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All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Impact of Soil Salinity on the Cowpea Nodule-Microbiome and the Isolation of Halotolerant PGPR Strains to Promote Plant Growth under Salinity Stress

Cowpea is one of the major legumes cultivated in arid and semiarid regions of the world. Four soil-microbial samples (SS-1 through SS-4) collected from semiarid soils in Punjab, Pakistan were planted with cowpea (Vigna unguiculata) crops, which were grown under salinity stress to analyze bacterial composition in the rhizosphere and within nodules using cultivation-dependent and -independent methods. Two varieties, 603 and the salt-tolerant CB 46, were each inoculated with or without the four different native soil samples or grown in medium either N-deficient (-N) or supplemented with N (+N). Plants inoculated with soil samples SS-2 and SS-4 grew better than plants inoculated with SS-1- and SS-3 and grew comparably with the +N controls. Environmental DNA (eDNA) was isolated from SS-1 and SS-4, and, by 16S ribosomal RNA sequencing, the soil microbiomes consisted mainly of Actinobacteria, Firmicutes, Proteobacteria, and other nonproteobacterial genera. However, analysis of eDNA isolated from cowpea nodules established by the trap plants showed that the nodule microbiome consisted almost exclusively of proteobacterial sequences, particularly species of Bradyrhizobium. Bacteria were isolated from both soils and nodules, and 34 of the 51 isolates tested positive for plant-growth-promoting rhizobacteria traits in plate assays. Many could serve as future inocula for crops in arid soils. The discrepancy between the types of bacteria isolated by culturing bacteria isolated from surface-sterilized cowpea nodules (proteobacteria and nonproteobacteria) versus those detected by sequencing DNA isolated from the nodules (proteobacteria) from cowpea nodules (proteobacteria and nonproteobacteria) versus those detected in the nodule microbiome (proteobacteria) needs further study.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Differentiation strategies of soil rare and abundant microbial taxa in response to changing climatic regimes

Despite the important roles of soil microbes, especially the most diverse rare taxa in maintaining community diversity and multifunctionality, how different climate regimes alter the stability and functions of the rare microbial biosphere remains unknown. We reciprocally transplanted field soils across a latitudinal gradient to simulate climate change and sampled the soils annually after harvesting the maize over the following 6 years (from 2005 to 2011). By sequencing microbial 16S ribosomal RNA gene amplicons, we found that changing climate regimes significantly altered the composition and dynamics of soil microbial communities. A continuous succession of the rare and abundant communities was observed. Rare microbial communities were more stable under changing climatic regimes, with lower variations in temporal dynamics, and higher stability and constancy of diversity. More nitrogen cycling genes were detected in the rare members than in the abundant members, including amoA, napA, nifH, nirK, nirS, norB and nrfA. Random forest analysis and receiver operating characteristics analysis showed that rare taxa may act as potential contributors to maize yield under changing climatics. The study indicates that the taxonomically and functionally diverse rare biosphere has the potential to increase functional redundancy and enhance the ability of soil communities to counteract environmental disturbances. With ongoing global climate change, exploring the succession process and functional changes of rare taxa may be important in elucidating the ecosystem stability and multifunctionality that are mediated by microbial communities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Development of DNA methylation‐based epigenetic age predictors in loblolly pine ( Pinus taeda )

Abstract Biological ageing is connected to life history variation across ecological scales and informs a basic understanding of age‐related declines in organismal function. Altered DNA methylation dynamics are a conserved aspect of biological ageing and have recently been modelled to predict chronological age among vertebrate species. In addition to their utility in estimating individual age, differences between chronological and predicted ages arise due to acceleration or deceleration of epigenetic ageing, and these discrepancies are linked to disease risk and multiple life history traits. Although evidence suggests that patterns of DNA methylation can describe ageing in plants, predictions with epigenetic clocks have yet to be performed. Here, we resolve the DNA methylome across CpG, CHG, and CHH‐methylation contexts in the loblolly pine tree ( Pinus taeda ) and construct epigenetic clocks capable of predicting ages in this species within 6% of its maximum lifespan. Although patterns of CHH‐methylation showed little association with age, both CpG and CHG‐methylation contexts were strongly associated with ageing, largely becoming hypomethylated with age. Among age‐associated loci were those in close proximity to malate dehydrogenase, NADH dehydrogenase, and 18S and 26S ribosomal RNA genes. This study reports one of the first epigenetic clocks in plants and demonstrates the universality of age‐associated DNA methylation dynamics which can inform conservation and management practices, as well as our ecological and evolutionary understanding of biological ageing in plants.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Assessing biogeographic survey gaps in bacterial diversity knowledge: A global synthesis of freshwaters

