Gut microbiome predictors of Escherichia coli sequence type 131 colonization and loss.

BACKGROUND: Escherichia coli sequence type 131 (ST131), specifically its fluoroquinolone-resistant H30R clade (ST131-H30R), is a global multidrug-resistant pathogen. The gut microbiome's role in ST131-H30R intestinal carriage is undefined. METHODS: Veterans and their household members underwent longitudinal fecal swab surveillance for ST131 in 2014-2018. The fecal microbiome was characterized by 16S rRNA qPCR and sequencing. We evaluated associations between ST131-H30R carriage and gut microbiome at baseline by random forest models to identify the most informative gut bacterial phyla and genera attributes for ST131 and ST131-H30R carriage status. Next, we assessed longitudinal associations between fecal microbiome and ST131-H30R carriage using a mixed-effects logistic regression with longitudinal measures. FINDINGS: Of the 519 participants, 78 were carriers of ST131, among whom 49 had ST131-H30R. At the baseline timepoint, H30R-positive participants had higher proportional abundances of Actinobacteria phylum (mean: 4.9% vs. 3.1%) than ST131-negative participants. H30R-positive participants also had higher abundances of Collinsella (mean: 2.3% vs. 1.1%) and lower abundances of Alistipes (mean: 2.1% vs. 2.6%) than ST131-negative participants. In the longitudinal analysis, Collinsella abundance correlated positively with ST131-H30R carriage status and negatively with the loss of ST131-H30R. Conversely, Alistipes corresponded with the loss and persistent absence of ST131-H30R even in the presence of a household exposure. INTERPRETATION: Abundances of specific fecal bacteria correlated with ST131-H30R carriage, persistence, and loss, suggesting their potential as targets for microbiome-based strategies to reduce carriage of ST131-H30R, a significant risk factor for invasive infections. FUNDING: This work was supported in part by National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award numbers R21AI117654 and UM1AI104681 and the Office of Research and Development, Department of Veterans Affairs. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Department of Veterans Affairs.

Microbial growth under drought is confined to distinct taxa and modified by potential future climate conditions.

Climate change increases the frequency and intensity of drought events, affecting soil functions including carbon sequestration and nutrient cycling, which are driven by growing microorganisms. Yet we know little about microbial responses to drought due to methodological limitations. Here, we estimate microbial growth rates in montane grassland soils exposed to ambient conditions, drought, and potential future climate conditions (i.e., soils exposed to 6 years of elevated temperatures and elevated CO2 levels). For this purpose, we combined 18O-water vapor equilibration with quantitative stable isotope probing (termed 'vapor-qSIP') to measure taxon-specific microbial growth in dry soils. In our experiments, drought caused >90% of bacterial and archaeal taxa to stop dividing and reduced the growth rates of persisting ones. Under drought, growing taxa accounted for only 4% of the total community as compared to 35% in the controls. Drought-tolerant communities were dominated by specialized members of the Actinobacteriota, particularly the genus Streptomyces. Six years of pre-exposure to future climate conditions (3 °C warming and + 300 ppm atmospheric CO2) alleviated drought effects on microbial growth, through more drought-tolerant taxa across major phyla, accounting for 9% of the total community. Our results provide insights into the response of active microbes to drought today and in a future climate, and highlight the importance of studying drought in combination with future climate conditions to capture interactive effects and improve predictions of future soil-climate feedbacks.

The predictive power of phylogeny on growth rates in soil bacterial communities.

