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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

The Near-Gapless Penicillium fuscoglaucum Genome Enables the Discovery of Lifestyle Features as an Emerging Post-Harvest Phytopathogen

Penicillium spp. occupy many diverse biological niches that include plant pathogens, opportunistic human pathogens, saprophytes, indoor air contaminants, and those selected specifically for industrial applications to produce secondary metabolites and lifesaving antibiotics. Recent phylogenetic studies have established Penicillium fuscoglaucum as a synonym for Penicillium commune, which is an indoor air contaminant and toxin producer and can infect apple fruit during storage. During routine culturing on selective media in the lab, we obtained an isolate of P. fuscoglaucum Pf_T2 and sequenced its genome. The Pf_T2 genome is far superior to available genomic resources for the species. Our assembly exhibits a length of 35.1 Mb, a BUSCO score of 97.9% complete, and consists of five scaffolds/contigs representing the four expected chromosomes. It was determined that the Pf_T2 genome was colinear with a type specimen P. fuscoglaucum and contained a lineage-specific, intact cyclopiazonic acid (CPA) gene cluster. For comparison, a highly virulent postharvest apple pathogen, P. expansum strain TDL 12.1, was included and showed a similar growth pattern in culture to our Pf_T2 isolate but was far more aggressive in apple fruit than P. fuscoglaucum. The genome of Pf_T2 serves as a major improvement over existing resources, has superior annotation, and can inform forthcoming omics-based work and functional genetic studies to probe secondary metabolite production and disparities in aggressiveness during apple fruit decay.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A method for achieving complete microbial genomes and improving bins from metagenomics data

Metagenomics facilitates the study of the genetic information from uncultured microbes and complex microbial communities. Assembling complete microbial genomes ( i.e ., circular with no misassemblies) from metagenomics data is difficult because most samples have high organismal complexity and strain diversity. Less than 100 circularized bacterial and archaeal genomes have been assembled from metagenomics data despite the thousands of datasets that are available. Circularized genomes are important for (1) building a reference collection as scaffolds for future assemblies, (2) providing complete gene content of a genome, (3) confirming little or no contamination of a genome, (4) studying the genomic context and synteny of genes, and (5) linking protein coding genes to ribosomal RNA genes to aid metabolic inference in 16S rRNA gene sequencing studies. We developed a method to achieve circularized genomes using iterative assembly, binning, and read mapping. In addition, this method exposes potential misassemblies from k-mer based assemblies. We chose species of the Candidate Phyla Radiation (CPR) to focus our initial efforts because they have small genomes and are only known to have one ribosomal RNA operon. We present 34 circular CPR genomes, one circular Margulisbacteria genome, and two circular megaphage genomes from 19 public and published datasets. We demonstrate findings that would likely be difficult without circularizing genomes, including that ribosomal genes are likely not operonic in the majority of CPR, and that some CPR harbor diverged forms of RNase P RNA. Code and a tutorial for this method is available at https://github.com/lmlui/Jorg .

Lui, Lauren↗
DOE OSTI2021

A method for achieving complete microbial genomes and improving bins from metagenomics data

Metagenomics facilitates the study of the genetic information from uncultured microbes and complex microbial communities. Assembling complete genomes from metagenomics data is difficult because most samples have high organismal complexity and strain diversity. Some studies have attempted to extract complete bacterial, archaeal, and viral genomes and often focus on species with circular genomes so they can help confirm completeness with circularity. However, less than 100 circularized bacterial and archaeal genomes have been assembled and published from metagenomics data despite the thousands of datasets that are available. Circularized genomes are important for (1) building a reference collection as scaffolds for future assemblies, (2) providing complete gene content of a genome, (3) confirming little or no contamination of a genome, (4) studying the genomic context and synteny of genes, and (5) linking protein coding genes to ribosomal RNA genes to aid metabolic inference in 16S rRNA gene sequencing studies. We developed a semi-automated method called Jorg to help circularize small bacterial, archaeal, and viral genomes using iterative assembly, binning, and read mapping. In addition, this method exposes potential misassemblies from k-mer based assemblies. We chose species of the Candidate Phyla Radiation (CPR) to focus our initial efforts because they have small genomes and are only known to have one ribosomal RNA operon. In addition to 34 circular CPR genomes, we present one circular Margulisbacteria genome, one circular Chloroflexi genome, and two circular megaphage genomes from 19 public and published datasets. We demonstrate findings that would likely be difficult without circularizing genomes, including that ribosomal genes are likely not operonic in the majority of CPR, and that some CPR harbor diverged forms of RNase P RNA. Code and a tutorial for this method is available at https://github.com/lmlui/Jorg and is available on the DOE Systems Biology KnowledgeBase as a beta app.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Adaptive modification of antiviral defense systems in microbial community under Cr-induced stress