Freshwaters account for 0.8% of Earth's surface area, yet support >10% of known plant and animal species making them disproportionately biodiverse. Modern molecular techniques have begun to reveal microbial diversity, but application of these approaches to address global microbial biogeography is relatively unknown in freshwaters. Our aim was to identify gaps in microbial data coverage along climatic and landscape disturbance gradients and among terrestrial biomes and hydrographic regions for all freshwater ecosystems and three freshwater habitat types: lakes and reservoirs (lentic); streams and rivers (lotic); and wetlands. We reviewed literature on microbial diversity in freshwaters surveyed using 16S ribosomal RNA sequencing which identify microbial taxa. We georeferenced survey locations and used a geographic information system to identify and map gaps in survey coverage using open-source data for climate, landscape disturbance, terrestrial biomes, and freshwater ecoregions. In our study, we compiled 3,425 georeferenced survey locations reported from 963 studies. Streams were surveyed most frequently (60.8% of survey locations), followed by lakes (33.5%) and wetlands (5.6%). Surveys were concentrated in North America, central and western Europe, and Southeast Asia; 35% of freshwater ecoregions were surveyed at least once across freshwater habitat types, whereas 23%, 23%, and 12% were surveyed at least once for lentic, lotic, and wetland habitat types, respectively. The climatic gap analysis indicated coverage is high for temperate regions but lacking in the tropics and Arctic, particularly for wetland ecosystems. Our assessment revealed high climatic coverage of freshwater microbial diversity knowledge, but expansive ecoregional gaps attributable to biased sampling near research institutions in North America, western Europe, and China. Future surveys should target ecoregions in Africa, South America, Central Asia, Australia, and Antarctica. An essential next step will be to curate and disseminate sequencing efforts to facilitate the study of processes driving global diversity patterns.

16S rRNA↗
DOE OSTI2021

Fire alters plant microbiome assembly patterns: integrating the plant and soil microbial response to disturbance

Summary It is increasingly evident that the plant microbiome is a strong determinant of plant health. While the ability to manipulate the microbiome in plants and ecosystems recovering from disturbance may be useful, our understanding of the plant microbiome in regenerating plant communities is currently limited. Using 16S ribosomal RNA (rRNA) gene and internal transcribed spacer (ITS) region amplicon sequencing, we characterized the leaf, stem, fine root, rhizome, and rhizosphere microbiome of < 1‐yr‐old aspen saplings and the associated bulk soil after a recent high‐intensity prescribed fire across a burn severity gradient. Consistent with previous studies, we found that soil microbiomes are responsive to fire. We extend these findings by showing that certain plant tissue microbiomes also change in response to fire. Differences in soil microbiome compositions could be attributed to soil chemical characteristics, but, generally, plant tissue microbiomes were not related to plant tissue elemental concentrations. Using source tracking modeling, we also show that fire influences the relative dominance of microbial inoculum and the vertical inheritance of the sapling microbiome from the parent tree. Overall, our results demonstrate how fire impacts plant microbiome assembly, diversity, and composition and highlights potential for further research towards increasing plant fitness and ecosystem recovery after fire events.

