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Soil microorganisms help transform plant inputs into mineral-associated soil organic carbon (SOC) – the largest and slowest-cycling pool of organic carbon on land. However, the microbial traits that influence this process are widely debated. While current theory and biogeochemical models have settled on carbon-use efficiency (CUE) and growth rate as positive predictors of mineral-associated SOC, empirical tests are sparse, with contradictory observations. Using 13C-labeling of an annual grass (Avena barbata) under two moisture regimes, we found that microbial traits associated with formation of 13C-mineral-associated SOC varied by soil habitat, as did active microbial taxa and SOC chemical composition. In the rhizosphere, bacterial-dominated communities with fast growth, high biomass, and high extracellular polymeric substance (EPS) production were positively associated with 13C-mineral-associated SOC. In contrast, the detritusphere held communities dominated by fungi and more filamentous bacteria, and with greater exoenzyme activity; there, 13C-mineral-associated SOC was associated with slower microbial growth and lower microbial biomass. CUE was a negative predictor of 13C-mineral-associated SOC in both habitats. Using 13C-quantitative stable isotope probing, we found that the majority of 13C assimilation in the rhizosphere and detritusphere at week 12 of the experiment was performed by very few bacterial and fungal taxa (3–5% of the total taxa that assimilated 13C). Several complementary chemical analyses (13C-NMR, FTICR-MS, and STXM-NEXAFS) suggested that SOC in the rhizosphere had a more oxidized chemical signature, while SOC in the detritusphere had a less oxidized, more lignin-like chemical signature. Our findings challenge current theory by demonstrating that microbial traits linked with mineral-associated SOC are not universal, but vary with soil habitat and moisture conditions, and are shaped by a small number of active taxa. Emerging SOC models that explicitly reflect these interactions may better predict SOC storage, since climate change causes shifts in soil moisture regimes and the ratio of living versus decaying roots.

Stable isotope probing (SIP) facilitates culture-independent identification of active microbial populations within complex ecosystems through isotopic enrichment of nucleic acids. Many DNA-SIP studies rely on 16S rRNA gene 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 gene 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 analytical 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. IMPORTANCE Answering the questions, "who is eating what?" and "who is active?" within complex microbial communities is paramount for our ability to model, predict, and modulate microbiomes for improved human and planetary health. These questions can be 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 microorganism's taxonomic identity and genome composition while providing quantitative estimates of the microorganism's 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.

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.

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.

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.

BackgroundThe transformation of plant photosynthate into soil organic carbon and its recycling to CO2 by soil microorganisms is one of the central components of the terrestrial carbon cycle. There are currently large knowledge gaps related to which soil-associated microorganisms take up plant carbon in the rhizosphere and the fate of that carbon.ResultsWe conducted an experiment in which common wild oats (Avena fatua) were grown in a 13CO2 atmosphere and the rhizosphere and non-rhizosphere soil was sampled for genomic analyses. Density gradient centrifugation of DNA extracted from soil samples enabled distinction of microbes that did and did not incorporate the 13C into their DNA. A 1.45-Mbp genome of a Saccharibacteria (TM7) was identified and, despite the microbial complexity of rhizosphere soil, curated to completion. The genome lacks many biosynthetic pathways, including genes required to synthesize DNA de novo. Rather, it requires externally derived nucleotides for DNA and RNA synthesis. Given this, we conclude that rhizosphere-associated Saccharibacteria recycle DNA from bacteria that live off plant exudates and/or phage that acquired 13C because they preyed upon these bacteria and/or directly from the labeled plant DNA. Isotopic labeling indicates that the population was replicating during the 6-week period of plant growth. Interestingly, the genome is ~ 30% larger than other complete Saccharibacteria genomes from non-soil environments, largely due to more genes for complex carbon utilization and amino acid metabolism. Given the ability to degrade cellulose, hemicellulose, pectin, starch, and 1,3-β-glucan, we predict that this Saccharibacteria generates energy by fermentation of soil necromass and plant root exudates to acetate and lactate. The genome also encodes a linear electron transport chain featuring a terminal oxidase, suggesting that this Saccharibacteria may respire aerobically. The genome encodes a hydrolase that could breakdown salicylic acid, a plant defense signaling molecule, and genes to interconvert a variety of isoprenoids, including the plant hormone zeatin.ConclusionsRhizosphere Saccharibacteria likely depend on other bacteria for basic cellular building blocks. We propose that isotopically labeled CO2 is incorporated into plant-derived carbon and then into the DNA of rhizosphere organisms capable of nucleotide synthesis, and the nucleotides are recycled into Saccharibacterial genomes.