Background The prokaryotic antiviral defense systems are crucial for mediating prokaryote-virus interactions that influence microbiome functioning and evolutionary dynamics. Despite the prevalence and significance of prokaryotic antiviral defense systems, their responses to abiotic stress and ecological consequences remain poorly understood in soil ecosystems. We established microcosm systems with varying concentrations of hexavalent chromium (Cr(VI)) to investigate the adaptive modifications of prokaryotic antiviral defense systems under abiotic stress. Results Utilizing hybrid metagenomic assembly with long-read and short-read sequencing, we discovered that anti- viral defense systems were more diverse and prevalent in heavily polluted soils, which was corroborated by meta-analyses of public datasets from various heavy metal-contaminated sites. As the Cr(VI) concentration increased, prokaryotes with defense systems favoring prokaryote-virus mutualism gradually supplanted those with defense systems incurring high adaptive costs. Additionally, as Cr(VI) concentrations increased, enriched antiviral defense systems exhibited synchronization with microbial heavy metal resistance genes. Furthermore, the proportion of antiviral defense systems carried by mobile genetic elements (MGEs), including plasmids and viruses, increased by approximately 43% and 39%, respectively, with rising Cr concentrations. This trend is conducive to strengthening the dissemination and sharing of defense resources within microbial communities. Conclusions Overall, our study reveals the adaptive modification of prokaryotic antiviral defense systems in soil ecosystems under abiotic stress, as well as their positive contributions to establishing prokaryote-virus mutualism and the evolution of microbial heavy metal resistance. These findings advance our understanding of microbial adaptation in stressful environments and may inspire novel approaches for microbiome manipulation and bioremediation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Transmission of Raccoon-Passaged Chronic Wasting Disease Agent to White-Tailed Deer

The transmission characteristics of prion diseases are influenced by host prion protein sequence and, therefore, the host species. Chronic wasting disease (CWD), a prion disease of cervids, has widespread geographical distribution throughout North America and occurs in both wild and farmed populations. CWD prions contaminate the environment through scattered excrement and decomposing carcasses. Fresh carcasses with CWD prions are accessible by free-ranging mesopredators such as raccoons and may provide a route of exposure. Previous studies demonstrated the susceptibility of raccoons to CWD from white-tailed deer. In this study, we demonstrate that white-tailed deer replicate raccoon-passaged CWD prions which results in clinical disease similar to intraspecies CWD transmission. Six white-tailed deer were oronasally inoculated with brain homogenate from a raccoon with CWD. All six deer developed clinical disease, had widespread lymphoid distribution of misfolded CWD prions (PrP Sc ), and had neuropathologic lesions with PrP Sc accumulation in the brain. The presence of PrP Sc was confirmed by immunohistochemistry, enzyme-linked immunoassay, and western blot. The western blot migration pattern of raccoon-passaged CWD was different from white-tailed deer CWD. Transmission of raccoon CWD back to white-tailed deer resulted in an interposed molecular phenotype that was measurably different from white-tailed deer CWD.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Assessing Microbial Communities Related to Mercury Transformations in Contaminated Streambank Soils

