Biology subjects

Malmstrom, R. R.

Publications and source records attributed to Malmstrom, R. R..

12 recordsLinked to original sources

bioRxiv2025

Activity-targeted metaproteomics enhances the ecophysiological characterization of cryptic syntrophic metabolisms

Syntrophic microbial consortia can contribute significantly to the activity and function of anoxic ecosystems, yet are often too rare to study their in situ physiologies using traditional molecular methods. Here, we combined bioorthogonal non-canonical amino acid tagging (BONCAT), stable isotope probing, and metaproteomics to improve the recovery of proteins from active members and track isotope incorporation in an anaerobic digestion community. Both click chemistry-enabled cell-sorting and direct protein pulldown coupled to metaproteomics improved recovery of isotopically labeled proteins during anaerobic acetate oxidation. Resulting labeled protein expression profiles revealed elevated activity of a rare and so-far uncharacterized syntrophic bacterium belonging to the family Natronincolaceae. BONCAT-based capture of newly translated proteins provided direct molecular evidence for the expression of a previously hypothesized oxidative glycine pathway for syntrophic acetate oxidation by this microorganism, showcasing the potential of targeted metaproteomics to characterize rare and active cells central to community metabolism in natural and engineered ecosystems.

microbiology↗

bioRxiv2025

Tracking Persistence and Dynamics of Active Soil Viruses with SIP-Viromics

Viruses are abundant and ecologically important in soils, yet the persistence and production dynamics of extracellular virions remain poorly understood. We applied a genome-resolved stable isotope probing viromics (SIP-viromics) approach, combining H 18O labeling with viral metagenomics, to track virion turnover in seasonally dry grassland soils following rewetting. We identified 354 viral populations (vOTUs) using individual-sample and combined metagenome assemblies. Only 22% of vOTUs exhibited significant 18O enrichment, indicating active replication and new virion production during the 1-week incubation; the majority (78%) persisted without detectable replication, consistent with a viral seed bank. Active vOTUs accounted for 4.76-5.15% of total virions per gram of soil, with viral loads ranging from 3.15 x 1010 to 6.59 x 1010 virions per gram. Probabilistic and deterministic sensitivity analyses spanning viral DNA fraction and genome length reinforced that persistent virions represented the majority of the extracellular viral pool post-wet-up, regardless of parameter assumptions. Host predictions linked both active and persistent vOTUs primarily to Actinomycetota and Pseudomonadota--bacterial groups known to rapidly resuscitate following rewetting--suggesting that some viruses exhibit rapid turnover while others persist over longer timescales, forming a stable viral pool capable of reinitiating infections during favorable conditions. These results demonstrate that SIP-viromics can distinguish newly produced from persistent virions and reveal host-associated patterns of lytic infection and virion production. Our findings advance understanding of soil virus-host interactions and highlight the ecological role of persistent virions as a genetic reservoir contributing to microbial turnover and biogeochemical cycling following environmental disturbance. ImportanceUnderstanding the persistence and production dynamics of soil viruses is critical for elucidating their roles in microbial community dynamics and nutrient cycling, yet these processes have remained largely uncharacterized due to methodological limitations. By integrating stable isotope probing with viromics, this study provides a robust framework for directly distinguishing newly produced from persistent virions in situ. Unlike conventional viromics, which only catalogs viral diversity, SIP-viromics enables quantification of active viral replication and persistence under natural soil conditions. Our results demonstrate that most virions in a seasonally dry soil persisted through a rewetting event, with active replication limited to a minority of viral populations. Persistent virions were primarily linked to dominant bacterial groups, indicating that host ecophysiology and environmental stability strongly influence lytic infection. Collectively, these findings highlight viruses as long-term reservoirs of genetic material, capable of shaping microbial dynamics and ecosystem processes over time. This work establishes SIP-viromics as a powerful approach for studying virus-host interactions and their ecological significance in terrestrial environments.