Predicting ecosystem function is critical to assess and mitigate the impacts of climate change. Quantitative predictions of microbially mediated ecosystem processes are typically uninformed by microbial biodiversity. Yet new tools allow the measurement of taxon-specific traits within natural microbial communities. There is mounting evidence of a phylogenetic signal in these traits, which may support prediction and microbiome management frameworks. We investigated phylogeny-based trait prediction using bacterial growth rates from soil communities in Arctic, boreal, temperate, and tropical ecosystems. Here we show that phylogeny predicts growth rates of soil bacteria, explaining an average of 31%, and up to 58%, of the variation within ecosystems. Despite limited overlap in community composition across these ecosystems, shared nodes in the phylogeny enabled ancestral trait reconstruction and cross-ecosystem predictions. Phylogenetic relationships could explain up to 38% (averaging 14%) of the variation in growth rates across the highly disparate ecosystems studied. Our results suggest that shared evolutionary history contributes to similarity in the relative growth rates of related bacteria in the wild, allowing phylogeny-based predictions to explain a substantial amount of the variation in taxon-specific functional traits, within and across ecosystems.

Using source-associated mobile genetic elements to identify zoonotic extraintestinal E. coli infections.

A one-health perspective may provide new and actionable information about Escherichia coli transmission. E. coli colonizes a broad range of vertebrates, including humans and food-production animals, and is a leading cause of bladder, kidney, and bloodstream infections in humans. Substantial evidence supports foodborne transmission of pathogenic E. coli strains from food animals to humans. However, the relative contribution of foodborne zoonotic E. coli (FZEC) to the human extraintestinal disease burden and the distinguishing characteristics of such strains remain undefined. Using a comparative genomic analysis of a large collection of contemporaneous, geographically-matched clinical and meat-source E. coli isolates (n = 3111), we identified 17 source-associated mobile genetic elements - predominantly plasmids and bacteriophages - and integrated them into a novel Bayesian latent class model to predict the origins of clinical E. coli isolates. We estimated that approximately 8 % of human extraintestinal E. coli infections (mostly urinary tract infections) in our study population were caused by FZEC. FZEC strains were equally likely to cause symptomatic disease as non-FZEC strains. Two FZEC lineages, ST131-H22 and ST58, appeared to have particularly high virulence potential. Our findings imply that FZEC strains collectively cause more urinary tract infections than does any single non-E. coli uropathogenic species (e.g., Klebsiella pneumoniae). Our novel approach can be applied in other settings to identify the highest-risk FZEC strains, determine their sources, and inform new one-health strategies to decrease the heavy public health burden imposed by extraintestinal E. coli infections.

Hyperactive nanobacteria with host-dependent traits pervade Omnitrophota.

Candidate bacterial phylum Omnitrophota has not been isolated and is poorly understood. We analysed 72 newly sequenced and 349 existing Omnitrophota genomes representing 6 classes and 276 species, along with Earth Microbiome Project data to evaluate habitat, metabolic traits and lifestyles. We applied fluorescence-activated cell sorting and differential size filtration, and showed that most Omnitrophota are ultra-small (~0.2 µm) cells that are found in water, sediments and soils. Omnitrophota genomes in 6 classes are reduced, but maintain major biosynthetic and energy conservation pathways, including acetogenesis (with or without the Wood-Ljungdahl pathway) and diverse respirations. At least 64% of Omnitrophota genomes encode gene clusters typical of bacterial symbionts, suggesting host-associated lifestyles. We repurposed quantitative stable-isotope probing data from soils dominated by andesite, basalt or granite weathering and identified 3 families with high isotope uptake consistent with obligate bacterial predators. We propose that most Omnitrophota inhabit various ecosystems as predators or parasites.

Distinct Growth Responses of Tundra Soil Bacteria to Short-Term and Long-Term Warming.