In nature, the bioaccumulative potent neurotoxin methylmercury (MeHg) is produced from inorganic mercury (Hg) predominantly by anaerobic microorganisms. Hg-contaminated soils are a potential source of MeHg due to microbial activity. We examine streambank soils collected from the contaminated East Fork Poplar Creek (EFPC) in Tennessee, USA, where seasonal variations in MeHg levels have been observed throughout the year, suggesting active microbial Hg methylation. In this study, we characterized the microbial community in contaminated bank soil samples collected from two locations over a period of one year and compared the results to soil samples from an uncontaminated reference site with similar geochemistry (n = 12). Microbial community composition and diversity were assessed by 16S rRNA gene amplicon sequencing. Furthermore, to isolate potential methylators from soils, enrichment cultures were prepared using selective media. A set of three clade-specific primers targeting the gene hgcA were used to detect Hg methylators among the δ-Proteobacteria in EFPC bank soils across all seasons. Two families among the δ-Proteobacteria that have been previously associated with Hg methylation, Geobacteraceae and Syntrophobacteraceae, were found to be predominant with relative abundances of 0.13% and 4.0%, respectively. However, in soil enrichment cultures, Firmicutes were predominant among families associated with Hg methylation. Specifically, Clostridiaceae and Peptococcaceae and their genera Clostridium and Desulfosporosinus were among the ten most abundant genera with relative abundances of 2.6% and 1.7%, respectively. These results offer insights into the role of microbial communities on Hg transformation processes in contaminated bank soils in EFPC. Identifying the biogeochemical drivers of MeHg production is critical for future remediation efforts.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

Deciphering Microbial Metal Toxicity Responses using RB-TnSeq and Activity-Based Metabolomics

To uncover metal toxicity targets and defense mechanisms of the facultative anaerobe Pantoea sp. strain MT58 (MT58), we used a multiomic strategy combining two global techniques, random bar code transposon site sequencing (RB-TnSeq) and activity-based metabolomics. MT58 is a metal-tolerant Oak Ridge Reservation (ORR) environmental isolate that was enriched in the presence of metals at concentrations measured in contaminated groundwater at an ORR nuclear waste site. The effects of three chemically different metals found at elevated concentrations in the ORR contaminated environment were investigated: the cation Al 3+ , the oxyanion CrO 4 2- , and the oxycation UO 2 2+ . Both global techniques were applied using all three metals under both aerobic and anaerobic conditions to elucidate metal interactions mediated through the activity of metabolites and key genes/proteins. These revealed that Al 3+ binds intracellular arginine, CrO 4 2- enters the cell through sulfate transporters and oxidizes intracellular reduced thiols, and membrane-bound lipopolysaccharides protect the cell from UO 2 2+ toxicity. In addition, the Tol outer membrane system contributed to the protection of cellular integrity from the toxic effects of all three metals. Likewise, we found evidence of regulation of lipid content in membranes under metal stress. Individually, RB-TnSeq and metabolomics are powerful tools to explore the impact various stresses have on biological systems. In this work, we show that together they can be used synergistically to identify the molecular actors and mechanisms of these pertubations to an organism, furthering our understanding of how living systems interact with their environment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Assessing the Ability of Developmentally Precocious Estrogen Signaling to Recapitulate Ovarian Transcriptomes and Follicle Dynamics in Alligators from a Contaminated Lake

Concern has grown in recent decades over anthropogenic contaminants that interfere with the functioning of endocrine hormones. However, mechanisms connecting developmental processes to pathologies associated with endocrine-disrupting chemical (EDC) exposure are poorly understood in naturally exposed populations. We sought to a) characterize divergence in ovarian transcriptomic and follicular profiles between alligators originating from a historically EDC-contaminated site, Lake Apopka, and a reference site; b) test the ability of developmentally precocious estrogen exposure to recapitulate site-associated patterns of divergence; and c) test whether treatment with exogenous follicle-stimulating hormone (FSH) is capable of rescuing phenotypes associated with contaminant exposure and/or embryonic estrogen treatment. Alligators eggs were collected from a contaminated site and a reference site, and a subset of eggs from the reference site were treated with estradiol (E2) during embryonic development prior to gonadal differentiation. After hatching, alligators were raised under controlled laboratory settings for 5 months. Juveniles from both sites were divided and treated with exogenous FSH. Histological analyses and RNA-sequencing were conducted to characterize divergence in ovarian follicle dynamics and transcriptomes between sites, between reference and E2-treated animals, and between FSH-treated and nontreated animals. We observed broad site-of-origin divergence in ovarian transcriptomes and reductions in ovarian follicle density between juvenile alligators from Lake Apopka and the reference site. Treating embryos from the reference site with E2 overwhelmingly recapitulated transcriptional and histological alterations observed in Lake Apopka juveniles. Ovarian phenotypes observed in Lake Apopka alligators or resulting from estrogen treatment were only partially rescued by treatment with exogenous FSH. Recapitulation of ovarian abnormalities by precocious E2 revealed a relatively simple mechanism underlying contaminant-induced pathologies in a historical example of environmental endocrine disruption. Findings reported here support a model where the developmental timing of estrogen signaling has the potential to permanently alter ovarian organization and function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Analysis of Previously Classified White Dwarf–Main-sequence Binaries Using Data from the APOGEE Survey