microbiology↗

bioRxiv2024

Soil redox drives virus-host community dynamics and plant biomass degradation in tropical rainforest soils

BackgroundWet tropical forest soils store a vast amount of organic carbon and cycle over a third of terrestrial net primary production. The microbiomes of these soils have a global impact on greenhouse gases and tolerate a remarkably dynamic redox environment--driven by high availability of reductant, high soil moisture, and fine-textured soils that limit oxygen diffusion. Yet tropical soil microbiomes, particularly virus-host interactions, remain poorly characterized, and we have little understanding of how they will shape future soil carbon cycling as high-intensity drought and precipitation events make soil redox conditions less predictable. ResultsTo investigate the effects of shifting soil redox conditions on active viral communities and virus-microbe interactions, we conducted a 44-day redox manipulation experiment using soils from the Luquillo Experimental Forest, Puerto Rico, amended with 13C-enriched plant biomass. We sequenced 10 bulk metagenomes and 85 stable isotope probing targeted metagenomes generated by extracting whole community DNA, performing density fractionation, and conducting shotgun sequencing. Viral and microbial genomes were assembled resulting in 5,420 viral populations (vOTUs) and 927 medium-to-high-quality metagenome-assembled genomes across 25 bacterial phyla. Notably, over half (54%) of the vOTUs were 13C-enriched, highlighting their active role in microbial degradation of plant litter. These active vOTUs primarily infected bacterial phyla Pseudomonadota, Acidobacteriota, and Actinomycetota, and 57% were unique to a particular redox treatment. The anoxic samples exhibited the most distinct viral communities, with an increased potential for modulating host metabolism by carrying redox-specific glycoside hydrolases. However, over a third of the vOTUs were present in all redox conditions, suggesting selection for cosmopolitan viruses occurs in these soils that naturally experience dynamic redox conditions. ConclusionsOur study demonstrates how redox conditions shape viral communities and virus-host interactions in soils. By applying different DNA assembly methods on stable isotope probing targeted metagenomes and incubating soils under various redox regimes, we identified distinct viral populations and observed significant variations in viral community composition and function. These findings highlight the specialized roles of viruses in microbial carbon degradation under diverse environmental conditions, providing important insights into their contributions to carbon cycling and the broader implications for climate change.

microbiology↗

bioRxiv2023

Spatial Co-transcriptomics Reveals Discrete Stages of the Arbuscular Mycorrhizal Symbiosis

The symbiotic interaction of plants with arbuscular mycorrhizal fungi (AM fungi) is ancient and widespread. Plants provide AM fungi with carbon in exchange for nutrients and water, making this interaction a prime target for crop improvement. However, plant-fungal interactions are restricted to a small subset of root cells, precluding the application of most conventional functional genomic techniques to study the molecular bases of these interactions. Here we used single-nucleus and spatial RNA sequencing to explore both M. truncatula and R. irregularis transcriptomes in AM symbiosis at cellular and spatial resolution. Integrated spatially-registered single-cell maps of interacting cells revealed major infected and uninfected plant root cell types. We observed that cortex cells exhibit distinct transcriptome profiles during different stages of colonization by AM fungi, indicating dynamic interplay between both organisms during establishment of the cellular interface enabling successful symbiosis. Our study provides insight into a symbiotic relationship of major agricultural and environmental importance and demonstrates a paradigm combining single-cell and spatial transcriptomics for the analysis of complex organismal interactions.

plant biology↗

bioRxiv2022

A standardized quantitative analysis strategy for stable isotope probing metagenomics