Increases in Arctic temperatures have thawed permafrost and accelerated tundra soil microbial activity, releasing greenhouse gases that amplify climate warming. Warming over time has also accelerated shrub encroachment in the tundra, altering plant input abundance and quality, and causing further changes to soil microbial processes. To better understand the effects of increased temperature and the accumulated effects of climate change on soil bacterial activity, we quantified the growth responses of individual bacterial taxa to short-term warming (3 months) and long-term warming (29 years) in moist acidic tussock tundra. Intact soil was assayed in the field for 30 days using 18O-labeled water, from which taxon-specific rates of 18O incorporation into DNA were estimated as a proxy for growth. Experimental treatments warmed the soil by approximately 1.5°C. Short-term warming increased average relative growth rates across the assemblage by 36%, and this increase was attributable to emergent growing taxa not detected in other treatments that doubled the diversity of growing bacteria. However, long-term warming increased average relative growth rates by 151%, and this was largely attributable to taxa that co-occurred in the ambient temperature controls. There was also coherence in relative growth rates within broad taxonomic levels with orders tending to have similar growth rates in all treatments. Growth responses tended to be neutral in short-term warming and positive in long-term warming for most taxa and phylogenetic groups co-occurring across treatments regardless of phylogeny. Taken together, growing bacteria responded distinctly to short-term and long-term warming, and taxa growing in each treatment exhibited deep phylogenetic organization. IMPORTANCE Soil carbon stocks in the tundra and underlying permafrost have become increasingly vulnerable to microbial decomposition due to climate change. The microbial responses to Arctic warming must be understood in order to predict the effects of future microbial activity on carbon balance in a warming Arctic. In response to our warming treatments, tundra soil bacteria grew faster, consistent with increased rates of decomposition and carbon flux to the atmosphere. Our findings suggest that bacterial growth rates may continue to increase in the coming decades as faster growth is driven by the accumulated effects of long-term warming. Observed phylogenetic organization of bacterial growth rates may also permit taxonomy-based predictions of bacterial responses to climate change and inclusion into ecosystem models.

Nutrients strengthen density dependence of per-capita growth and mortality rates in the soil bacterial community.

Density dependence in an ecological community has been observed in many macro-organismal ecosystems and is hypothesized to maintain biodiversity but is poorly understood in microbial ecosystems. Here, we analyze data from an experiment using quantitative stable isotope probing (qSIP) to estimate per-capita growth and mortality rates of bacterial populations in soils from several ecosystems along an elevation gradient which were subject to nutrient addition of either carbon alone (glucose; C) or carbon with nitrogen (glucose + ammonium-sulfate; C + N). Across all ecosystems, we found that higher population densities, quantified by the abundance of genomes per gram of soil, had lower per-capita growth rates in C + N-amended soils. Similarly, bacterial mortality rates in C + N-amended soils increased at a significantly higher rate with increasing population size than mortality rates in control and C-amended soils. In contrast to the hypothesis that density dependence would promote or maintain diversity, we observed significantly lower bacterial diversity in soils with stronger negative density-dependent growth. Here, density dependence was significantly but weakly responsive to nutrients and was not associated with higher bacterial diversity.

Life history strategies among soil bacteria-dichotomy for few, continuum for many.

Study of life history strategies may help predict the performance of microorganisms in nature by organizing the complexity of microbial communities into groups of organisms with similar strategies. Here, we tested the extent that one common application of life history theory, the copiotroph-oligotroph framework, could predict the relative population growth rate of bacterial taxa in soils from four different ecosystems. We measured the change of in situ relative growth rate to added glucose and ammonium using both 18O-H2O and 13C quantitative stable isotope probing to test whether bacterial taxa sorted into copiotrophic and oligotrophic groups. We saw considerable overlap in nutrient responses across most bacteria regardless of phyla, with many taxa growing slowly and few taxa that grew quickly. To define plausible life history boundaries based on in situ relative growth rates, we applied Gaussian mixture models to organisms' joint 18O-13C signatures and found that across experimental replicates, few taxa could consistently be assigned as copiotrophs, despite their potential for fast growth. When life history classifications were assigned based on average relative growth rate at varying taxonomic levels, finer resolutions (e.g., genus level) were significantly more effective in capturing changes in nutrient response than broad taxonomic resolution (e.g., phylum level). Our results demonstrate the difficulty in generalizing bacterial life history strategies to broad lineages, and even to single organisms across a range of soils and experimental conditions. We conclude that there is a continued need for the direct measurement of microbial communities in soil to advance ecologically realistic frameworks.