16rRNA↗
DOE OSTI2023

Targeted rRNA depletion enables efficient mRNA sequencing in diverse bacterial species and complex co-cultures

Bacterial mRNA sequencing is inefficient due to the abundance of ribosomal RNA that is challenging to deplete. While commercial kits target rRNA from common bacterial species, they are frequently inefficient when applied to divergent species, including those from environmental isolates. Similarly, other methods typically employ large probe sets that tile the entire length of rRNAs; however, such approaches are infeasible when applied to many species. Therefore, we present EMBR-seq+, which requires fewer than 10 oligonucleotides per rRNA by combining rRNA blocking primers with RNase H-mediated depletion to achieve rRNA removal efficiencies of up to 99% in diverse bacterial species. Furthermore, in more complex microbial co-cultures between Fibrobacter succinogenes strain UWB7 and anaerobic fungi, EMBR-seq+ depleted both bacterial and fungal rRNA, with a fourfold improvement in bacterial rRNA depletion compared with a commercial kit, thereby demonstrating that the method can be applied to non-model microbial mixtures. Notably, for microbes with unknown rRNA sequences, EMBR-seq+ enables rapid iterations in probe design without requiring to start experiments from total RNA. Finally, efficient depletion of rRNA enabled systematic quantification of the reprogramming of the bacterial transcriptome when cultured in the presence of the anaerobic fungi Anaeromyces robustus or Caecomyces churrovis. We observed that F. succinogenes strain UWB7 downregulated several lignocellulose-degrading carbohydrate-active enzymes in the presence of anaerobic gut fungi, suggesting close interactions between two cellulolytic species that specialize in different aspects of biomass breakdown. Thus, EMBR-seq+ enables efficient, cost-effective, and rapid quantification of the transcriptome to gain insights into non-model microbial systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Inferring microbial co-occurrence networks from amplicon data: a systematic evaluation

Microbes commonly organize into communities consisting of hundreds of species involved in complex interactions with each other. 16S ribosomal RNA (16S rRNA) amplicon profiling provides snapshots that reveal the phylogenies and abundance profiles of these microbial communities. These snapshots, when collected from multiple samples, can reveal the co-occurrence of microbes, providing a glimpse into the network of associations in these communities. However, the inference of networks from 16S data involves numerous steps, each requiring specific tools and parameter choices. Moreover, the extent to which these steps affect the final network is still unclear. In this study, we perform a meticulous analysis of each step of a pipeline that can convert 16S sequencing data into a network of microbial associations. Through this process, we map how different choices of algorithms and parameters affect the co-occurrence network and identify the steps that contribute substantially to the variance. We further determine the tools and parameters that generate robust co-occurrence networks and develop consensus network algorithms based on benchmarks with mock and synthetic data sets. The Microbial Co-occurrence Network Explorer, or MiCoNE (available at https://github.com/segrelab/MiCoNE) follows these default tools and parameters and can help explore the outcome of these combinations of choices on the inferred networks. We envisage that this pipeline could be used for integrating multiple data sets and generating comparative analyses and consensus networks that can guide our understanding of microbial community assembly in different biomes.

16S rRNA↗
DOE OSTI2023

Fecal microbiota transplantation ameliorates type 2 diabetes via metabolic remodeling of the gut microbiota in db/db mice

Gut microbiome (GM) deregulation has been implicated in major conditions such as obesity and type 2 diabetes (T2DM). Our previous prospective study indicated that fecal microbiota transplantation (FMT) successfully improved patients with T2DM. We hypothesized that FMT may be a potential therapeutic method for T2DM, but its precise mechanisms in T2DM remains to be elucidated. Eight db/m mice were FMT donors and control mice, and 16 genetically diabetic db/db mice were equally divided into two groups (db/db+phosphate-buffered saline (PBS) group, db/db+FMT group). The db/db+FMT group was administered fresh fecal suspension (0.2 mL/mice) daily for 4 weeks. Analysis of the GM and serum metabolome was carried out by 16S ribosomal RNA sequencing and liquid chromatogram-mass spectrometry, respectively. Effects of FMT on the gut barrier and pancreas were assessed using protein assays, messenger RNA, immunohistology and clinical indicators testing. Our results showed that FMT treatment of db/db mice relieves a series of clinical indicators, including fasting plasma glucose, serum insulin and oral glucose tolerance test among others. Compared with non-diabetic control mice, db/db+PBS mice exhibited decreased abundance of Ruminococaceae, Porphyromonadaceae and increased abundance of Rikenellaceae and Lactobacillaceae. FMT treatment reversed this effect on the microbiome. Eleven metabolites were changed between the db/db+PBS and db/db+FMT groups. Correlation analysis showed that the structural changes of the GM were correlated with host metabolite levels. We further showed that FMT treatment of db/db mice improved intestinal barrier function, reduced inflammation and caused an alteration in the number of circulating immune cells. FMT-mediated changes in the GM, serum metabolites, intestinal epithelial barrier, inflammation and circulating immune cells play an important role in the efficacy of FMT on T2DM disease progression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Chickpea NCR13 disulfide cross-linking variants exhibit profound differences in antifungal activity and modes of action