Stable isotope probing (SIP) facilitates culture-independent identification of active microbial populations within complex ecosystems through isotopic enrichment of nucleic acids. Many SIP studies rely on 16S rRNA sequences to identify active taxa but connecting these sequences to specific bacterial genomes is often challenging. Here, we describe a standardized laboratory and analysis framework to quantify isotopic enrichment on a per-genome basis using shotgun metagenomics instead of 16S rRNA sequencing. To develop this framework, we explored various sample processing and analysis approaches using a designed microbiome where the identity of labeled genomes, and their level of isotopic enrichment, were experimentally controlled. With this ground truth dataset, we empirically assessed the accuracy of different analytic models for identifying active taxa, and examined how sequencing depth impacts the detection of isotopically labeled genomes. We also demonstrate that using synthetic DNA internal standards to measure absolute genome abundances in SIP density fractions improves estimates of isotopic enrichment. In addition, our study illustrates the utility of internal standards to reveal anomalies in sample handling that could negatively impact SIP metagenomic analyses if left undetected. Finally, we present SIPmg, an R package to facilitate the estimation of absolute abundances and perform statistical analyses for identifying labeled genomes within SIP metagenomic data. This experimentally validated analysis framework strengthens the foundation of DNA-SIP metagenomics as a tool for accurately measuring the in situ activity of environmental microbial populations and assessing their genomic potential. ImportanceAnswering the question of who is eating what? within complex microbial communities is paramount for our ability to model, predict, and modulate microbiomes for improved human and planetary health. This question is often pursued using stable isotope probing to track the incorporation of labeled compounds into cellular DNA during microbial growth. However, with traditional stable isotope methods, it is challenging to establish links between an active microorganisms taxonomic identity and genome composition, while providing quantitative estimates of the microorganisms isotope incorporation rate. Here, we report an experimental and analytical workflow that lays the foundation for improved detection of metabolically active microorganisms and better quantitative estimates of genome-resolved isotope incorporation, which can be used to further refine ecosystem-scale models for carbon and nutrient fluxes within microbiomes.

microbiology↗

bioRxiv2020

Host population diversity as a driver of viral infection cycle in wild populations of green sulfur bacteria with long standing virus-host interactions

Viral infections of bacterial hosts range from highly lytic to lysogenic, where highly lytic viruses undergo viral replication and immediately lyse their hosts, and lysogenic viruses have a latency period before replication and host lysis. While both types of infections are routinely observed in the environment, the ecological and evolutionary processes that regulate these different viral dynamics are still not well understood. In this study, we identify and characterize the long-term dynamics of uncultivated viruses infecting green sulfur bacteria (GSB) in a model freshwater lake sampled from 2005-2018. Because of the additional requirements for the laboratory cultivation of anaerobes like GSB, viruses infecting GSB have yet to be formally identified, leaving their diversity and impact on natural populations of GSB virtually unknown. In this study, we used two approaches to identify viruses infecting GSB; one in vitro based on flow cytometry cell sorting, the other in silico based on CRISPR spacer sequences. We then took advantage of existing bulk metagenomes derived from Trout Bog Lake covering the 2005-2018 period to examine the interactions between GSB hosts and their viruses across multiple years and seasons. From our data, GSB populations in Trout Bog Lake were found to be concurrently infected with at least 2-8 viruses each, many of which were lysogenic viruses; one GSB host population in particular was consistently associated with two lysogens with a nearly 100% infection rate for over 10 years. We illustrate with a theoretical infection model that such an interaction can be stable over multiple years given a low, but persistent level of lysogen induction in host populations with already high infection rates. Overall, our data suggest that single GSB populations are typically infected by multiple viruses at the same time, that lytic and lysogenic viruses can readily co-infect the same host population in the same ecosystem, and that host strain-level diversity might be an important factor controlling the lytic/lysogeny switch.

microbiology↗

bioRxiv2020

Measurement error and resolution in quantitative stable isotope probing: implications for experimental design