We present analyses of near-infrared spectroscopic data from the Apache Point Observatory Galactic Evolution Experiment (APOGEE) survey for 45 previously confirmed or candidate white dwarf–main-sequence (WDMS) binaries identified by the optical Sloan Digital Sky Survey (SDSS) and LAMOST surveys. Among these 45 systems, we classify three as having red giant primaries in the LAMOST sample and 14 as young stellar object contaminants in the photometrically identified SDSS sample. From among the subsample of 28 systems that we confirm to have MS primaries, we derive and place limits on orbital periods and velocity amplitudes for 14. Seven systems have significant velocity variations that warrant a post-common-envelope (PCE) binary classification, four of which are newly classified, three of which are newly confirmed, and five for which we can derive full orbital parameters. If confirmed, one of these newly discovered systems (2M14544500+4626456) will have the second-longest orbital period reported for a typical compact PCE WDMS binary (P = 15.1 days). In addition to the seven above, we also recover and characterize with APOGEE data the well-known PCE WDMS systems EG UMa and HZ 9. We also investigate the overall metallicity distribution of the WDMS sample, which is a parameter space not often explored for these systems. Of note, we find one system (2M14244053+4929580) to be extremely metal-poor ([Fe/H] = − 1.42) relative to the rest of the near-solar sample. Additionally, the PCE systems in our sample are found to be, on average, higher in metallicity than their wide-binary counterparts, though we caution that with this small number of systems, the sample may not be representative of the overall distribution of WDMS systems.

36 MATERIALS SCIENCE↗
DOE OSTI2025

Metagenome-assembled genomes from topsoils along a hillslope water gradient across early snowmelt to late summer in East River, CO

Drought is changing the American Mountain West at unprecedented rates with unknown consequences to soil microbiome composition and function. As a part of LBNL Watershed Science Focus Area (SFA), we investigated shifts in microbial community and transcriptional activity on a subalpine conifer-meadow transition zone throughout the summer of 2023 as soil dried down. This work took place in Crested Butte, CO on Snodgrass mountain, using a proxy for drought conditions.Here we present metagenome assembled genomes (MAGs) for the bacterial and archaeal community at 0-10cm from three sites along a hillslope water gradient across five timepoints from early snowmelt to late summer. 42 metagenomes were sequenced at Joint Genome Institute (JGI) and can be found under the JGI GOLD (Genomes Online Database) sequencing project Gs0166660. Metagenomes were assembled through an inhouse pipeline (see methods), binned using four autobinners (concoct, maxbin2, metabat2, and vamb) and consolidated using dastool. The consolidated bins from all metagenomes were pooled, filtered by completeness (>70%) and contamination (<10%), and dereplicated at 95% ANI using drep. This dataset (1) a zip file of 157 MAGs (as fasta files, Gs0166660_bins_tar.gz), (2) sample metadata file with sample IGSNs (International Generic Sample Numbers) (samples.csv), (3) bounding box coordinates for the sampled locations (Gs0166660.kml), (4) metagenome assembly and coassembly metadata file listing IMG/M (Integrated Microbial Genomes/Metagenomes) metagenome accessions linking samples to metagenomes (EastRiver_Drought_ESSDive_Metadata.csv), (5) location metadata file (locations.csv), (6) file-level metadata file (flmd.csv) and (7) data dictionary (dd.csv) file.This work was supported by the Watershed Function Science Focus Area at Lawrence Berkeley National Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-05CH11231.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Montane Conifer, Aspen, Meadow, and Sagebrush Metagenome Resolved Genomes and Traits in East River Watershed, Colorado, USA

