Biology subjects

Thrash, J. C.

Publications and source records attributed to Thrash, J. C..

12 recordsLinked to original sources

bioRxiv2025

Detergent-based separation of microbes from marine particles

Marine particles, typically composed of organic detritus and cellular debris, harbor microbial communities that are distinct from the planktonic, or free-living, communities in the pelagic ocean. However, without being first separated from the particle and microbial consortia, these microbes are inaccessible to further investigation via single-cell microbiology methods like flow cytometry, cell-sorting, and dilution-based isolation. To confront this obstacle, we compared the dissociative effects of two commonly used detergents, Tween20 and Tween80, on particle-associated marine microbial communities. The ability of Tween treatments to liberate cells from particles, and to maintain cell integrity, was quantified by flow cytometry from multiple communities across seasons and locations. Both Tween20 and Tween80, at 185 RPM shaking, gently dissociated microbes from their particles, causing very little cell mortality. Additionally, Tween80 liberated a greater number of particle-associated cells into the free-living fraction. We also analyzed the effects of Tween treatments on the microbial community composition for one of these collections via 16S rRNA gene amplicon sequencing of the particle-associated and free-living fractions relative to unamended controls. Tween20 and Tween80 were both effective for microbial dissolution from particles, however Tween80 treatments displayed greater uniformity in the dissociated communities and significantly enriched for the most abundant particle-associated members. Together these data indicate that Tween80 was most effective at gently dissociating particle-associated cells. ImportanceMicrobes that reside on marine particulate organic matter are vital facets of marine biogeochemistry. As they degrade the particle on which they reside, the resulting concentrated region of activity influences surrounding biogeochemistry and redox gradients, making particle-associated microbes significant to overall marine ecology. To understand single-cell activities amidst the microbial assemblage on the particle, cells must first be removed from the substrate for downstream analyses. Methods for microbial dissociation from solid surfaces or sediment communities have been described, however, analogous methods for more ephemeral particles, that also maintain cell viability and preserve DNA for next-generation sequencing, are understudied. Here we optimized a method that leveraged detergents to dissociate microbes from marine particles. We evaluated effectiveness through filter size-fractionation, flow cytometry, and community composition analyses, and provide recommendations to gently and effectively remove microbes from marine particles.

microbiology↗

bioRxiv2025

Culture-supported ecophysiology of the SAR116 clade demonstrates metabolic and spatial niche partitioning

Marine SAR116 bacterioplankton are ubiquitous in surface waters across global oceans and form their own order, Puniceispirillales, within the Alphaproteobacteria. To date no comparative physiology among diverse SAR116 isolates has been performed to capture the functional diversity within the clade, and further, diversity through the lens of metabolic potential and environmental preferences via clade-wide pangenomics remains poorly constrained. Using high-throughput dilution-to-extinction cultivation, we isolated and genome sequenced five new and diverse SAR116 isolates from the northern Gulf of Mexico. Here we present a comparative physiological analysis of these SAR116 isolates, along with a pangenomic investigation of the SAR116 clade using a combination of metagenome-assembled genomes (MAGs, n=258), single-amplified genomes (SAGs, n=84), previously existing (n=2), and new isolate genomes (n=5), totaling 349 SAR116 genomes. Phylogenomic investigation supported the division of SAR116 into three distinct subclades, each with additional structure totalling 15 monophyletic groups. Our SAR116 isolates belonged to three groups within subclade I representing distinct genera with different morphologies and varied phenotypic responses to salinity and temperature. Overall, SAR116 genomes encoded differences in vitamin and amino acid synthesis, trace metal transport, and osmolyte synthesis and transport. They also had genetic potential for diverse sulfur oxidation metabolisms, placing SAR116 at the confluence of the organic and inorganic sulfur pools. SAR116 subclades showed distinct patterns in habitat preferences across open ocean, coastal, and estuarine environments, and three of our isolates represented the most abundant coastal and estuarine subclade. This investigation provides the most comprehensive exploration of SAR116 to date anchored by new culture genomes and physiology.

microbiology↗

bioRxiv2024

Polyhydroxybutyrate production in freshwater SAR11 (LD12)