Small cysteine-rich antifungal peptides with multi-site modes of action (MoA) have potential for development as biofungicides. In particular, legumes of the inverted repeat-lacking clade express a large family of nodule-specific cysteine-rich (NCR) peptides that orchestrate differentiation of nitrogen-fixing bacteria into bacteroids. These NCRs can form two or three intramolecular disulfide bonds and a subset of these peptides with high cationicity exhibits antifungal activity. However, the importance of intramolecular disulfide pairing and MoA against fungal pathogens for most of these plant peptides remains to be elucidated. Our study focused on a highly cationic chickpea NCR13, which has a net charge of +8 and contains six cysteines capable of forming three disulfide bonds. NCR13 expression in Pichia pastoris resulted in formation of two peptide folding variants, NCR13_PFV1 and NCR13_PFV2, that differed in the pairing of two out of three disulfide bonds despite having an identical amino acid sequence. The NMR structure of each PFV revealed a unique three-dimensional fold with the PFV1 structure being more compact but less dynamic. Surprisingly, PFV1 and PFV2 differed profoundly in the potency of antifungal activity against several fungal plant pathogens and their multi-faceted MoA. PFV1 showed significantly faster fungal cell-permeabilizing and cell entry capabilities as well as greater stability once inside the fungal cells. Additionally, PFV1 was more effective in binding fungal ribosomal RNA and inhibiting protein translation in vitro. Furthermore, when sprayed on pepper and tomato plants, PFV1 was more effective in reducing disease symptoms caused by Botrytis cinerea, causal agent of gray mold disease in fruits, vegetables, and flowers. In conclusion, our work highlights the significant impact of disulfide pairing on the antifungal activity and MoA of NCR13 and provides a structural framework for design of novel, potent antifungal peptides for agricultural use.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Depth-resolved sagebrush root metabolomics, rhizosphere microbial communities, and geochemistry at the East River Watershed

This data set consists of results from soil nutrient profile, untargeted metabolomics, mass spec imaging, and amplicon sequencing. Data for soil nutrient profile includes common cations (Ca, Mg, Na, and K etc.) extracted from 3 digesting steps – ammonia acetate (for exchangeable cations), nitric acid (for acid dissolved fraction), and hydrofluoric acid/perchloric acid (HF/HClO4) for whole soil digestion. It also includes concentration of organic carbon, inorganic nitrogen (ammonia and nitrate) and phosphorus (Bray-1 P and nitric acid extract), and total nitrogen and phosphorus. Data for untargeted metabolomics includes metabolomic profile for root exudate/tissues and soil extracts from depths at surface soil to saprolite, that were measured using gas chromatography – mass spectrometry (GC-MS), and liquid chromatography – tandem mass spectrometry (LC-MS/MS). Data for mass spec imaging includes spatial distribution of metabolites that were detected and annotated with Fourier transformation ion cyclotron resonance mass spectrometer (FTICR-MS). Data for amplicon sequencing includes the base paired 16S and ITS ribosomal RNA sequences from Miseq Illumina sequencing. All samples were collected from 2 sampling campaign October 2022 and June 2023. Collectively, these datasets enable a mechanistic evaluation of how nutrient acquisition, especially nitrogen and phosphorus, differs between shallow roots operating in soil and deep roots functioning within the fractured bedrock zone. All files are provided as comma-separated values (CSV) fies (.csv) and (GZIP) file (.gz). The compressed .gz FASTQ files can be read directly in R using the dada2 package as part of the amplicon sequence analysis workflow. 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. This research was performed on a project award 60563 (https://dx.doi.org/10.46936/expl.proj.2022.60563/60008727) from the Environmental Molecular Sciences Laboratory, a DOE Office of Science User Facility sponsored by the Biological and Environmental Research program under Contract No. DE-AC05-76RL01830.