Climate change is driving vegetation shifts in mountain watersheds, with unknown impacts on biogeochemical cycles. We hypothesize that these shifts will reshape soil microbiomes and associated biogeochemical processes. As a part of Lawrence Berkeley National Laboratory (LBNL) Watershed Science Focus Area (SFA), we assessed microbiome and microbial functional trait differences between soils under conifer, aspen, forby meadows, and sagebrush across the East River Watershed, CO, controlling for elevation and aspect.Here we present metagenome assembled genomes (MAGs) for the bacterial and archaeal communities from soils 0-20cm in depth across three locations in the watershed—Headwaters, Upper Reaches, and Lower Reaches from August 3-11th 2016. Each location was further subdivided into two blocks, with one block on a west facing aspect, and two on the east aspect of the valley. Within blocks, two samples per vegetation type were taken (one at each depth). This resulted in 66 samples, which were sequenced at JGI and can be found under the Joint Genome Institute (JGI) Genomes Online Database (GOLD) sequencing project Gs0118068. Metagenomes were assembled through an inhouse pipeline (see methods), binned using four autobinners (concoct, maxbin2, metabat2, and vamb) and consolidated using dastool. The consolidated bins from all metagenomes were pooled, filtered by completeness (>75%) and contamination (<25%), and dereplicated at 95% ANI using drep. The dataset includes a zip file of 687 genomes (Vegtype_MAGS.zip), the accession numbers for the underlying metagenomes, a csv file with MAG quality metrics and taxonomy from Genome Taxonomy Database (GTDB) and National Center for Biotechnology Information (NCBI) taxonomic representative genome proteins (EastRiver_Vegtype_drep_genome_info.csv), and a file containing MAG quality metrics and taxonomy (gtdb_drep_bin_taxonomy.csv). The dataset additionally includes a sample metadata file (EastRiver_Vegtype_sample_metadata.csv), a metadata file used to register associated samples with IGSNs (International Generic Sample Numbers) (samples.csv), a Google KML file for the sampled locations (sample_collection_sites.kml), a location metadata file (locations.csv), a file-level metadata file (flmd.csv), and a data dictionary (dd.csv) file.This work was supported by the Watershed Function Science Focus Area at Lawrence Berkeley National Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-05CH11231.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Lessons Learned from Demolition of Hanford's Plutonium Finishing Plant - 20507

U.S. Department of Energy (DOE) contractor CH2M Hill Plateau Remediation Company (CHPRC), a Jacobs owned company, is making significant progress demolishing the Plutonium Finishing Plant (PFP), which has long been known as one of the most hazardous buildings in the DOE Environmental Management Complex (DOE Complex). The facility is located on the Hanford Site and produced plutonium metal during the Cold War. Production was stopped in 1989, the facility was formally shut down in 1996, and material processing was completed in 2004. After approximately 25 years of demolition preparations, open-air demolition of the main portions of the Hazard Category II nuclear facility began in November 2016. To prepare the PFP for demolition, CHPRC employees performed some of the most hazardous work across the DOE Complex. In addition to extensive demolition preparations, including a wide-reaching communication strategy across the Hanford Site, CHPRC implemented robust controls and monitoring during demolition, and when necessary, adjusted demolition practices and sequence to maintain employee safety and project efficiency. Demolition of the main PFP building began in November 2016. The technical complexity, high hazards and radiological concerns have all led to a difficult demolition environment. Following a spread of contamination at the project site beyond the radiological boundaries in December 2017, work was halted and a recovery plan was generated to allow the demolition work to continue. After the implementation of additional controls and concurrence from regulatory agencies and DOE, the demolition was ready to resume work and reinitiated in August 2018. There are a number of lessons learned from the demolition activities and recovery plan process. This paper allows CHPRC and DoE's Richland Operations Office (RL) to share lessons learned and progress to date with other challenging and hazardous projects across the DOE Complex. Understanding the risk acceptance level is critical to execution of any open-air demolition project and is unique to each facility. Four key lessons learned from this project include: 1) plan execution issues, 2) stakeholder communication issues, 3) watching for summit fever and 4) minimizing exposed contaminated surfaces during demolition. (authors)