SAR11 bacteria (order Pelagibacterales) are oligotrophs and often the most abundant bacterioplankton in aquatic environments. A subset of sequenced SAR11 genomes, predominantly in the brackish and freshwater SAR11 subclades, contain homologs of pha genes, which in other organisms confer the ability to store carbon and energy via polyhydroxyalkanoate (PHA) polymers. Here, we investigated the relevance of PHA production to SAR11 biology. Phylogenetics showed that Pha proteins occurred on a long branch and provided evidence for origin at the common ancestor of the brackish IIIa and freshwater LD12 subclades, followed by horizontal transfer within SAR11. Using the LD12 representative "Candidatus Fonsibacter ubiquis" strain LSUCC0530, we found that a large minority of LSUCC0530 cells contained a single Nile red-staining granule, confirmed that the cells produced polyhydroxybutyrate (PHB), and estimated the total PHB content in the cells. We heterologously expressed the LSUCC0530 phaCAB locus in Escherichia coli, finding it to be functional and the likely origin of the PHB. We also determined that, irrespective of changes to carbon, nitrogen, and phosphorus concentrations, a similar fraction of LSUCC0530 cells generated PHB granules and expression of the phaCAB locus remained constant. We suggest that PHB synthesis in LSUCC0530 may be constitutively active due to the slow growth dynamics and minimal regulation that characterize SAR11 bacteria. This is the first characterization of polymer storage in SAR11, providing new insights into the likely fitness advantage for cells harboring this metabolism.

microbiology↗

bioRxiv2024

Emergent Metabolic Niches for Marine Heterotrophs

Ocean microbial communities are made up of thousands of diverse taxa whose metabolic demands set the rates of both biomass production and degradation. Thus, these microscopic organisms play a critical role in ecosystem dynamics, global carbon cycling, and climate. While we have frameworks for relating phytoplankton diversity to rates of carbon fixation, our knowledge of how variations in heterotrophic microbial populations drive changes in carbon cycling is in its infancy. Here, we leverage global metagenomic datasets and metabolic models to identify a set of metabolic niches with distinct growth strategies. These groupings provide a simplifying framework for describing microbial communities in different oceanographic regions and for understanding how heterotrophic microbial populations function. This framework, predicated directly on metabolic capability rather than taxonomy, enables us to tractably link heterotrophic diversity directly to biogeochemical rates in large scale ecosystem models.

ecology↗

bioRxiv2024

New isolates refine the ecophysiology of the Roseobacter CHAB-I-5 lineage

The CHAB-I-5 cluster is a pelagic lineage that can comprise a significant proportion of all roseobacters in surface oceans and have predicted roles in biogeochemical cycling via heterotrophy, aerobic anoxygenic photosynthesis (AAnP), CO oxidation, DMSP degradation, and other metabolisms. Though cultures of CHAB-I-5 have been reported, none have been explored and the best known representative, strain SB2, was lost from culture after obtaining the genome sequence. We have isolated two new CHAB-I-5 representatives, strains US3C007 and FZCC0083, and assembled complete, circularized genomes with 98.7% and 92.5% average nucleotide identities with the SB2 genome. Comparison of these three with 49 other unique CHAB-I-5 metagenome-assembled and single-cell genomes indicated that the cluster represents a genus with two species, and we identified subtle differences in genomic content between the two species subclusters. Metagenomic recruitment from over fourteen hundred samples expanded their known global distribution and highlighted both isolated strains as representative members of the clade. FZCC0083 grew over twice as fast as US3C007 and over a wider range of temperatures. The axenic culture of US3C007 occurs as pleomorphic cells with most exhibiting a coccobacillus/vibrioid shape. We propose the name Thalassovivens spotae, gen nov., sp. nov. for the type strain US3C007T.

microbiology↗

bioRxiv2023

The AEGEAN-169 clade of bacterioplankton is synonymous with SAR11 subclade V (HIMB59) and metabolically distinct