Quantitative stable isotope probing (qSIP) estimates the degree of incorporation of an isotope tracer into nucleic acids of metabolically active organisms and can be applied to microorganisms growing in complex communities, such as the microbiomes of soil or water. As such, qSIP has the potential to link microbial biodiversity and biogeochemistry. As with any technique involving quantitative estimation, qSIP involves measurement error; a more complete understanding of error, precision and statistical power will aid in the design of qSIP experiments and interpretation of qSIP data. We used several existing qSIP datasets of microbial communities found in soil and water to evaluate how variance in the estimate of isotope incorporation depends on organism abundance and on the resolution of the density fractionation scheme. We also assessed statistical power for replicated qSIP studies, and sensitivity and specificity for unreplicated designs. We found that variance declines as taxon abundance increases. Increasing the number of density fractions reduces variance, although the benefit of added fractions declines as the number of fractions increases. Specifically, nine fractions appear to be a reasonable tradeoff between cost and precision for most qSIP applications. Increasing replication improves power and reduces the minimum detectable threshold for inferring isotope uptake to 5 atom%. Finally, we provide evidence for the importance of internal standards to calibrate the %GC to mean weighted density regression per sample. These results should benefit those designing future SIP experiments, and provide a reference for metagenomic SIP applications where financial and computational limitations constrain experimental scope. ImportanceOne of the biggest challenges in microbial ecology is correlating the identity of microorganisms with the roles they fulfill in natural environmental systems. Studies of microbes in pure culture reveal much about genomic content and potential functions, but may not reflect an organisms activity within its natural community. Culture-independent studies supply a community-wide view of composition and function in the context of community interactions, but fail to link the two. Quantitative stable isotope probing (qSIP) is a method that can link the identity and function of specific microbes within a naturally occurring community. Here we explore how the resolution of density-gradient fractionation affects the error and precision of qSIP results, how they may be improved via additional replication, and cost-benefit balanced scenarios for SIP experimental design.

microbiology↗

bioRxiv2019

Niche differentiation is spatially and temporally regulated in the rhizosphere

The rhizosphere is a hotspot for microbial C transformations, and the origin of root polysaccharides and polymeric carbohydrates that are important precursors to soil organic matter. However, the ecological mechanisms that underpin rhizosphere carbohydrate depolymerization are poorly understood. Using Avena fatua, a common annual grass, we analyzed time-resolved metatranscriptomes to compare microbial function in rhizosphere, detritusphere, and combined rhizosphere-detritusphere habitats. Population transcripts were binned with a unique reference database generated from soil isolate and single amplified genomes, metagenomes, and stable isotope probing metagenomes. While soil habitat significantly affected both community composition and overall gene expression, succession of microbial functions occurred at a faster time scale than compositional changes. Using hierarchical clustering of upregulated decomposition gene expression, we identified four distinct microbial guilds populated by taxa whose functional succession patterns suggest specialization for substrates provided by fresh growing roots, decaying root detritus, the combination of live and decaying root biomass, or aging root material. Carbohydrate depolymerization genes were consistently upregulated in the rhizosphere, and both taxonomic and functional diversity were high in the combined rhizosphere-detritusphere--suggesting coexistence of rhizosphere guilds is facilitated by niche differentiation. Metatranscriptome-defined guilds provide a framework to model rhizosphere succession and its consequences for soil carbon cycling.

ecology↗

bioRxiv2018

Study of Oak Ridge soils using BONCAT-FACS-Seq reveals that a large fraction of the soil microbiome is active