EARTH SCIENCE > AGRICULTURE > SOILS > CARBON↗
DOE OSTI2021

Morphological and molecular characterization of Paratylenchus beltsvillensis n. sp. (Tylenchida: Paratylenchidae) from the rhizosphere of pine tree ( Pinus virginiana Mill) in Maryland, USA

The pin nematode, Paratylechus beltsvillensis sp. collected from rhizosphere soil of a Virginia pine tree (Pinus virginiana Mill) growing in Little Paint Branch Park, Beltsville, Prince George’s County, Maryland, USA, is described and illustrated along with light and scanning electron photomicrographs. Females, males, and juveniles of this new species were recovered from soil samples using the sugar centrifugal flotation and Baermann funnel extraction methods. Morphologically, females are short, body length ranging from 245 to 267 μm, stylet from 70 to 75 μm long with anchor shaped knobs, vulva located at 70–73% and small vulval flap, spermatheca large, and ovoid filled with sperms. Lateral field with three incisures, of which the outer two are prominent. Tail slender, having a rounded tail terminus. Males without stylet and have a degenerated pharynx, spicules = 17–20 µm and gubernaculum = 5.0–5.5 µm. Both morphological observations and molecular analysis of ITS and partial 28S ribosomal RNA gene sequences indicated that the specimens collected from the soil at Beltsville Park from rhizosphere soil samples from Virginia pine represents a new pin nematode species.

59 BASIC BIOLOGICAL SCIENCES↗
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 OSTI2022

Multiple Cases of Bacterial Sequence Erroneously Incorporated Into Publicly Available Chloroplast Genomes

Public sequencing databases are invaluable resources to biological researchers, but assessing data veracity as well as the curation and maintenance of such large collections of data can be challenging. Genomes of eukaryotic organelles, such as chloroplasts and other plastids, are particularly susceptible to assembly errors and misrepresentations in these databases due to their close evolutionary relationships with bacteria, which may co-occur within the same environment, as can be the case when sequencing plants. Here, based on sequence similarities with bacterial genomes, we identified several suspicious chloroplast assemblies present in the National Institutes of Health (NIH) Reference Sequence (RefSeq) collection. Investigations into these chloroplast assemblies reveal examples of erroneous integration of bacterial sequences into chloroplast ribosomal RNA (rRNA) loci, often within the rRNA genes, presumably due to the high similarity between plastid and bacterial rRNAs. The bacterial lineages identified within the examined chloroplasts as the most likely source of contamination are either known associates of plants, or co-occur in the same environmental niches as the examined plants. Modifications to the methods used to process untargeted ‘raw’ shotgun sequencing data from whole genome sequencing efforts, such as the identification and removal of bacterial reads prior to plastome assembly, could eliminate similar errors in the future.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Improved Microbial Community Characterization of 16S rRNA via Metagenome Hybridization Capture Enrichment

Environmental microbial diversity is often investigated from a molecular perspective using 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics. While amplicon methods are fast, low-cost, and have curated reference databases, they can suffer from amplification bias and are limited in genomic scope. In contrast, shotgun metagenomic methods sample more genomic regions with fewer sequence acquisition biases, but are much more expensive (even with moderate sequencing depth) and computationally challenging. Here, we develop a set of 16S rRNA sequence capture baits that offer a potential middle ground with the advantages from both approaches for investigating microbial communities. These baits cover the diversity of all 16S rRNA sequences available in the Greengenes (v. 13.5) database, with no sequence having <78% sequence identity to at least one bait for all segments of 16S. The use of our baits provide comparable results to 16S amplicon libraries and shotgun metagenomic libraries when assigning taxonomic units from 16S sequences within the metagenomic reads. We demonstrate that 16S rRNA capture baits can be used on a range of microbial samples (i.e., mock communities and rodent fecal samples) to increase the proportion of 16S rRNA sequences (average > 400-fold) and decrease analysis time to obtain consistent community assessments. Furthermore, our study reveals that bioinformatic methods used to analyze sequencing data may have a greater influence on estimates of community composition than library preparation method used, likely due in part to the extent and curation of the reference databases considered. Thus, enriching existing aliquots of shotgun metagenomic libraries and obtaining modest numbers of reads from them offers an efficient orthogonal method for assessment of bacterial community composition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Substrate Utilization and Competitive Interactions Among Soil Bacteria Vary With Life-History Strategies

