Biology subjects

Hofmockel, K.

Publications and source records attributed to Hofmockel, K..

10 recordsLinked to original sources

bioRxiv2026

Mobile genetic elements are active and responsive to community context in model microbial consortium

Insertion and excision of genomic islands (GIs), chromosomally-integrated mobile genetic elements (MGEs), are major sources of microbial genome plasticity and can impact gene expression and phenotype of the host organism. GI mobilization also influences microbial communities beyond the host organism as GI excision generates MGEs that can be transferred between community members through horizontal gene transfer and induction of prophages can kill host populations, which impacts community structure. Established computational methods now enable precise GI mapping in genomes, as well as highly sensitive detection of GI excision from deep-genome sequencing data. We applied these approaches to metagenomic datasets from a defined soil microbial consortium grown on glass beads under hydration stress and compared GI activity with that observed in monoculture. Under these environmentally structured community growth conditions, GI excision was more abundant and involved a broader range of host species and GI types than under isolate growth conditions. Combined analysis with metatranscriptomic and metaproteomic data identified patterns of GI gene expression associated with induction. Three GIs showed particularly high excision together with strong transcription, numerous detected proteins, and evidence of association with potential transfer particles, including phages or vesicles. These results indicate that isolate studies can miss a substantial environmentally responsive layer of GI activity. More broadly, this work establishes a framework for mining existing community multi-omic datasets to quantify dynamic genome restructuring, identify active but poorly understood GIs, and generate mechanistic hypotheses about the processes that shape microbial genome plasticity and gene flow.

microbiology↗

bioRxiv2026

A truncated soil phage catechol 1,2-dioxygenase illustrates how viruses preserve and disseminate auxiliary catalytic functions in the soil microbiome

Bacteriophages can rewire host chemistry via auxiliary viral genes (AVGs). Using metagenomic and metatranscriptomic data from the native soil microbiome, we identified transcriptionally active AVGs, including a viral catechol 1,2-dioxygenase (V-C12DO). V-C12DO shares [~]40% sequence identity with its nearest bacterial homologs and lacks the helical dimerization domain. Despite truncation, V-C12DO retains more than [~]25% of the global consensus residues compared to C12DOs across domains of life, including the two tyrosines and two histidines that coordinate the non-heme Fe(III) active site. A 1.7 [A] crystal structure also showed the conservation of the canonical {beta}-sandwich scaffold for the iron. We next confirmed that V-C12DO cleaves catechol and remains highly active across a broad range of temperatures (30-60 {degrees}C), pH (5.5-9), and salinity (up to 2 M), exceeding those of known bacterial CD12Os. This work shows that truncated phage enzymes preserve the core catalytic chemistry and potentially further expand host metabolic versatility across dynamic environmental conditions.

microbiology↗

bioRxiv2024

Environmental matrix and moisture are key determinants of microbial phenotypes expressed in a reduced complexity soil-analog

Soil moisture and porosity regulate microbial metabolism by influencing factors such as redox conditions, substrate availability, and soil connectivity. However, the inherent biological, chemical, and physical heterogeneity of soil complicates laboratory investigations into microbial phenotypes that mediate community metabolism. This difficulty arises from challenges in accurately representing the soil environment and in establishing a tractable microbial community that limits confounding variables. To address these challenges in our investigation of community metabolism, we use a reduced-complexity microbial consortium grown in a soil analog using a glass-bead matrix amended with chitin. Long-read and short-read metagenomes, metatranscriptomes, metaproteomes, and metabolomes were analyzed to test the effects of soil structure and moisture on chitin degradation. Our soil structure analog system greatly altered microbial expression profiles compared to the liquid-only incubations, emphasizing the importance of incorporating environmental parameters, like pores and surfaces, for understanding microbial phenotypes relevant to soil ecosystems. These changes were mainly driven by differences in overall expression of chitin-degrading Streptomyces species and stress-tolerant Ensifer. Our findings suggest that the success of Ensifer in a structured environment is likely related to its ability to repurpose carbon via the glyoxylate shunt while potentially using polyhydroxyalkanoate granules as a C source. We also identified traits like motility, stress resistance, and biofilm formation that underlie the degradation of chitin across our treatments and inform how they may ultimately alter carbon use efficiency. Together our results demonstrate that community functions like decomposition are sensitive to environmental conditions and more complex than the multi-enzyme pathways involved in depolymerization. ImportanceSoil moisture and porosity are critical mediators of microbial metabolism by influencing factors such as redox conditions, substrate availability, and soil connectivity. However, identifying how microbial community metabolism shifts in response to varying levels of moisture and porosity remains a challenging frontier. This difficulty arises from challenges in accurately representing the soil environment and in establishing tractable microbial communities that limit confounding variables. Moreover, inferring phenotypes based on "key" genes often fails to predict complex phenotypes that arise from cellular interactions. Here, we establish a tractably complex microbial community in a soil analog system amended with chitin and leverage it to understand how microorganisms respond to changes in porosity and moisture. By using genome-resolved metagenomics, metatranscriptomics, and metaproteomics, we report on the microbial lifestyle strategies that underpin changes in community expression like carbon conservation, biofilm production, and stress response.