The ability to link soil microbial diversity to soil processes requires technologies that differentiate active subpopulations of microbes from so-called relic DNA and dormant cells. Measures of microbial activity based on various techniques including DNA labelling have suggested that most cells in soils are inactive, a fact that has been difficult to reconcile with observed high levels of bulk soil activities. We hypothesized that measures of in situ DNA synthesis may be missing the soil microbes that are metabolically active but not replicating, and we therefore applied BONCAT (Bioorthogonal Non Canonical Amino Acid Tagging) i.e. a proxy for activity that does not rely on cell division, to measure translationally active cells in soils. We compared the active population of two soil depths from Oak Ridge (TN) incubated under the same conditions for up to seven days. Depending on the soil, a maximum of 25 - 70% of the cells were active, accounting for 3-4 million cells per gram of soil type, which is an order of magnitude higher than previous estimates. The BONCAT positive cell fraction was recovered by fluorescence activated cell sorting (FACS) and identified by 16S rDNA amplicon sequencing. The diversity of the active fraction was a selected subset of the bulk soil community. Excitingly, some of the same members of the community were recruited at both depths independently from their abundance rank. On average, 86% of sequence reads recovered from the active community shared >97% sequence similarity with cultured isolates from the field site. Our observations are in line with a recent report that, of the few taxa that are both abundant and ubiquitous in soil, 45% are also cultured - and indeed some of these ubiquitous microorganisms were found to be translationally active. The use of BONCAT on soil microbiomes provides evidence that a large portion of the soil microbes can be active simultaneously. We conclude that BONCAT coupled to FACS and sequencing is effective for interrogating the active fraction of soil microbiomes in situ and provides new perspectives to link metabolic capacity to overall soil ecological traits and processes.

microbiology↗

bioRxiv2017

An Oxidative Pathway of Deoxyribose Catabolism

Using genome-wide mutant fitness assays in diverse bacteria, we identified novel oxidative pathways for the catabolism of 2-deoxy-D-ribose and 2-deoxy-D-ribonate. We propose that deoxyribose is oxidized to deoxyribonate, oxidized to ketodeoxyribonate, and cleaved to acetyl-CoA and glyceryl-CoA. We have genetic evidence for this pathway in three genera of bacteria, and we confirmed the oxidation of deoxyribose to ketodeoxyribonate in vitro. In Pseudomonas simiae, the expression of enzymes in the pathway is induced by deoxyribose or deoxyribonate, while in Paraburkholderia bryophila and in Burkholderia phytofirmans, the pathway proceeds in parallel with the known deoxyribose 5-phosphate aldolase pathway. We identified another oxidative pathway for the catabolism of deoxyribonate, with acyl-CoA intermediates, in Klebsiella michiganensis. Of these four bacteria, only P. simiae relies entirely on an oxidative pathway to consume deoxyribose. The deoxyribose dehydrogenase of P. simiae is either non-specific or evolved recently, as this enzyme is very similar to a novel vanillin dehydrogenase from Pseudomonas putida that we identified. So, we propose that these oxidative pathways evolved primarily to consume deoxyribonate, which is a waste product of metabolism.\n\nImportanceDeoxyribose is one of the building blocks of DNA and is released when cells die and their DNA degrades. We identified a bacterium that can grow with deoxyribose as its sole source of carbon even though its genome does not encode any of the known genes for breaking down deoxyribose. By growing many mutants of this bacterium together on deoxyribose and using DNA sequencing to measure the change in the mutants abundance, we identified multiple protein-coding genes that are required for growth on deoxyribose. Based on the similarity of these proteins to enzymes of known function, we propose a 6-step pathway in which deoxyribose is oxidized and then cleaved. Diverse bacteria use a portion of this pathway to break down a related compound, deoxyribonate, which is a waste product of human metabolism and is present in urine. Our study illustrates the utility of large-scale bacterial genetics to identify previously unknown metabolic pathways.

microbiology↗

bioRxiv2017

Ecophysiology of freshwater Verrucomicrobia inferred from genomes recovered through time-series metagenomics