Quantitative Stable-Isotope Probing (qSIP) with Metagenomics Links Microbial Physiology and Activity to Soil Moisture in Mediterranean-Climate Grassland Ecosystems.

The growth and physiology of soil microorganisms, which play vital roles in biogeochemical cycling, are shaped by both current and historical soil environmental conditions. Here, we developed and applied a genome-resolved metagenomic implementation of quantitative stable isotope probing (qSIP) with an H218O labeling experiment to identify actively growing soil microorganisms and their genomic capacities. qSIP enabled measurement of taxon-specific growth because isotopic incorporation into microbial DNA requires production of new genome copies. We studied three Mediterranean grassland soils across a rainfall gradient to evaluate the hypothesis that historic precipitation levels are an important factor controlling trait selection. We used qSIP-informed genome-resolved metagenomics to resolve the active subset of soil community members and identify their characteristic ecophysiological traits. Higher year-round precipitation levels correlated with higher activity and growth rates of flagellar motile microorganisms. In addition to heavily isotopically labeled bacteria, we identified abundant isotope-labeled phages, suggesting phage-induced cell lysis likely contributed to necromass production at all three sites. Further, there was a positive correlation between phage activity and the activity of putative phage hosts. Contrary to our expectations, the capacity to decompose the diverse complex carbohydrates common in soil organic matter or oxidize methanol and carbon monoxide were broadly distributed across active and inactive bacteria in all three soils, implying that these traits are not highly selected for by historical precipitation. IMPORTANCE Soil moisture is a critical factor that strongly shapes the lifestyle of soil organisms by changing access to nutrients, controlling oxygen diffusion, and regulating the potential for mobility. We identified active microorganisms in three grassland soils with similar mineral contexts, yet different historic rainfall inputs, by adding water labeled with a stable isotope and tracking that isotope in DNA of growing microbes. By examining the genomes of active and inactive microorganisms, we identified functions that are enriched in growing organisms, and showed that different functions were selected for in different soils. Wetter soil had higher activity of motile organisms, but activity of pathways for degradation of soil organic carbon compounds, including simple carbon substrates, were comparable for all three soils. We identified many labeled, and thus active bacteriophages (viruses that infect bacteria), implying that the cells they killed contributed to soil organic matter. The activity of these bacteriophages was significantly correlated with activity of their hosts.

Life and death in the soil microbiome: how ecological processes influence biogeochemistry.

Soil microorganisms shape global element cycles in life and death. Living soil microorganisms are a major engine of terrestrial biogeochemistry, driving the turnover of soil organic matter - Earth's largest terrestrial carbon pool and the primary source of plant nutrients. Their metabolic functions are influenced by ecological interactions with other soil microbial populations, soil fauna and plants, and the surrounding soil environment. Remnants of dead microbial cells serve as fuel for these biogeochemical engines because their chemical constituents persist as soil organic matter. This non-living microbial biomass accretes over time in soil, forming one of the largest pools of organic matter on the planet. In this Review, we discuss how the biogeochemical cycling of organic matter depends on both living and dead soil microorganisms, their functional traits, and their interactions with the soil matrix and other organisms. With recent omics advances, many of the traits that frame microbial population dynamics and their ecophysiological adaptations can be deciphered directly from assembled genomes or patterns of gene or protein expression. Thus, it is now possible to leverage a trait-based understanding of microbial life and death within improved biogeochemical models and to better predict ecosystem functioning under new climate regimes.

Decreased growth of wild soil microbes after 15 years of transplant-induced warming in a montane meadow.