Microorganisms have evolved various life-history strategies to survive fluctuating resource conditions in soils. However, it remains elusive how the life-history strategies of microorganisms influence their processing of organic carbon, which may affect microbial interactions and carbon cycling in soils. Here, we characterized the genomic traits, exometabolite profiles, and interactions of soil bacteria representing copiotrophic and oligotrophic strategists. Isolates were selected based on differences in ribosomal RNA operon ( rrn ) copy number, as a proxy for life-history strategies, with pairs of “high” and “low” rrn copy number isolates represented within the Micrococcales, Corynebacteriales, and Bacillales. We found that high rrn isolates consumed a greater diversity and amount of substrates than low rrn isolates in a defined growth medium containing common soil metabolites. We estimated overlap in substrate utilization profiles to predict the potential for resource competition and found that high rrn isolates tended to have a greater potential for competitive interactions. The predicted interactions positively correlated with the measured interactions that were dominated by negative interactions as determined through sequential growth experiments. This suggests that resource competition was a major force governing interactions among isolates, while cross-feeding of metabolic secretion likely contributed to the relatively rare positive interactions observed. By connecting bacterial life-history strategies, genomic features, and metabolism, our study advances the understanding of the links between bacterial community composition and the transformation of carbon in soils.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genomic Characterization and Wetland Occurrence of a Novel Campylobacter Isolate from Canada Geese

Populations of resident, non-migratory Canada geese are rapidly increasing. Canada geese are known to transmit viral and bacterial diseases, posing a possible threat to human health. The most prevalent pathogens vectored by geese are Campylobacter species, yet the current understanding of the identity and virulence of these pathogens is limited. In our previous study, we observed a high prevalence of Campylobacter spp. in the Banklick Creek wetland—a constructed treatment wetland (CTW) located in northern KY (USA) used to understand sources of fecal contamination originating from humans and waterfowl frequenting the area. To identify the types of Campylobacter spp. found contaminating the CTW, we performed genetic analyses of Campylobacter 16s ribosomal RNA amplified from CTW water samples and collected fecal material from birds frequenting those areas. Our results showed a high occurrence of a Campylobacter canadensis-like clade from the sampling sites. Whole-genome sequence analyses of an isolate from Canada goose fecal material, called MG1, were used to confirm the identity of the CTW isolates. Further, we examined the phylogenomic position, virulence gene content, and antimicrobial resistance gene profile of MG1. Lastly, we developed an MG1-specific real-time PCR assay and confirmed the presence of MG1 in Canada goose fecal samples surrounding the CTW. Our findings reveal that the Canada goose-vectored Campylobacter sp. MG1 is a novel isolate compared to C. canadensis that possesses possible zoonotic potential, which may be of human health concern.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Storage and Algal Association of Bacteria That Protect Microchloropsis salina from Grazing by Brachionus plicatilis

Loss of algal production from the crashes of algal mass cultivation systems represents a significant barrier to the economic production of microalgal-based biofuels. Current strategies for crash prevention can be too costly to apply broadly as prophylaxis. Bacteria are ubiquitous in microalgal mass production cultures, however few studies investigate their role and possible significance in this particular environment. Previously, we demonstrated the success of selected protective bacterial communities to save Microchloropsis salina cultures from grazing by the rotifer Brachionus plicatilis. In the current study, these protective bacterial communities were further characterized by fractionation into rotifer-associated, algal-associated, and free-floating bacterial fractions. Small subunit ribosomal RNA amplicon sequencing was used to identify the bacterial genera present in each of the fractions. Here, we show that Marinobacter, Ruegeria, and Boseongicola in algae and rotifer fractions from rotifer-infected cultures likely play key roles in protecting algae from rotifers. Several other identified taxa likely play lesser roles in protective capability. The identification of bacterial community members demonstrating protective qualities will allow for the rational design of microbial communities grown in stable co-cultures with algal production strains in mass cultivation systems. Such a system would reduce the frequency of culture crashes and represent an essentially zero-cost form of algal crop protection.

59 BASIC BIOLOGICAL SCIENCES↗