microbiology↗

bioRxiv2024

Improving the visualization of viruses in soil

Viruses are numerically the most abundant forms on Earth, and most are present in soil. Even though viruses are highly abundant in soil and critical to rhizosphere function, visualizing the diverse morphotypes within soil has been challenging. The difficulty is primarily due to the heterogenous nature of isolated suspensions that typically contain nanometer to micron scale debris which renders protein crystallography for structural studies unfeasible and hinders cryo-electron microscopy due to ice thickness and contrast issues. Here we employed and compared a simple spin filtration method to cleanup solutions of extracted viruses for direct observation with cryo-electron microscopy. Although relatively simple, the method employs common physical biochemical separation steps to remove large and small debris which dramatically improves image quality and preservation of structural features to permit visualizing morphotypes not typically seen with conventional negative stain approaches. In addition to tailed and non-tailed polyhedral phages, several under reported or novel morphotypes of soil viruses are directly visualized as a particle library with both 2D and 3D information.

microbiology↗

bioRxiv2024

Unveiling lipid chemodiversity in root exudates: A comprehensive characterizaton of the exudate metabo-lipidome in Tall Wheatgrass

The rhizosphere, where plant roots meet soil, is a hub of biogeochemical activity with ecosystem impacts on carbon stocks. Root derived carbon has been found to contribute more to soil carbon stocks than aboveground litter. Nonetheless, the molecular chemodiversity of root exudates remains poorly understood due to limited characterization and annotation. Here our goal was to discover the molecular chemodiversity of metabolites and lipids in root exudates to advance our understanding of plant root inputs belowground. We worked with mature, field-grown tall wheatgrass (Thinopyrum ponticum) and optimized exudate collection protocols to enable the capture of non-polar lipids in addition to polar and semi-polar metabolites. Rates of carbon input via hydrophobic exudates were approximately double that of aqueous exudates and carbon/nitrogen ratios were markedly higher in hydrophobic compared to aqueous exudates, emphasizing the importance of lipids, due to their high carbon content. To maximize molecular coverage of exudate chemodiversity, we used liquid chromatography coupled tandem mass-spectrometry for paired untargeted metabolomics and lipidomics or metabo-lipidomics. We substantially increased the characterization of exudate chemodiversity by employing both tandem mass spectral library searching and deep learning-based chemical class assignment. Notably, in this unprecedented characterization of intact lipids in root exudates, we discovered a diverse variety of lipids, including substantial levels of triacylglycerols ([~]19 g/g fresh root per min), fatty acyls, sphingolipids, sterol lipids, and glycerophospholipids. Comparison of the root exudate and tissue lipidomes revealed minimum glycerophospholipids in exudates, suggesting the exudate protocol did not extract lipids from root cell membranes.

plant biology↗

bioRxiv2023

Using methyl bromide for interspecies cell-cell signaling and as a reporter in a model soil consortium

Soil microbial communities with reduced complexity are emerging as model systems for studying consortia-scale phenotypes. To establish synthetic biology tools for studying these communities in hard-to-image environmental materials, we evaluated whether a single member of a model soil consortium (MSC) can be programmed to report on gene expression without requiring matrix disruption. For these studies, we targeted a five member MSC that includes Dyadobacter fermentans, Ensifer adhaerens, Rhodococcus sp003130705, Streptomyces sp001905665, and Variovorax beijingensis. By coupling the expression of a methyl halide transferase to a constitutive promoter, we show that Variovorax beijingensis can be programmed to synthesize methyl halides that accumulate in the soil headspace at levels that are [≥]24-fold higher than all other MSC members across a range of environmentally-relevant hydration conditions. We find that methyl halide production can report on a MSC promoter that is activated by changes in water potential, and we demonstrate that a synthetic gas signal can be read out directly using gas chromatography and indirectly using a soil-derived Methylorubrum that is programmed to produce a visual output in response to methyl halides. These tools will be useful for future studies that investigate how MSC respond to dynamic hydration conditions, such as drought and flood events induced by climate change, which can alter soil water potential and induce the release of stored carbon.

synthetic biology↗

bioRxiv2022

Bulk and spatially resolved extracellular metabolomics of free-living nitrogen fixation