The carbon stored in soil exceeds that of plant biomass and atmospheric carbon and its stability can impact global climate. Growth of decomposer microorganisms mediates both the accrual and loss of soil carbon. Growth is sensitive to temperature and given the vast biological diversity of soil microorganisms, the response of decomposer growth rates to warming may be strongly idiosyncratic, varying among taxa, making ecosystem predictions difficult. Here, we show that 15 years of warming by transplanting plant-soil mesocosms down in elevation, strongly reduced the growth rates of soil microorganisms, measured in the field using undisturbed soil. The magnitude of the response to warming varied among microbial taxa. However, the direction of the response-reduced growth-was universal and warming explained twofold more variation than did the sum of taxonomic identity and its interaction with warming. For this ecosystem, most of the growth responses to warming could be explained without taxon-specific information, suggesting that in some cases microbial responses measured in aggregate may be adequate for climate modeling. Long-term experimental warming also reduced soil carbon content, likely a consequence of a warming-induced increase in decomposition, as warming-induced changes in plant productivity were negligible. The loss of soil carbon and decreased microbial biomass with warming may explain the reduced growth of the microbial community, more than the direct effects of temperature on growth. These findings show that direct and indirect effects of long-term warming can reduce growth rates of soil microbes, which may have important feedbacks to global warming.

Microbes on decomposing litter in streams: entering on the leaf or colonizing in the water?

When leaves fall in rivers, microbial decomposition commences within hours. Microbial assemblages comprising hundreds of species of fungi and bacteria can vary with stream conditions, leaf litter species, and decomposition stage. In terrestrial ecosystems, fungi and bacteria that enter soils with dead leaves often play prominent roles in decomposition, but their role in aquatic decomposition is less known. Here, we test whether fungi and bacteria that enter streams on senesced leaves are growing during decomposition and compare their abundances and growth to bacteria and fungi that colonize leaves in the water. We employ quantitative stable isotope probing to identify growing microbes across four leaf litter species and two decomposition times. We find that most of the growing fungal species on decomposing leaves enter the water with the leaf, whereas most growing bacteria colonize from the water column. Results indicate that the majority of bacteria found on litter are growing, whereas the majority of fungi are dormant. Both bacterial and fungal assemblages differed with leaf type on the dried leaves and throughout decomposition. This research demonstrates the importance of fungal species that enter with the leaf on aquatic decomposition and the prominence of bacteria that colonize decomposing leaves in the water.

Soil minerals affect taxon-specific bacterial growth.

Secondary minerals (clays and metal oxides) are important components of the soil matrix. Clay minerals affect soil carbon persistence and cycling, and they also select for distinct microbial communities. Here we show that soil mineral assemblages-particularly short-range order minerals-affect both bacterial community composition and taxon-specific growth. Three soils with different parent material and presence of short-range order minerals were collected from ecosystems with similar vegetation and climate. These three soils were provided with 18O-labeled water and incubated with or without artificial root exudates or pine needle litter. Quantitative stable isotope probing was used to determine taxon-specific growth. We found that the growth of bacteria varied among soils of different mineral assemblages but found the trend of growth suppression in the presence of short-range order minerals. Relative growth of bacteria declined with increasing concentration of short-range order minerals between 25-36% of taxa present in all soils. Carbon addition in the form of plant litter or root exudates weakly affected relative growth of taxa (p = 0.09) compared to the soil type (p < 0.01). However, both exudate and litter carbon stimulated growth for at least 34% of families in the soils with the most and least short-range order minerals. In the intermediate short-range order soil, fresh carbon reduced growth for more bacterial families than were stimulated. These results highlight how bacterial-mineral-substrate interactions are critical to soil organic carbon processing, and how growth variation in bacterial taxa in these interactions may contribute to soil carbon persistence and loss.

Stable-Isotope-Informed, Genome-Resolved Metagenomics Uncovers Potential Cross-Kingdom Interactions in Rhizosphere Soil.