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗
DOE OSTI2022

Photometric calibration in u -band using blue halo stars

ABSTRACT We develop a method to calibrate u-band photometry based on the observed colour of blue Galactic halo stars. The Galactic halo stars belong to an old stellar population of the Milky Way and have relatively low metallicity. The ‘blue tip’ of the halo population – the main sequence turn-off (MSTO) stars – is known to have a relatively uniform intrinsic edge u-g colour with only slow spatial variation. In SDSS data, the observed variation is correlated with Galactic Latitude, which we attribute to contamination by higher metallicity disc stars and fit with an empirical curve. This curve can then be used to calibrate u-band imaging if g-band imaging of matching depth is available. Our approach can be applied to single-field observations at |b| > 30°, and removes the need for standard star observations or overlap with calibrated u-band imaging. We include in our method the calibration of g-band data with ATLAS-Refcat2. We test our approach on stars in KiDS DR 4, ATLAS DR 4, and DECam imaging from the NOIRLab Source Catalog (NSC DR2), and compare our calibration with SDSS. For this process, we use synthetic magnitudes to derive the colour equations between these data sets, in order to improve zero-point accuracy. We find an improvement for all data sets, reaching a zero-point precision of 0.016 mag for KiDS (compared to the original 0.033 mag), 0.020 mag for ATLAS (originally 0.027 mag), and 0.016 mag for DECam (originally 0.041 mag). Thus, this method alone reaches the goal of 0.02 mag photometric precision in u-band for the Rubin Observatory’s Legacy Survey of Space and Time (LSST).

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2022

Disc dichotomy signature in the vertical distribution of [Mg/Fe] and the delayed gas infall scenario

Context. Analysis of the Apache Point Observatory Galactic Evolution Experiment project (APOGEE) data suggests the existence of a clear distinction between two sequences of disc stars in the [α/Fe] versus [Fe/H] abundance ratio space, known as the high- and low-α sequence, respectively. This dichotomy also emerges from an analysis of the vertical distribution of the [α/Fe] abundance ratio. Aims. We aim to test whether the revised two-infall chemical evolution models designed to reproduce the low- and high-α sequences in the [α/Fe] versus [Fe/H] ratios in the solar neighbourhood are also capable of predicting the disc bimodality observed in the vertical distribution of [Mg/Fe] in APOGEE DR16 data. Methods. Along with the chemical composition of the simple stellar populations born at different Galactic times predicted by our reference chemical evolution models in the solar vicinity, we provide their maximum vertical height above the Galactic plane |zmax| computed assuming the relation between the vertical action and stellar age in APOGEE thin-disc stars. Result. The vertical distribution of the [Mg/Fe] abundance ratio predicted by the reference chemical evolution models is in agreement with that observed when combining the APOGEE DR16 data (chemical abundances) with the astroNN catalogue (stellar ages, orbital parameters) for stars younger than 8 Gyr (only low-α sequence stars). Including the high-α disc component, the dichotomy in the vertical [Mg/Fe] abundance distribution is reproduced considering the observational cut in the Galactic height of |z|< 2 kpc. However, our model predicts an overly flat (almost constant) growth of the maximum vertical height |zmax| quantity as a function of [Mg/Fe] for high-α objects in contrast with the median values from APOGEE data. Possible explanations for such a tension are that: (i) the APOGEE sample with |z|< 2 kpc is more likely than ours to be contaminated by halo stars, causing the median values to be kinematically hotter, and (ii) external perturbations – such as minor mergers – that the Milky Way experienced in the past could have heated up the disc, and the heating of the orbits cannot be modeled by only scattering processes. Assuming a disc dissection based on chemistry for APOGEE-DR16 stars (|z|< 2 kpc), the observed |zmax| distributions for high-α and low-α sequences are in good agreement with our model predictions if we consider the errors in the vertical action estimates in the calculation. Moreover, a better agreement between predicted and observed stellar distributions at different Galactic vertical heights is achieved if asteroseismic ages are included as a constraint in the best-fit model calculations. Conclusions. The signature of a delayed gas infall episode, which gives rise to a hiatus in the star formation history of the Galaxy, are imprinted both in the [Mg/Fe] versus [Fe/H] relation and in vertical distribution of [Mg/Fe] abundances in the solar vicinity.