Bacterioplankton of the SAR11 clade are the most abundant marine microorganisms and consist of numerous subclades spanning Order level divergence (Pelagibacterales). The assignment of the earliest diverging subclade V (a.k.a. HIMB59) to the Pelagibacterales is highly controversial, with multiple recent phylogenetic studies placing them completely separate from SAR11. Other than through phylogenomics, subclade V has not received detailed examination due to limited genomes from this group. Here, we assessed the ecogenomic characteristics of subclade V to better understand the role of this group in comparison to the Pelagibacterales. We used a new isolate genome, recently released single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs), and previously established SAR11 genomes to perform a comprehensive comparative genomics analysis. We paired this analysis with recruitment of metagenomes spanning open ocean, coastal, and brackish systems. Phylogenomics, average amino acid identity, and 16S rRNA gene phylogeny indicate that SAR11 subclade V is synonymous with the ubiquitous AEGEAN-169 clade, and support the contention that this group represents a taxonomic Family. AEGEAN-169 shared many bulk genome qualities with SAR11, such as streamlining and low GC content, but genomes were generally larger. AEGEAN-169 had overlapping distributions with SAR11 but was metabolically distinct from SAR11 in its potential to transport and utilize a broader range of sugars as well as in transport of trace metals and thiamin. Thus, regardless of the ultimate phylogenetic placement of AEGEAN-169, these organisms have distinct metabolic capacities that likely allow them to differentiate their niche from canonical SAR11 taxa. IMPORTANCEOne goal of marine microbiologists is to uncover the roles various microorganisms are playing in biogeochemical cycles. Success in this endeavor relies on differentiating groups of microbes and circumscribing their relationships. An early-diverging group (subclade V) of the most abundant bacterioplankton, SAR11, has recently been proposed as a separate lineage that does not share a most recent common ancestor. But beyond phylogenetics, little has been done to evaluate how these organisms compare with SAR11. Our work leverages dozens of new genomes to demonstrate the similarities and differences between subclade V and SAR11. In our analysis, we also establish that subclade V is synonymous with a group of bacteria established from 16S rRNA gene sequences, AEGEAN-169. Subclade V/AEGEAN-169 has clear metabolic distinctions from SAR11 and their shared traits point to remarkable convergent evolution if they do not share a most recent common ancestor.

microbiology↗

bioRxiv2021

Ecophysiology of the cosmopolitan OM252 bacterioplankton (Gammaproteobacteria)

Among the thousands of species that comprise marine bacterioplankton communities, most remain functionally obscure. One key cosmopolitan group in this understudied majority is the OM252 clade of Gammaproteobacteria. Although frequently found in sequence data and even previously cultured, the diversity, metabolic potential, physiology, and distribution of this clade has not been thoroughly investigated. Here we examined these features of OM252 bacterioplankton using a newly isolated strain and genomes from publicly available databases. We demonstrated that this group constitutes a globally distributed novel genus (Candidatus Halomarinus), sister to Litoricola, comprising two subclades and multiple distinct species. OM252 organisms have small genomes (median 2.21 Mbp) and are predicted obligate aerobes capable of alternating between chemoorganoheterotrophic and chemolithotrophic growth using reduced sulfur compounds as electron donors, with subclade I genomes encoding the Calvin-Benson-Bassham cycle for carbon fixation. One representative strain of subclade I, LSUCC0096, had extensive halotolerance but a mesophilic temperature range for growth, with a maximum of 0.36 doublings/hr at 35{degrees}C. Cells were curved rod/spirillum-shaped, ~1.5 x 0.2 m. Growth on thiosulfate as the sole electron donor under autotrophic conditions was roughly one third that of heterotrophic growth, even though calculations indicated similar Gibbs energies for both catabolisms. These phenotypic data show that some Ca. Halomarinus organisms can switch between serving as carbon sources or sinks and indicate the likely anabolic cost of lithoautotrophic growth. Our results thus provide new hypotheses about the roles of these organisms in global biogeochemical cycling of carbon and sulfur. ImportanceMarine microbial communities are teeming with understudied taxa due to the sheer numbers of species in any given sample of seawater. One group, the OM252 clade of Gammaproteobacteria, has been identified in gene surveys from myriad locations, and one isolated organism has even been genome sequenced (HIMB30). However, further study of these organisms has not occurred. Using another isolated representative (strain LSUCC0096) and publicly available genome sequences from metagenomic and single-cell genomic datasets, we examined the diversity within the OM252 clade, the distribution of these taxa in the worlds oceans, reconstructed the predicted metabolism of the group, and quantified growth dynamics in LSUCC0096. Our results generate new knowledge about the previously enigmatic OM252 clade and point towards the importance of facultative chemolithoautotrophy for supporting some clades of ostensibly "heterotrophic" taxa.