The functioning, health, and productivity of soil are 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 nonrhizosphere soil at 6 and 9 weeks of plant growth and extracted DNA that was then separated by density using ultracentrifugation. Thirty-two fractions from each of five samples were grouped by density, sequenced, assembled, and binned to generate 55 unique bacterial genomes that were ≥70% complete. We also identified complete 18S rRNA sequences of several 13C-enriched microeukaryotic bacterivores and fungi. We generated 10 circularized bacteriophage (phage) genomes, some of which were the most labeled entities in the rhizosphere, suggesting that phage may be important agents of turnover of plant-derived C in soil. 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 genome bins for traits known to be used in interactions involving bacteria, microeukaryotes, and plant roots and found DNA from heavily 13C-labeled bacterial genes thought to be involved in modulating plant signaling hormones, plant pathogenicity, and defense against microeukaryote grazing. Stable-isotope-informed, genome-resolved metagenomics indicated that phage can be important agents of turnover of plant-derived carbon in soil. IMPORTANCE Plants grow in intimate association with soil microbial communities; these microbes can facilitate the availability of essential resources to plants. Thus, plant productivity commonly depends on interactions with rhizosphere bacteria, viruses, and eukaryotes. Our work is significant because we identified the organisms that took up plant-derived organic C in rhizosphere soil and determined that many of the active bacteria are plant pathogens or can impact plant growth via hormone modulation. Further, by showing that bacteriophage accumulate CO2-derived carbon, we demonstrated their vital roles in redistribution of plant-derived C into the soil environment through bacterial cell lysis. The use of stable-isotope probing (SIP) to identify consumption (or lack thereof) of root-derived C by key microbial community members within highly complex microbial communities opens the way for assessing manipulations of bacteria and phage with potentially beneficial and detrimental traits, ultimately providing a path to improved plant health and soil carbon storage.

Nutrients cause consolidation of soil carbon flux to small proportion of bacterial community.

Nutrient amendment diminished bacterial functional diversity, consolidating carbon flow through fewer bacterial taxa. Here, we show strong differences in the bacterial taxa responsible for respiration from four ecosystems, indicating the potential for taxon-specific control over soil carbon cycling. Trends in functional diversity, defined as the richness of bacteria contributing to carbon flux and their equitability of carbon use, paralleled trends in taxonomic diversity although functional diversity was lower overall. Among genera common to all ecosystems, Bradyrhizobium, the Acidobacteria genus RB41, and Streptomyces together composed 45-57% of carbon flow through bacterial productivity and respiration. Bacteria that utilized the most carbon amendment (glucose) were also those that utilized the most native soil carbon, suggesting that the behavior of key soil taxa may influence carbon balance. Mapping carbon flow through different microbial taxa as demonstrated here is crucial in developing taxon-sensitive soil carbon models that may reduce the uncertainty in climate change projections.

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 nonpredatory 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 nonpredatory 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 wolf-pack hunting behavior. Added carbon substrates disproportionately stimulated growth of obligate predators, with responses 63% higher than those of nonpredators for the Bdellovibrionales and 81% higher for the Vampirovibrionales, whereas responses of facultative predators to substrate addition were no different from those of nonpredators. This finding supports the 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.IMPORTANCE The word "predator" may conjure images of leopards killing and eating impala on the African savannah or of great white sharks attacking elephant seals off the coast of California. But microorganisms are also predators, including bacteria that kill and eat other bacteria. While predatory bacteria have been found in many environments, it has been challenging to document their importance in nature. This study quantified the growth of predatory and nonpredatory bacteria in soils (and one stream) by tracking isotopically labeled substrates into newly synthesized DNA. Predatory bacteria were more active than nonpredators, and obligate predators, such as Bdellovibrionales and Vampirovibrionales, increased in growth rate in response to added substrates at the base of the food chain, strong evidence of trophic control. This work provides quantitative measures of predator activity and suggests that predatory bacteria-along with protists, nematodes, and phages-are active and important in microbial food webs.

The temperature sensitivity of soil: microbial biodiversity, growth, and carbon mineralization.

