Biology subjects

Firestone, M. K.

Publications and source records attributed to Firestone, M. K..

30 records · Page 2Linked to original sources

bioRxiv2021

Community RNA-Seq: Multi-kingdom responses to living versus decaying root inputs in soil

Roots are the primary source of organic carbon inputs to most soils. Decomposition is a multi-trophic process involving multiple kingdoms of microbial life, but typically microbial ecology studies focus on one or two major lineages in isolation. We used Illumina shotgun RNA sequencing to conduct PCR-independent SSU rRNA community analysis ("community RNA-Seq") to simultaneously study the bacteria, archaea, fungi, and microfauna surrounding both living and decomposing roots of the annual grass, Avena fatua. Plants were grown in 13CO2-labeled microcosms amended with 15N-root litter. We identified rhizosphere substrate preferences for 13C-exudates versus 15N-litter using NanoSIMS microarray imaging (Chip-SIP). When litter was available, rhizosphere and bulk soil had significantly more Amoebozoa, which are potentially important yet often overlooked top-down drivers of detritusphere community dynamics and nutrient cycling. Bulk soil containing litter was depleted in Actinobacteria but had significantly more Bacteroidetes and Proteobacteria. While Actinobacteria were abundant in the rhizosphere, Chip-SIP showed Actinobacteria preferentially incorporated litter relative to root exudates, indicating this groups more prominent role in detritus elemental cycling in the rhizosphere. Our results emphasize that decomposition is a multi-trophic process involving cross-kingdom interactions, and the trajectory of carbon through this soil food web likely impacts the fate of carbon in soil.

microbiology↗

bioRxiv2020

Methane-derived carbon flow through host-virus trophic networks in soil

The concentration of atmospheric methane continues to increase with microbial communities controlling soil-atmosphere fluxes. While there is substantial knowledge of the diversity and function of organisms regulating methane production and consumption, the frequency and impact of interactions with viruses on their activity in soil is unknown. Metagenomic sequencing of soil microbial communities has enabled identification of linkages between viruses and hosts. However, determining host-virus linkages through sequencing does not determine whether a virus or a host are active. In this study, we identified active individual interactions in situ by following the transfer of assimilated carbon from active hosts to viruses. Using DNA stable-isotope probing combined with metagenomic analyses, we characterized methane-fueled microbial networks in acidic and neutral pH soils, specifically primary and secondary utilisers of carbon, together with the recent transfer of methane-derived carbon to viruses. Sixty-three percent of viral contigs from replicated soil incubations contained genes associated with known methanotrophic bacteria. Genomic sequences from 13C-enriched viruses were present in clustered regularly interspaced short palindromic repeats (CRISPR) arrays of multiple, closely-related Methylocystis populations, revealing differences in their history of viral interaction. Viruses infecting non-methanotrophic methylotrophs and heterotrophic predatory bacteria were also identified through the analysis of shared homologous genes, demonstrating that carbon is transferred to a diverse range of viruses associated with methane-fueled microbial food networks.

microbiology↗

bioRxiv2020

Transcription of protease and chitinase genes provides a window onto macromolecular organic nitrogen decomposition in soil

Nitrogen (N) commonly limits terrestrial plant growth partly because most soil-N is present as macromolecular organic compounds and not directly available to plants. Soil microbes degrade these large N-containing substrates to gradually release plant-available inorganic-N throughout the growing season, potentially meeting plant demand. Knowing which microbes are responsible for release of organic N, as well as their spatiotemporal patterns of activity, can enable microbial management strategies that increase plant access to soil-N and reduce dependency on fertilizer-N. We used time-resolved metatranscriptomes to follow taxonomy-resolved differential expression of N-depolymerizing enzymes. Taxonomic groups show adaptations based on extracellular proteases to specialized habitats in soil characterized by presence (Betaproteobacteria) or absence (Thaumarcheota) of live roots and root detritus (Deltaproteobacteria and Fungi). A similar increase of eukaryotic chitinases near root detritus hints at predation of fungi. Others demonstrate temporal patterns such as increasing expression over time, implying increased competitiveness with substrate depletion (Chloroflexi). Phylotypes from the same genus can have different potential benefits to the plant based on protease expression (e.g., Janthinobacterium), which should be considered when designing bioaugmentation. We identify one Janthinobacterium phylotype and two Burkholderiales that may be candidates for bioaugmentation near young roots and a Rhizobacter which could benefit mature roots.

microbiology↗

bioRxiv2020

Routes to Roots: Direct Evidence of Water Transport by Arbuscular Mycorrhizal Fungi to Host Plants

Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with 80% of surveyed land plant species and are well-recognized for accessing and transferring nutrients to plants1. Yet AMF also perform other essential functions, notably improving plant-water relations2. Some research attributes the role of AMF in plant-water relations solely to enhancing plant nutrition and osmoregulation for plants partnered with AMF3,4,5, while indirect evidence suggests AMF may transport water to plants1,6,7. Here, we used isotopically-labeled water and a fluorescent dye to directly track and quantify water transport by AMF to plants in a greenhouse experiment. We specifically assessed whether AMF can access water in soil unavailable to plants and transport it across an air gap to host plants. Plants grown with AMF that had access to a physically separated 18O-labeled water source transpired twice as much, and this transpired water contained three times as much label compared to plants with AMF with no access to the separated labeled water source. We estimated that water transported by AMF could explain 46.2% of the water transpired. In addition, a fluorescent dye indicated that water was transported via an extracytoplasmic hyphal pathway.

microbiology↗

bioRxiv2020

Stable isotope informed genome-resolved metagenomics uncovers potential trophic interactions in rhizosphere soil

The functioning, health, and productivity of soil is intimately tied to a complex network of interactions, particularly in plant root-associated rhizosphere soil. We conducted a stable isotope-informed, genome-resolved metagenomic study to trace carbon from Avena fatua grown in a 13CO2 atmosphere into soil. We collected paired rhizosphere and non-rhizosphere soil at six and nine weeks of plant growth and extracted DNA that was then separated by density using gradient centrifugation. Thirty-two fractions from each sample were grouped by density, sequenced, assembled, and binned to generate 55 unique microbial genomes that were >70% complete. The complete 18S rRNA sequences of several micro-eukaryotic bacterivores and fungi were enriched in 13C. We generated several circularized bacteriophage (phage) genomes, some of which were the most labelled entities in the rhizosphere. CRISPR locus targeting connected one of these phage to a Burkholderiales host predicted to be a plant pathogen. Another highly labeled phage is predicted to replicate in a Catenulispora sp., a possible plant growth-promoting bacterium. We searched the genomes for traits known to be used in interactions involving bacteria, micro-eukaryotes and plant roots and found that heavily isotopically-labeled bacteria have the ability to modulate plant signaling hormones, possess numerous plant pathogenicity factors, and produce toxins targeting micro-eukaryotes. Overall, 13C stable isotope-informed genome-resolved metagenomics revealed that very active bacteria often have the potential for strong interactions with plants and directly established that phage can be important agents of turnover of plant-derived carbon in soil.

microbiology↗

bioRxiv2020

Unexpected diversity of CPR bacteria and nanoarchaea in the rare biosphere of rhizosphere-associated grassland soil

Candidate Phyla Radiation (CPR) bacteria and nanoarchaea populate most ecosystems, but are rarely detected in soil. We concentrated particles less than 0.2 m from grassland soil, enabling targeted metagenomic analysis of these organisms, which are almost totally unexplored in soil. We recovered a diversity of CPR bacteria and some nanoarchaea sequences, but no sequences from other cellular organisms. The sampled sequences include Doudnabacteria (SM2F11) and Pacearchaeota, organisms not previously reported in soil, as well as Saccharibacteria, Parcubacteria and Microgenomates. CPR and DPANN (an acronym of the names of the first included archaea phyla) enrichments of 100-1000-fold were achieved compared to bulk soil, in which we estimate these organisms comprise about 1 to 100 cells per gram of soil. Like most CPR and DPANN sequenced to date, we predict these microorganisms live symbiotic, anaerobic lifestyles. However, Saccharibacteria, Parcubacteria, and Doudnabacteria genomes sampled here also encode ubiquinol oxidase operons that may have been acquired from other bacteria, likely during adaptation to aerobic soil environments. We posit that although present at low abundance, CPR bacteria and DPANN archaea could impact overall soil microbial community function by modulating host organism abundances and activity.

microbiology↗

bioRxiv2020

Conversion of marginal land into switchgrass conditionally accrues soil carbon and reduces methane consumption

Switchgrass (Panicum virgatum L.) is a perennial C4 grass native to tallgrass prairies of the Central US, and a promising bioenergy feedstock. Switchgrass can be cultivated on soils with low nutrient contents and its rooting depth, of up to 2 m, has brought attention to the crop as a potential mechanism to sequester and build soil carbon (C). Switchgrass, therefore, offers multifaceted benefits on degraded soils by enhancing soil organic matter content. However, to evaluate the sustainability of switchgrass-based biofuel production, it is crucial to understand the impacts of land conversion and switchgrass establishment on biotic/abiotic characteristics of various soils. In this study, we characterized the ecosystem-scale consequences of switchgrass growing at two highly-eroded, Dust Bowl remnant field sites from Oklahoma US, with silt-loam (SL) or clay-loam (CL) soil textures having low nitrogen (N), phosphorus (P), and C contents. Paired plots at each site, including fallow control and switchgrass-cultivated, were assessed. Our results indicated that switchgrass significantly increased soil C at the SL site and reduced microbial diversity at the CL site. The CL site exhibited significantly higher CO2 flux and higher respiration from switchgrass plots. Strikingly, switchgrass significantly reduced the CH4 consumption by an estimated 39% for the SL site and 47% for the CL site. Structural equation modeling identified soil temperature, P content, and soil moisture levels as the most influential factors regulating both CO2 and CH4 fluxes. CO2 flux was also influenced by microbial biomass while CH4 flux was influenced by microbial diversity. Together, our results suggest that site selection by soil type is a crucial factor in improving soil C stocks and mitigating greenhouse gas (GHG) fluxes, especially considering our finding that switchgrass reduced methane consumption, implying that carbon balance considerations should be accounted for to fully evaluate the sustainability of switchgrass cultivation.

