Resource partitioning and amino acid assimilation in a terrestrial geothermal spring.

High-temperature geothermal springs host simplified microbial communities; however, the activities of individual microorganisms and their roles in the carbon cycle in nature are not well understood. Here, quantitative stable isotope probing (qSIP) was used to track the assimilation of 13C-acetate and 13C-aspartate into DNA in 74 °C sediments in Gongxiaoshe Hot Spring, Tengchong, China. This revealed a community-wide preference for aspartate and a tight coupling between aspartate incorporation into DNA and the proliferation of aspartate utilizers during labeling. Both 13C incorporation into DNA and changes in the abundance of taxa during incubations indicated strong resource partitioning and a significant phylogenetic signal for aspartate incorporation. Of the active amplicon sequence variants (ASVs) identified by qSIP, most could be matched with genomes from Gongxiaoshe Hot Spring or nearby springs with an average nucleotide similarity of 99.4%. Genomes corresponding to aspartate primary utilizers were smaller, near-universally encoded polar amino acid ABC transporters, and had codon preferences indicative of faster growth rates. The most active ASVs assimilating both substrates were not abundant, suggesting an important role for the rare biosphere in the community response to organic carbon addition. The broad incorporation of aspartate into DNA over acetate by the hot spring community may reflect dynamic cycling of cell lysis products in situ or substrates delivered during monsoon rains and may reflect N limitation.

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.

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.

Responses of Biocrust and Associated Soil Bacteria to Novel Climates Are Not Tightly Coupled.

Climate change is expanding drylands even as land use practices degrade them. Representing ∼40% of Earth's terrestrial surface, drylands rely on biological soil crusts (biocrusts) for key ecosystem functions including soil stability, biogeochemical cycling, and water capture. Understanding how biocrusts adapt to climate change is critical to understanding how dryland ecosystems will function with altered climate. We investigated the sensitivity of biocrusts to experimentally imposed novel climates to track changes in productivity and stability under both warming and cooling scenarios. We established three common gardens along an elevational-climate gradient on the Colorado Plateau. Mature biocrusts were collected from each site and reciprocally transplanted intact. Over 20 months we monitored visible species composition and cover, chlorophyll a, and the composition of soil bacterial communities using high throughput sequencing. We hypothesized that biocrusts replanted at their home site would show local preference, and biocrusts transplanted to novel environments would maintain higher cover and stability at elevations higher than their origin, compared to at elevations lower than their origin. We expected responses of the visible biocrust cover and soil bacterial components of the biocrust community to be coupled, with later successional taxa showing higher sensitivity to novel environments. Only high elevation sourced biocrusts maintained higher biocrust cover and community stability at their site of origin. Biocrusts from all sources had higher cover and stability in the high elevation garden. Later successional taxa decreased cover in low elevation gardens, suggesting successional reversal with warming. Visible community composition was influenced by both source and transplant environment. In contrast, soil bacterial community composition was not influenced by transplant environments but retained fidelity to the source. Thus, responses of the visible and soil bacterial components of the biocrust community were not coupled. Synthesis: Our results suggest biocrust communities are sensitive to climate change, and loss of species and function can be expected, while associated soil bacteria may be buffered against rapid change.

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.

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.

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.

Stable Isotope Probing of Microorganisms in Environmental Samples with H218O.

We describe a protocol for investigating microbial growth in environmental samples via stable isotope probing (SIP) with H218O. Water is a universal substrate for all microorganisms and replication is required for DNA to become labeled with 18O. By measuring how much the DNA of each taxon becomes enriched with 18O when an environmental sample is incubated with H218O, it is feasible to quantify that population's DNA replication rate, which is a proxy for growth.

Microbial Taxon-Specific Isotope Incorporation with DNA Quantitative Stable Isotope Probing.

Quantitative stable isotope probing (qSIP) measures rates of taxon-specific element assimilation in intact microbial communities, utilizing substrates labeled with a heavy isotope.The laboratory protocol for qSIP is nearly identical to that for conventional stable isotope probing, with two key additions: (1) in qSIP, qPCR measurements are conducted on each density fraction recovered after isopycnic separation, and (2) in qSIP, multiple density fractions are sequenced spanning the entire range of densities over which nucleic acids were recovered. qSIP goes beyond identifying taxa assimilating a substrate, as it also allows for measuring that assimilation for each taxon within a given microbial community. Here, we describe an analysis process necessary to determine atom fraction excess of a heavy stable isotope added to an environmental sample for a given taxon's DNA.

Evolutionary history constrains microbial traits across environmental variation.

Organisms influence ecosystems, from element cycling to disturbance regimes, to trophic interactions and to energy partitioning. Microorganisms are part of this influence, and understanding their ecology in nature requires studying the traits of these organisms quantitatively in their natural habitats-a challenging task, but one which new approaches now make possible. Here, we show that growth rate and carbon assimilation rate of soil microorganisms are influenced more by evolutionary history than by climate, even across a broad climatic gradient spanning major temperate life zones, from mixed conifer forest to high-desert grassland. Most of the explained variation (~50% to ~90%) in growth rate and carbon assimilation rate was attributable to differences among taxonomic groups, indicating a strong influence of evolutionary history, and taxonomic groupings were more predictive for organisms responding to resource addition. With added carbon and nitrogen substrates, differences among taxonomic groups explained approximately eightfold more variance in growth rate than did differences in ecosystem type. Taxon-specific growth and carbon assimilation rates were highly intercorrelated across the four ecosystems, constrained by the taxonomic identity of the organisms, such that plasticity driven by environment was limited across ecosystems varying in temperature, precipitation and dominant vegetation. Taken together, our results suggest that, similar to multicellular life, the traits of prokaryotes in their natural habitats are constrained by evolutionary history to a greater degree than environmental variation.