Abstract Predicted shifts in precipitation patterns could impact soil microbial activity, and thereby terrestrial ecosystem functioning. In a Mediterranean grassland soil which had been subjected to contrasting precipitation patterns, we investigated the response of active and inactive bacterial and fungal communities to rewetting over time, using 18O stable isotope probing. Altered precipitation patterns prior to rewetting had little impact on the trajectories over time of the active and inactive bacterial communities after rewetting, as bacteria died or were recruited from the inactive to the active community. The duration of the dry summer conditions affected the diversity and phylogenetic clustering of the inactive microbial community and its functional potential, likely indicating long-lasting effects on ecosystem stability. Highlights • Inactive bacterial community structure changed over time after rewetting. • Precipitation history had little effect on bacterial community trajectories over time. • The diversity of inactive bacterial community changed over time after rewetting. • Active and inactive fungal communities did not differ after rewetting. [ABSTRACT FROM AUTHOR]

The rapid increase in niicrobial activity that occurs when a dry soil is rewetted has been well documented and is of great interest due to implications of changing precipitation patterns on soil C dynamics. Several studies have shown minor net changes in microbial population diversity or abundance following wet-up, but the gross population dynamics of bacteria and fungi resulting from soil wet-up are virtually unknown. Here we applied DNA stable isotope probing with H218O coupled with quantitative PCR to characterize new growth, survival, and mortality of bacteria and fungi following the rewetting of a seasonally dried California annual grassland soil. Microbial activity, as determined by CO2 production, increased significantly within three hours of wet-up, yet new growth was not detected until after three hours, suggesting a pulse of nongrowth activity immediately following wet-up, likely due to osmo-regulation and resuscitation from dormancy in response to the rapid change in water potential. Total microbial abundance revealed little change throughout the seven-day post-wet incubation, but there was substantial turnover of both bacterial and fungal populations (49% and 52%, respectively). New growth was linear between 24 and 168 hours for both bacteria and fungi, with average growth rates of 2.3 × 108 bacterial 16S rRNA gene copies-[g dry mass]-1.h-1 and 4.3 × 107 fungal ITS copies[g dry mass]-1.h-1. While bacteria and fungi differed in their mortality and survival characteristics during the seven-day incubation, mortality that occurred within the first three hours was similar, with 25% and 27% of bacterial and fungal gene copies disappearing from the pre-wet community, respectively. The rapid disappearance of gene copies indicates that cell death, occurring either during the extreme dry down period (preceding five months) or during the rapid change in water potential due to wetup, generates a significant pool of available C that likely contributes to the large pulse in CO2 associated with wet-up. A dynamic assemblage of growing and dying organisms controlled the CO2 pulse, but the balance between death and growth resulted in relatively stable total population abundances, even after a profound and sudden change in environment. [ABSTRACT FROM AUTHOR]

Soil microorganisms help transform plant inputs into mineral-associated soil organic carbon (SOC) – the largest and slowest-cycling pool of organic carbon on land. However, the microbial traits that influence this process are widely debated. While current theory and biogeochemical models have settled on carbon-use efficiency (CUE) and growth rate as positive predictors of mineral-associated SOC, empirical tests are sparse, with contradictory observations. Using 13C-labeling of an annual grass (Avena barbata) under two moisture regimes, we found that microbial traits associated with formation of 13C-mineral-associated SOC varied by soil habitat, as did active microbial taxa and SOC chemical composition. In the rhizosphere, bacterial-dominated communities with fast growth, high biomass, and high extracellular polymeric substance (EPS) production were positively associated with 13C-mineral-associated SOC. In contrast, the detritusphere held communities dominated by fungi and more filamentous bacteria, and with greater exoenzyme activity; there, 13C-mineral-associated SOC was associated with slower microbial growth and lower microbial biomass. CUE was a negative predictor of 13C-mineral-associated SOC in both habitats. Using 13C-quantitative stable isotope probing, we found that the majority of 13C assimilation in the rhizosphere and detritusphere at week 12 of the experiment was performed by very few bacterial and fungal taxa (3–5% of the total taxa that assimilated 13C). Several complementary chemical analyses (13C-NMR, FTICR-MS, and STXM-NEXAFS) suggested that SOC in the rhizosphere had a more oxidized chemical signature, while SOC in the detritusphere had a less oxidized, more lignin-like chemical signature. Our findings challenge current theory by demonstrating that microbial traits linked with mineral-associated SOC are not universal, but vary with soil habitat and moisture conditions, and are shaped by a small number of active taxa. Emerging SOC models that explicitly reflect these interactions may better predict SOC storage, since climate change causes shifts in soil moisture regimes and the ratio of living versus decaying roots. • Microbial traits associated with mineral-associated SOC varied by soil habitat. • In the rhizosphere, fast growth and high yield were positively associated with SOC. • The detritusphere community had slower growth and greater exoenzyme activity than the rhizosphere community. • 13C-qSIP showed most C assimilation was performed by very few active taxa. • Chemical composition of SOC was more oxidized in the rhizosphere than the detritusphere. [ABSTRACT FROM AUTHOR]