analyzed to determine whether they belonged to new species. Taxonomy was assigned using GTDB-Tk (v1.7.0) in KBase. The identity of 16S rRNA genes to other organisms was found using EzBioCloud (5). Values of digital DNA-DNA hybridization (dDDH) were found with Type (Strain) Genome Server (6). These analyses suggest that Lachnospiraceae sp. C1.1 and Porphyromonadaceae sp. W3.11 represent novel species or genera. GTDB-Tk assigned Lachnospiracae sp. C1.1 to family Lachnospiraceae and genus NK4A144, which contains no type strains. It assigned Porphyromonadaceae sp. W3.11 to Porphyromonadaceae and genus Porphyromonas_A. Values of 16S rRNA identity and dDDH with respect to type strains were low (table S4). Although more phenotypic data are needed, available evidence supports assignment of genomes to new species or genera. Related publication Hackmann TJ, Zhang B. The phenotype and genotype of fermentative prokaryotes. Sci Adv. 2023 Sep 29;9(39):eadg8687. doi: 10.1126/sciadv.adg8687. Epub 2023 Sep 27. PMID: 37756392; PMCID: PMC10530074.

Hackmann, Timothy↗

DOE OSTI2021

Comparison of two culture methods for the enumeration of Legionella pneumophila from potable water samples

Legionella infections have steadily increased in the United States over the last 20 years, and most of these infections have been attributed to contaminated water. The gold standard for confirmation of Legionella presence in water is culturing with Buffered Charcoal Yeast Extract (BCYE) agar. Following many modifications, this method is still time-consuming, expensive, and can take longer than 10 days for full confirmation. The Legiolert is a newer and simpler culture product that is claimed to be able to quantify Legionella pneumophila in 7 days with high sensitivity and specificity and does not need further confirmation for the presence of L. pneumophila. This study compared the culturability of L. pneumophila occurring in a simulated home plumbing system using both Legiolert and BCYE agar methods. Out of 185 water samples, Legiolert and BCYE method detected L. pneumophila in 83 and 85% of the samples, respectively. The two methods were determined to be statistically equivalent for culturability of L. pneumophila, though the detected levels by Legiolert were slightly higher than the BCYE method. The molecular confirmation of positive (n = 254) and negative wells (n = 82) with Legiolert also showed a high specificity of 96.5% (i.e., 3.5% false positives (9/254) and 0% false negatives (0/82)).

Boczek, Laura A.↗