Soil microorganisms drive ecosystem function, but challenges of scale between microbe and ecosystem hinder our ability to accurately quantify and predictively model the soil microbe-ecosystem function relationship. Quantifying this relationship necessitates studies that systematically characterize multi-omics of soil microorganisms and their activity across sampling scales from spatially resolved to bulk measures, and structural complexity, from liquid pure culture to in situ. To address this need, we cultured two diazotrophic bacteria in liquid and solid media, with and without nitrogen (N) to quantify differences in extracellular metabolites associated with nitrogen fixation under increasing environmental structural complexity. We also quantified extracellular metabolites across sampling scales including bulk sampling via GC-MS analysis and spatially resolved analysis via MALDI mass spectrometry imaging. We found extracellular production of inorganic and organic N during free-living nitrogen fixation activity, highlighting a key mechanism of terrestrial N contributions from this process. Additionally, our results emphasize the need to consider the structural complexity of the environment and spatial scale when quantifying microbial activity. We found differences in metabolite profiles between culture conditions, supporting previous work indicating environmental structure influences microbial function, and across scales, underscoring the need to quantify microbial scale conditions to accurately interpret microbial function. ImportanceStudying soil microorganisms, both who is present and what they are doing, is a challenge because of vast differences in scale between microorganism and ecosystem and because of inherent complexities of the soil system (e.g., opacity, chemical complexity). This makes measuring and predicting important ecosystem processes driven by soil microorganisms, like free-living nitrogen fixation, difficult. Free-living nitrogen fixing bacteria play a key role in terrestrial nitrogen contributions and may represent a significant, yet overlooked, nitrogen source in agricultural systems like bioenergy crops. However, we still know very little about how free-living nitrogen fixation contributes nitrogen to terrestrial systems. Our work provides key insight by hierarchically increasing structural complexity (liquid vs. solid culture) and scale (spatially resolved vs. bulk) to address the impact of environmental structure and sampling scale on detection of free-living nitrogen fixation and to identify the forms of nitrogen contributed to terrestrial systems by free-living nitrogen bacteria.

microbiology↗

bioRxiv2021

The Functional Significance of Bacterial Predators

Predation structures food webs, influences energy flow, and alters rates and pathways of nutrient cycling through ecosystems, effects that are well documented for macroscopic predators. In the microbial world, predatory bacteria are common, yet little is known about their rates of growth and roles in energy flows through microbial food webs, in part because these are difficult to quantify. Here, we show that growth and carbon uptake were higher in predatory bacteria compared to non-predatory bacteria, a finding across 15 sites, synthesizing 82 experiments and over 100,000 taxon-specific measurements of element flow into newly synthesized bacterial DNA. Obligate predatory bacteria grew 36% faster and assimilated carbon at rates 211% higher than non-predatory bacteria. These differences were less pronounced for facultative predators (6% higher growth rates, 17% higher carbon assimilation rates), though high growth and carbon assimilation rates were observed for some facultative predators, such as members of the genera Lysobacter and Cytophaga, both capable of gliding motility and wolfpack hunting behavior. Added carbon substrates disproportionately stimulated growth of obligate predators, with responses 63% higher than non-predators for the Bdellovibrionales and 81% higher for the Vampirovibrionales, whereas responses of facultative predators to substrate addition were no different from non-predators. This finding supports ecological theory that higher productivity increases predator control of lower trophic levels. These findings also indicate that the functional significance of bacterial predators increases with energy flow, and that predatory bacteria influence element flow through microbial food webs.

ecology↗

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↗

bioRxiv2018

High Genetic Potential for Proteolytic Decomposition in Northern Peatland Ecosystems

AbstractNitrogen (N) is a scarce nutrient commonly limiting primary productivity. Microbial decomposition of complex carbon (C) into small organic molecules (e.g., free amino acids) has been suggested to supplement biologically-fixed N in high latitude peatlands. We evaluated the microbial (fungal, bacterial, and archaeal) genetic potential for organic N depolymerization in peatlands at Marcell Experimental Forest (MEF) in northern Minnesota. We used guided gene assembly to examine the abundance and diversity of protease genes; and further compared to those of N-fixing (nifH) genes in shotgun metagenomic data collected across depth at two distinct peatland environments (bogs and fens). Microbial proteases greatly outnumbered nifH genes with the most abundant gene families (archaeal M1 and bacterial Trypsin) each containing more sequences than all sequences attributed to nifH. Bacterial protease gene assemblies were diverse and abundant across depth profiles, indicating a role for bacteria in releasing free amino acids from peptides through depolymerization of older organic material and contrasting the paradigm of fungal dominance in depolymerization in forest soils. Although protease gene assemblies for fungi were much less abundant overall than for bacteria, fungi were prevalent in surface samples and therefore may be vital in degrading large soil polymers from fresh plant inputs during early stage of depolymerization. In total, we demonstrate that depolymerization enzymes from a diverse suite of microorganisms, including understudied bacterial and archaeal lineages, are likely to play a substantial role in C and N cycling within northern peatlands.\n\nImportanceNitrogen (N) is a common limitation on primary productivity, and its source remains unresolved in northern peatlands that are vulnerable to environmental change. Decompositionof complex organic matter into free amino acids has been proposed as an important N source, but the genetic potential of microorganisms mediating this process has not been examined. Such information can elucidate possible responses of northern peatlands to environmental change. We show high genetic potential for microbial production of free amino acids across a range of microbial guilds. In particular, the abundance and diversity of bacterial genes encoding proteolytic activity suggests a predominant role for bacteria in regulating productivity and contrasts a paradigm of fungal dominance of organic N decomposition. Our results expand our current understanding of coupled carbon and nitrogen cycles in north peatlands and indicate that understudied bacterial and archaeal lineages may be central in this ecosystems response to environmental change.

ecology↗