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2020

Metagenome-Guided Proteomic Quantification of Reductive Dehalogenases in the Dehalococcoides mccartyi -Containing Consortium SDC-9

At groundwater sites contaminated with chlorinated ethenes, fermentable substrates are often added to promote reductive dehalogenation by indigenous or augmented microorganisms. Contemporary bioremediation performance monitoring relies on nucleic acid biomarkers of key organohalide-respiring bacteria, such as Dehalococcoides mccartyi (Dhc). In this work, metagenome sequencing of the commercial, Dhc-containing consortium, SDC-9, identified 12 reductive dehalogenase (RDase) genes, including pceA (two copies), vcrA, and tceA, and allowed for specific detection and quantification of RDase peptides using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Shotgun (i.e., untargeted) proteomics applied to the SDC-9 consortium grown with tetrachloroethene (PCE) and lactate identified 143 RDase peptides, and 36 distinct peptides that covered greater than 99% of the protein-coding sequences of the PceA, TceA, and VcrA RDases. Quantification of RDase peptides using multiple reaction monitoring (MRM) assays with 13 C-/ 15 N-labeled peptides determined 1.8 × 10 3 TceA and 1.2 × 10 2 VcrA RDase molecules per Dhc cell. Ultimately, the MRM mass spectrometry approach allowed for sensitive detection and accurate quantification of relevant Dhc RDases and has potential utility in bioremediation monitoring regimes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A multirate mass transfer model to represent the interaction of multicomponent biogeochemical processes between surface water and hyporheic zones (SWAT-MRMT-R 1.0)

Abstract. Surface water quality along river corridors can be modulated by hyporheic zones (HZs) that are ubiquitous and biogeochemically active. Watershed management practices often ignore the potentially important role of HZs as a natural reactor. To investigate the effect of hydrological exchange and biogeochemical processes on the fate of nutrients in surface water and HZs, a novel model, SWAT-MRMT-R, was developed coupling the Soil and Water Assessment Tool (SWAT) watershed model and the reaction module from a flow and reactive transport code (PFLOTRAN). SWAT-MRMT-R simulates concurrent nonlinear multicomponent biogeochemical reactions in both the channel water and its surrounding HZs, connecting the channel water and HZs through hyporheic exchanges using multirate mass transfer (MRMT) representation. Within the model, HZs are conceptualized as transient storage zones with distinguished exchange rates and residence times. The biogeochemical processes within HZs are different from those in the channel water. Hyporheic exchanges are modeled as multiple first-order mass transfers between the channel water and HZs. As a numerical example, SWAT-MRMT-R is applied to the Hanford Reach of the Columbia River, a large river in the United States, focusing on nitrate dynamics in the channel water. Major nitrate contaminants entering the Hanford Reach include those from the legacy waste, irrigation return flows (irrigation water that is not consumed by crops and runs off as point sources to the stream), and groundwater seepage resulting from irrigated agriculture. A two-step reaction sequence for denitrification and an aerobic respiration reaction is assumed to represent the biogeochemical transformations taking place within the HZs. The spatially variable hyporheic exchange rates and residence times in this example are estimated with the basin-scale Networks with EXchange and Subsurface Storage (NEXSS) model. Our simulation results show that (1), given a residence time distribution, how the exchange fluxes to HZs are approximated when using MRMT can significantly change the amount of nitrate consumption in HZs through denitrification and (2) source locations of nitrate have a different impact on surface water quality due to the spatially variable hyporheic exchanges.