Predictive genomic traits for bacterial growth in culture versus actual growth in soil.

Relationships between microbial genes and performance are often evaluated in the laboratory in pure cultures, with little validation in nature. Here, we show that genomic traits related to laboratory measurements of maximum growth potential failed to predict the growth rates of bacteria in unamended soil, but successfully predicted growth responses to resource pulses: growth increased with 16S rRNA gene copy number and declined with genome size after substrate addition to soils, responses that were repeated in four different ecosystems. Genome size best predicted growth rate in response to addition of glucose alone; adding ammonium with glucose weakened the relationship, and the relationship was absent in nutrient-replete pure cultures, consistent with the idea that reduced genome size is a mechanism of nutrient conservation. Our findings demonstrate that genomic traits of soil bacteria can map to their ecological performance in nature, but the mapping is poor under native soil conditions, where genomic traits related to stress tolerance may prove more predictive. These results remind that phenotype depends on environmental context, underscoring the importance of verifying proposed schemes of trait-based strategies through direct measurement of performance in nature, an important and currently missing foundation for translating microbial processes from genes to ecosystems.

Quantitative stable isotope probing with H218O reveals that most bacterial taxa in soil synthesize new ribosomal RNA.

Most soil bacterial taxa are thought to be dormant, or inactive, yet the extent to which they synthetize new rRNA is poorly understood. We analyzed 18O composition of RNA extracted from soil incubated with H218O and used quantitative stable isotope probing to characterize rRNA synthesis among microbial taxa. RNA was not fully labeled with 18O, peaking at a mean of 23.6 ± 6.8 atom percent excess (APE) 18O after eight days of incubation, suggesting some ribonucleotides in soil were more than eight days old. Microbial taxa varied in the degree they incorporated 18O into their rRNA over time and there was no correlation between the APE 18O of bacterial rRNA and their rRNA to DNA ratios, suggesting that the ratios were not appropriate to measure ribonucleotide synthesis. Our study indicates that, on average, 94% of soil taxa produced new rRNA and therefore were metabolically active.

Developing climate-smart restoration: Can plant microbiomes be hardened against heat waves?

Heat waves are increasing in frequency and intensity, presenting a challenge for the already difficult practice of ecological restoration. We investigated whether pre-heating locally sourced rhizosphere soil (inoculum) could acclimatize plants to a field-imposed heat wave in a restoration setting. Soil heating in the laboratory caused a marked shift in rhizosphere bacterial community composition, accompanied by an increase in species evenness. Furthermore, pre-heated rhizosphere soil reduced plant height, number of leaves, and shoot mass of the C4 grass, blue grama (Bouteloua gracilis), and it reduced the shoot mass of the C3 grass, Arizona fescue (Festuca arizonica) in the glasshouse. Following transplantation and the application of a field heat wave, pre-heated inoculum did not influence heat wave survival for either plant species. However, there were strong species-level responses to the field heat wave. For instance, heat wave survivorship was over four times higher in blue grama (92%) than in Arizona fescue (22%). These results suggest that the use of C4 seeds may be preferable for sites exhibiting high heat wave risk. Further research is needed to understand whether inocula are more effective in highly degraded soil in comparison with partially degraded soils.

Taxonomic patterns in the nitrogen assimilation of soil prokaryotes.

Nitrogen (N) is frequently a limiting nutrient in soil; its availability can govern ecosystem functions such as primary production and decomposition. Assimilation of N by microorganisms impacts the availability of N in soil. Despite its established ecological significance, the contributions of microbial taxa to N assimilation are unknown. Here we measure N uptake and use by microbial phylotypes and taxonomic groups within a diverse assemblage of soil microbes through quantitative stable isotope probing (qSIP) with 15 N. Following incubation with 15 NH4+, distinct patterns of 15 N assimilation among taxonomic groups were observed. For instance, glucose addition stimulated 15 N assimilation in most members of Actinobacteria and Proteobacteria but generally decreased 15 N use by Firmicutes and Bacteriodetes. While NH4+ is considered a preferred and universal source of N to prokaryotes, the majority (> 80%) of N assimilation in our soils could be attributed to a handful of active orders. Characterizing N assimilation of taxonomic groups with 15 N qSIP may provide a basis for understanding how microbial community composition influences N availability in the environment.

Bacterial carbon use plasticity, phylogenetic diversity and the priming of soil organic matter.

Microorganisms perform most decomposition on Earth, mediating carbon (C) loss from ecosystems, and thereby influencing climate. Yet, how variation in the identity and composition of microbial communities influences ecosystem C balance is far from clear. Using quantitative stable isotope probing of DNA, we show how individual bacterial taxa influence soil C cycling following the addition of labile C (glucose). Specifically, we show that increased decomposition of soil C in response to added glucose (positive priming) occurs as a phylogenetically diverse group of taxa, accounting for a large proportion of the bacterial community, shift toward additional soil C use for growth. Our findings suggest that many microbial taxa exhibit C use plasticity, as most taxa altered their use of glucose and soil organic matter depending upon environmental conditions. In contrast, bacteria that exhibit other responses to glucose (reduced growth or reliance on glucose for additional growth) clustered strongly by phylogeny. These results suggest that positive priming is likely the prototypical response of bacteria to sustained labile C addition, consistent with the widespread occurrence of the positive priming effect in nature.