molecular biology↗

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↗

bioRxiv2020

A quantitative framework reveals the ecological drivers of grassland soil microbial community assembly in response to warming

Unraveling the drivers controlling community assembly is a central issue in ecology. Selection, dispersal, diversification and drift are conceptually accepted as major community assembly processes. Defining their relative importance in governing biodiversity is compellingly needed, but very challenging. Here, we present a novel framework to quantitatively infer community assembly mechanisms by phylogenetic bin-based null model analysis (iCAMP). Our results with simulated microbial communities showed that iCAMP had high accuracy (0.93 - 0.99), precision (0.80 - 0.94), sensitivity (0.82 - 0.94), and specificity (0.95 - 0.98), which were 10-160% higher than those from the entire community-based approach. Applying it to grassland microbial communities in response to experimental warming, our analysis showed that homogeneous selection (38%) and "drift" (59%) played dominant roles in controlling grassland soil microbial community assembly. Interestingly, warming enhanced homogeneous selection, but decreased "drift" over time. Warming-enhanced selection was primarily imposed on Bacillales in Firmicutes, which were strengthened by increased drought and reduced plant productivity. This general framework should also be useful for plant and animal ecology.

ecology↗

bioRxiv2019

Microbial extracellular polysaccharide production and aggregate stability controlled by Switchgrass (Panicum virgatum) root biomass and soil water potential

Deep-rooting perennial grasses are promising feedstock for biofuel production, especially in marginal soils lacking organic material, nutrients, and/or that experience significant water stress. Perennial grass roots can alter surrounding soil conditions and influence microbial activities, particularly the production of extracellular polymeric substances composed primarily of extracellular polysaccharides (EPS). These polymers can alleviate cellular moisture and nutrient stress, and enhance soil characteristics through improved water retention and aggregate stability, the latter of which may in turn enhance carbon persistence. In this study we used a 13CO2 tracer greenhouse experiment to examine the effect of switchgrass cultivation on the production and origin of EPS in a marginal soil with five fertilization/water treatments (control, +N, +NP, +P, low water). Soils with both added nitrogen and phosphorus had the highest root biomass, EPS and percentage of water-stable soil aggregates. Multiple linear regression analyses revealed root biomass was the most important determinant for soil EPS production, potentially by controlling carbon supply and diurnal changes in soil water potential. Path analysis highlighted the role of soil water potential were and EPS on with water-stable soil aggregates, indicating that EPS concentration and soil aggregation have similar drivers in this soil. High mannose content confirmed the microbial origin of EPS. 13CO2 labeling indicated that 0.18% of newly fixed plant carbon was incorporated into EPS. Analysis of field samples suggests that EPS is significantly enhanced under long-term switchgrass cultivation. Our results demonstrate that switchgrass cultivation can promote microbial production of EPS, providing a mechanism to enhance sustainability of marginal soils.

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↗

bioRxiv2019

Metatranscriptomic reconstruction reveals RNA viruses with the potential to shape carbon cycling in soil

Viruses impact nearly all organisms on Earth, with ripples of influence in agriculture, health and biogeochemical processes. However, very little is known about RNA viruses in an environmental context, and even less is known about their diversity and ecology in the most complex microbial system, soil. Here, we assembled 48 individual metatranscriptomes from four habitats within a soil sampled over a 22-day time series: rhizosphere alone, detritosphere alone, a combination of the two, and unamended soil (four time points and three biological replicates per time point). We resolved the RNA viral community, uncovering a high diversity of viral sequences. We also investigated possible host organisms by analyzing metatranscriptome marker gene content. Based on viral phylogeny, much of the diversity was Narnaviridae that parasitize fungi or Leviviridae that infect Proteobacteria. Both host and viral communities appear to be highly dynamic, and rapidly diverged depending on experimental conditions. The viral communities were structured based on the presence of litter, while putative hosts appeared to be impacted by both the presence of litter and roots. A clear time signature from Leviviridae and their hosts indicated that viruses were replicating. With this time-resolved analysis, we show that RNA viruses are diverse, abundant and active in soil. Their replication causes host cell death, mobilizing carbon in a process that represents a largely overlooked component of carbon cycling in soil.

microbiology↗