58 GEOSCIENCES↗
DOE OSTI2023

Virulence factors and antimicrobial resistance profiles of Campylobacter isolates recovered from consecutively reused broiler litter

ABSTRACT Campylobacterinfections are a leading cause of bacterial diarrhea in humans globally. Infections are due to consumption of contaminated food products and are highly associated with chicken meat, with chickens being an important reservoir forCampylobacter. Here, we characterized the genetic diversity ofCampylobacter jejuni(C. jejuni) andCampylobacter coli(C. coli) detected in broiler chicken litter over three consecutive flocks and determined their antimicrobial resistance (ARM) and virulence factor (VF) profiles.Campylobacterwas detected in 9.38% (27/288) of litter samples collected. Antimicrobial susceptibility testing and whole genome sequencing were performed onC. jejuni(n= 39) andC. coli(n= 5) isolates.Campylobactervirulence factors differed within and across broiler houses but were explained by the broiler flock cohort raised on litter,Campylobacterspecies andCampylobactermultilocus sequence type (MLST). Virulence factors involved in the ability to invade and colonize host tissues and evade host defenses were present inC. jejuniisolates (ST-464) from flock cohorts 1 and 2 but absent inC. jejuniisolates (ST-48) from flock cohort 3.C. jejuniisolates from house three harbored a significantly higher proportion of virulence genes with functions related to glycosylation and immune evasion thanC. jejuniisolates from houses 1 and 2 (P< 0.01). AllC. jejuniisolates were susceptible to all antibiotics tested whileC. coli(n= 4) were resistant to tetracycline and harbored the tetracycline resistant ribosomal protection protein (TetO). Our results suggest that house environment and broiler management practices imposed selective pressures on virulence factors and antimicrobial resistance genes ofCampylobacter. IMPORTANCE Campylobacteris a leading cause of foodborne illness in the United States due to consumption of contaminated or mishandled food products, often associated with chicken meat.Campylobacteris common in the microbiota of avian and mammalian gut; however, acquisition of antimicrobial resistance genes (ARGs) and virulence factors (VFs) may result in strains that pose significant threat to public health. Although there are studies investigating the genetic diversity ofCampylobacterstrains isolated from post-harvest chicken samples, there are limited data on the genome characteristics of isolates recovered from preharvest broiler production. Here, we show thatCampylobacter jejuniandCampylobacter colidiffer in their carriage of antimicrobial resistance and virulence factors may also differ in their ability to persist in litter during consecutive grow-out of broiler flocks. We found that presence/absence of virulence factors needed for evasion of host defense mechanisms and gut colonization played an integral role in differentiatingCampylobacterstrains.

Microbiology↗
DOE OSTI2023

Trimming and Decontamination of Metagenomic Data can Significantly Impact Assembly and Binning Metrics, Phylogenomic and Functional Analysis

Background: Investigators using metagenomic sequencing to study microbiomes often trim and decontaminate reads without knowing their effect on downstream analyses. Objective: This study was designed to evaluate the impacts JGI trimming and decontamination procedures have on assembly and binning metrics, placement of MAGs into species trees, and functional profiles of MAGs extracted from complex rhizosphere metagenomes, as well as how more aggressive trimming impacts these binning metrics. Methods: Twenty-three Miscanthus x giganteus rhizosphere metagenomes were subjected to different combinations and thresholds of force, kmer, and quality trimming and decontamination using BBDuk. Reads were assembled and binned in KBase. Phylogenomic and statistical analyses were applied to evaluate the effects of trimming and decontamination on downstream analyses. Results: We found that JGI trimmed and decontaminated reads had significant impacts on assembly and binning metrics compared to raw reads, including significantly higher total contig counts, more contigs greater than 10k bp in length, and larger total lengths of raw assemblies compared to QC assemblies, and 2.0% lower average contamination of QC MAGs compared to raw MAGs. We also found that differences in the placement of MAGs in species trees increased with decreasing completeness and contamination thresholds. Furthermore, aggressive trimming (Q20) was found to significantly reduce MAG counts. Conclusion: Trimming and decontamination of metagenomics reads prior to assembly can change an investigator’s answer to the questions, “Who is there and what are they doing?” However, mild trimming and decontamination of metagenomic reads with high-quality scores are recommended for removing sample processing and sequencing artifacts.

Whitham, Jason M.↗