Microbes are critical in carbon and nutrient cycling in freshwater ecosystems. Members of the Verrucomicrobia are ubiquitous in such systems, yet their roles and ecophysiology are not well understood. In this study, we recovered 19 Verrucomicrobia draft genomes by sequencing 184 time-series metagenomes from a eutrophic lake and a humic bog that differ in carbon source and nutrient availabilities. These genomes span four of the seven previously defined Verrucomicrobia subdivisions, and greatly expand the known genomic diversity of freshwater Verrucomicrobia. Genome analysis revealed their potential role as (poly)saccharide-degraders in freshwater, uncovered interesting genomic features for this life style, and suggested their adaptation to nutrient availabilities in their environments. Between the two lakes, Verrucomicrobia populations differ significantly in glycoside hydrolase gene abundance and functional profiles, reflecting the autochthonous and terrestrially-derived allochthonous carbon sources of the two ecosystems respectively. Interestingly, a number of genomes recovered from the bog contained gene clusters that potentially encode a novel porin-multiheme cytochrome c complex and might be involved in extracellular electron transfer in the anoxic humic-rich environment. Notably, most epilimnion genomes have large numbers of so-called \"Planctomycete-specific\" cytochrome c-containing genes, which exhibited nearly opposite distribution patterns with glycoside hydrolase genes, probably associated with the different environmental oxygen availability and carbohydrate complexity between lakes/layers. Overall, the recovered genomes are a major step towards understanding the role, ecophysiology and distribution of Verrucomicrobia in freshwater.\n\nIMPORTANCEFreshwater Verrucomicrobia are cosmopolitan in lakes and rivers, yet their roles and ecophysiology are not well understood, as cultured freshwater Verrucomicrobia are restricted to one subdivision of this phylum. Here, we greatly expand the known genomic diversity of this freshwater lineage by recovering 19 Verrucomicrobia draft genomes from 184 metagenomes collected from a eutrophic lake and a humic bog across multiple years. Most of these genomes represent first freshwater representatives of several Verrucomicrobia subdivisions. Genomic analysis revealed Verrucomicrobia as potential (poly)saccharide-degraders, and suggested their adaptation to carbon source of different origins in the two contrasting ecosystems. We identified putative extracellular electron transfer genes and so-called \"Planctomycete-specific\" cytochrome c-containing genes, and found their distinct distribution patterns between the lakes/layers. Overall, our analysis greatly advances the understanding of the function, ecophysiology and distribution of freshwater Verrucomicrobia, while highlighting their potential role in freshwater carbon cycling.

microbiology↗

bioRxiv2017

High-Throughput Metabolic Network Analysis and Metatranscriptomics of a Cosmopolitan and Streamlined Freshwater Lineage

An explosion in the number of available genome sequences obtained through metagenomics and single-cell genomics has enabled a new view of the diversity of microbial life, yet we know surprisingly little about how microbes interact with each other or their environment. In fact, the majority of microbial species remain uncultivated, while our perception of their ecological niches is based on reconstruction of their metabolic potential. In this work, we demonstrate how the \"seed set framework\", which computes the set of compounds that an organism must acquire from its environment (1), enables computational analysis of metabolic reconstructions, while providing new insights into a microbes metabolic capabilities, such as nutrient use and auxotrophies. We apply this framework to members of the ubiquitous freshwater Actinobacterial lineage acI, confirming and extending previous experimental and genomic observations implying that acI bacteria are heterotrophs reliant on peptides and saccharides. We also present the first metatranscriptomic study of the acI lineage, revealing high expression of transport proteins and the light-harvesting protein actinorhodopsin. Putative transport proteins complement predictions of nutrients and essential metabolites while providing additional support to the hypothesis that members of the acI are photoheterotrophs.\n\nImportanceThe metabolic activity of uncultivated microorganisms contributes to numerous ecosystem processes, ranging from nutrient cycling in the environment to influencing human health and disease. Advances in sequencing technology have enabled the assembly of genomes for these microorganisms, but our ability to generate reference genomes far outstrips our ability to analyze them. Common approaches to analyzing microbial metabolism require reconstructing the entirety of an organisms metabolic pathways, or performing targeted searches for genes involved in a specific process. This paper presents a third approach, in which draft metabolic reconstructions are used to identify compounds through which an organism may interact with its environment. These compounds can then guide more intensive metabolic reconstruction efforts, and also provide new hypotheses about the specific contributions microbes make to ecosystem-scale metabolic processes.

microbiology↗