microbiology↗

bioRxiv2018

Environmental DNA reveals the structure of phytoplankton assemblages along a 2900-km transect in the Mississippi River

The environmental health of aquatic ecosystems is critical to society, yet traditional assessments of water quality have limited utility for some bodies of water such as large rivers. Sequencing of environmental DNA (eDNA) has the potential to complement if not replace traditional sampling of biotic assemblages for the purposes of reconstructing aquatic assemblages and, by proxy, assessing water quality. Despite this potential, there has been little testing of the ability of eDNA to reconstruct assemblages and their absolute and relative utility to infer water quality metrics. Here, we reconstruct phytoplankton communities by amplifying and sequencing DNA from a portion of the 23S rRNA region from filtered water samples along a 2900-km transect in the Mississippi River. Across the entire length, diatoms dominated the assemblage (72.6%) followed by cryptophytes (8.7%) and cyanobacteria (7.0%). There were no general trends in the abundances of these major taxa along the length of the river, but individual taxon abundance peaked in different regions. For example, the abundance of taxa genetically similar to Melosira tropica peaked at approximately 60% of all reads 2750 km upstream from the Gulf of Mexico, while taxa similar to Skeletonema marinoi began to increase below the confluence with the Missouri River until it reached approximately 30% of the reads at the Gulf of Mexico. There were four main clusters of samples based on phytoplankton abundance, two above the confluence with the Missouri and two below. Phytoplankton abundance was a poor predictor of NH4 + concentrations in the water, but predicted 61% and 80% of the variation in observed NO3 -and PO4 3-concentrations, respectively. Phytoplankton richness increased with increasing distance along the river, but was best explained by phosphate concentrations and water clarity. Along the Mississippi transect, there was similar structure to phytoplankton and bacterial assemblages, indicating that the two sets of organisms are responding to similar environmental factors. In all, the research here demonstrates the potential utility of metabarcoding for reconstructing aquatic assemblages, which might aid in conducting water quality assessments.

ecology↗

bioRxiv2017

The CURE for Culturing Fastidious Microbes

Course-based Undergraduate Research Experiences (CUREs) expand the scientific educational benefits of research to large groups of students in a course setting. As part of an ongoing effort to integrate CUREs into first-year biology labs, we developed a microbiology CURE (mCURE) that uses a modified dilution-to-extinction high throughput culturing protocol for isolating abundant yet fastidious aquatic bacterioplankton during one semester. Students learn common molecular biology techniques like nucleic acid extraction, PCR, and molecular characterization; read and evaluate scientific literature; and receive training in scientific communication through written and oral exercises that incorporate social media elements. In the first three semesters, the mCUREs achieved similar cultivability success as implementation of the protocol in a standard laboratory setting. Our modular framework facilitates customization of the curriculum for use in multiple settings and we provide classroom exercises, assignments, assessment tools, and examples of student output to assist with implementation.

scientific communication and education↗

bioRxiv2016

Decoding bacterioplankton metabolism in the northern Gulf of Mexico Dead Zone

Marine regions that have seasonal to long-term low dissolved oxygen (DO) concentrations, sometimes called dead zones, are increasing in number and severity around the globe with deleterious effects on ecology and economics. One of the largest of these coastal dead zones occurs on the continental shelf of the northern Gulf of Mexico (nGOM), which results from eutrophication-enhanced bacterioplankton respiration and strong seasonal stratification. Previous research in this dead zone revealed the presence of multiple cosmopolitan bacterioplankton lineages that have eluded cultivation, and thus their metabolic roles in this ecosystem remain unknown. We used a coupled shotgun metagenomic and metatranscriptomic approach to determine the metabolic potential of Marine Group II Euryarchaeota, SAR406, and SAR202. We recovered multiple high-quality, nearly complete genomes from all three groups as well as those belonging to Candidate Phyla usually associated with anoxic environments-Parcubacteria (OD1) and Peregrinibacteria. Two additional groups with putative assignments to ACD39 and PAUC34f supplement the metabolic contributions by uncultivated taxa. Our results indicate active metabolism in all groups, including prevalent aerobic respiration, with concurrent expression of genes for nitrate reduction in SAR406 and SAR202, and dissimilatory nitrite reduction to ammonia and sulfur reduction by SAR406. We also report a variety of active heterotrophic carbon processing mechanisms, including degradation of complex carbohydrate compounds by SAR406, SAR202, ACD39, and PAUC34f. Together, these data help constrain the metabolic contributions from uncultivated groups in the nGOM during periods of low DO and suggest roles for these organisms in the breakdown of complex organic matter.\n\nImportanceDead zones receive their name primarily from the reduction of eukaryotic macrobiota (demersal fish, shrimp, etc.) that are also key coastal fisheries. Excess nutrients contributed from anthropogenic activity such as fertilizer runoff result in algal blooms and therefore ample new carbon for aerobic microbial metabolism. Combined with strong stratification, microbial respiration reduces oxygen in shelf bottom waters to levels unfit for many animals (termed hypoxia). The nGOM shelf remains one of the largest eutrophication-driven hypoxic zones in the world, yet despite its potential as a model study system, the microbial metabolisms underlying and resulting from this phenomenon--many of which occur in bacterioplankton from poorly understood lineages--have received only preliminary study. Our work details the metabolic potential and gene expression activity for uncultivated lineages across several low DO sites in the nGOM, improving our understanding of the active biogeochemical cycling mediated by these \"microbial dark matter\" taxa during hypoxia.