Microorganisms drive soil carbon mineralization and changes in their activity with increased temperature could feedback to climate change. Variation in microbial biodiversity and the temperature sensitivities (Q10) of individual taxa may explain differences in the Q10 of soil respiration, a possibility not previously examined due to methodological limitations. Here, we show phylogenetic and taxonomic variation in the Q10 of growth (5-35 °C) among soil bacteria from four sites, one from each of Arctic, boreal, temperate, and tropical biomes. Differences in the temperature sensitivities of taxa and the taxonomic composition of communities determined community-assembled bacterial growth Q10, which was strongly predictive of soil respiration Q10 within and across biomes. Our results suggest community-assembled traits of microbial taxa may enable enhanced prediction of carbon cycling feedbacks to climate change in ecosystems across the globe.

Measurement Error and Resolution in Quantitative Stable Isotope Probing: Implications for Experimental Design.

Quantitative stable isotope probing (qSIP) estimates isotope tracer incorporation into DNA of individual microbes and can link microbial biodiversity and biogeochemistry in complex communities. As with any quantitative estimation technique, qSIP involves measurement error, and a fuller understanding of error, precision, and statistical power benefits qSIP experimental design and data interpretation. We used several qSIP data sets-from soil and seawater microbiomes-to evaluate how variance in isotope incorporation estimates depends on organism abundance and resolution of the density fractionation scheme. We assessed statistical power for replicated qSIP studies, plus sensitivity and specificity for unreplicated designs. As a taxon's abundance increases, the variance of its weighted mean density declines. Nine fractions appear to be a reasonable trade-off between cost and precision for most qSIP applications. Increasing the number of density fractions beyond that reduces variance, although the magnitude of this benefit declines with additional fractions. Our analysis suggests that, if a taxon has an isotope enrichment of 10 atom% excess, there is a 60% chance that this will be detected as significantly different from zero (with alpha 0.1). With five replicates, isotope enrichment of 5 atom% could be detected with power (0.6) and alpha (0.1). Finally, we illustrate the importance of internal standards, which can help to calibrate per sample conversions of %GC to mean weighted density. These results should benefit researchers designing future SIP experiments and provide a useful reference for metagenomic SIP applications where both financial and computational limitations constrain experimental scope.IMPORTANCE One 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 their genomic content and potential functions but may not reflect an organism's activity within its natural community. Culture-independent studies supply a community-wide view of composition and function in the context of community interactions but often fail to link the two. Quantitative stable isotope probing (qSIP) is a method that can link the identity and functional activity 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 experimental replication, and discuss cost-benefit balanced scenarios for SIP experimental design.

Taxon-specific microbial growth and mortality patterns reveal distinct temporal population responses to rewetting in a California grassland soil.

Microbial activity increases after rewetting dry soil, resulting in a pulse of carbon mineralization and nutrient availability. The biogeochemical responses to wet-up are reasonably well understood and known to be microbially mediated. Yet, the population level dynamics, and the resulting changes in microbial community patterns, are not well understood as ecological phenomena. Here, we used sequencing of 16S rRNA genes coupled with heavy water (H218O) DNA quantitative stable isotope probing to estimate population-specific rates of growth and mortality in response to a simulated wet-up event in a California annual grassland soil. Bacterial growth and mortality responded rapidly to wet-up, within 3 h, and continued throughout the 168 h incubation, with patterns of sequential growth observed at the phylum level. Of the 37 phyla detected in the prewet community, growth was found in 18 phyla while mortality was measured in 26 phyla. Rapid growth and mortality rates were measurable within 3 h of wet-up but had contrasting characteristics; growth at 3 h was dominated by select taxa within the Proteobacteria and Firmicutes, whereas mortality was taxonomically widespread. Furthermore, across the community, mortality exhibited density-independence, consistent with the indiscriminate shock resulting from dry-down and wet-up, whereas growth was density-dependent, consistent with control by competition or predation. Total aggregated growth across the community was highly correlated with total soil CO2 production. Together, these results illustrate how previously "invisible" population responses can translate quantitatively to emergent observations of ecosystem-scale biogeochemistry.