microbiology↗

bioRxiv2016

Insights on the importance of salinity from the first cultured freshwater SAR11 (LD12) representative

Evolutionary transitions between fresh and salt water happen infrequently among bacterioplankton. Within the ubiquitous and highly abundant heterotrophic Alphaproteobacteria order Pelagibacterales (SAR11), most members live in marine habitats, but the LD12 subclade has evolved as a unique freshwater lineage. LD12 cells occur as some of the most dominant freshwater bacterioplankton, yet this group has remained elusive to cultivation, hampering a more thorough understanding of its biology. Here, we report the first successful isolation of an LD12 representative, strain LSUCC0530, using high throughput dilution to extinction cultivation methods, and its complete genome sequence. Growth experiments corroborate ecological data suggesting active populations of LD12 in brackish water up to salinities of ~5. LSUCC0530 has the smallest closed genome thus far reported for a SAR11 strain (1.16 Mbp). The genome affirms many previous metabolic predictions from cultivation-independent analyses, like a complete Embden-Meyerhof-Parnas glycolysis pathway, but also provides novel insights, such as the first isocitrate dehydrogenase in LD12, a likely homologous recombination of malate synthase from outside of the SAR11 clade, and analogous substitutions of ion transporters with others that occur throughout the rest of the SAR11 clade. Growth data support metagenomic recruitment results suggesting temperature-based ecotype diversification within LD12. Key gene losses for osmolyte uptake provide a succinct hypothesis for the evolutionary transition of LD12 from salt to fresh water. For strain LSUCC0530, we propose the provisional nomenclature Candidatus Fonsibacter ubiquis.

microbiology↗

bioRxiv2016

Microbial regime changes and indicators of eutrophication on the Mississippi River identified via a human-powered 2900 km transect

Draining 31 states and roughly 3 million km2, the Mississippi River (MSR) and its tributaries constitute an essential resource to millions of people for clean drinking water, transportation, agriculture, and industry. Since the turn of the 20th century, MSR water quality has continually rated poorly due to human activity. Acting as first responders, microorganisms can mitigate, exacerbate, and/or serve as predictors for water quality, yet we know little about their community structure or ecology at the whole river scale for large rivers. We collected both biological (16S and 18S rRNA gene amplicons) and physicochemical data from 38 MSR sites over nearly 3000 km from Minnesota to the Gulf of Mexico. Our results revealed a microbial community composed of similar taxa to other rivers but with unique trends in the relative abundance patterns among phyla, OTUs, and the core microbiome. Furthermore, we observed a separation in microbial communities that mirrored the transition from an 8th to 10th Strahler order river at the Missouri River confluence, marking a different start to the lower MSR than the historical distinction at the Ohio River confluence in Cairo, IL. Within MSR microbial assemblages we identified subgroups of OTUs from the phyla Acidobacteria, Bacteroidetes, Oomycetes, and Heterokonts that were associated with, and predictive of, the important eutrophication nutrients nitrate and phosphate. This study offers the most comprehensive view of MSR microbiota to date, provides important groundwork for higher resolution microbial studies of river perturbation, and identifies potential microbial indicators of river health related to eutrophication.

microbiology↗