Measurements of soil protist richness and community composition are influenced by primer pair, annealing temperature, and bioinformatics choices.

Protists are a diverse and understudied group of microbial eukaryotic organisms especially in terrestrial environments. Advances in molecular methods are increasing our understanding of the distribution and functions of these creatures; however, there is a vast array of choices researchers make including barcoding genes, primer pairs, PCR settings, and bioinformatic options that can impact the outcome of protist community surveys. Here, we tested four commonly used primer pairs targeting the V4 and V9 regions of the 18S rRNA gene using different PCR annealing temperatures and processed the sequences with different bioinformatic parameters in 10 diverse soils to evaluate how primer pair, amplification parameters, and bioinformatic choices influence the composition and richness of protist and non-protist taxa using Illumina sequencing. Our results showed that annealing temperature influenced sequencing depth and protist taxon richness for most primer pairs, and that merging forward and reverse sequencing reads for the V4 primer pairs dramatically reduced the number of sequences and taxon richness of protists. The data sets of primers that targeted the same 18S rRNA gene region (e.g., V4 or V9) had similar protist community compositions; however, data sets from primers targeting the V4 18S rRNA gene region detected a greater number of protist taxa compared to those prepared with primers targeting the V9 18S rRNA region. There was limited overlap of protist taxa between data sets targeting the two different gene regions (80/549 taxa). Together, we show that laboratory and bioinformatic choices can substantially affect the results and conclusions about protist diversity and community composition using metabarcoding.IMPORTANCEEcosystem functioning is driven by the activity and interactions of the microbial community, in both aquatic and terrestrial environments. Protists are a group of highly diverse, mostly unicellular microbes whose identity and roles in terrestrial ecosystem ecology have been largely ignored until recently. This study highlights the importance of choices researchers make, such as primer pair, on the results and conclusions about protist diversity and community composition in soils. In order to better understand the roles protist taxa play in terrestrial ecosystems, biases in methodological and analytical choices should be understood and acknowledged.

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.

Isotopic labeling of metabolic water with 18 O2.

RATIONALE: Water is the medium of life, is involved in biochemical reactions, and is exchanged among internal pools and with the water in the external environment of organisms. Understanding these processes can be improved by isotopically labeling the metabolic water that is produced inside the cells of organisms during aerobic respiration. METHODS: Here we describe a new method for isotopically labeling cellular water by incubating microbes and plant tissues in air enriched in 18 O2 . As oxygen gas is reduced during respiration, H2 18 O is produced. The rate of H2 18 O production and the synthesis of biomolecules that incorporate 18 O from H2 18 O can be quantified using cavity ringdown spectrometry and isotope ratio mass spectrometry. RESULTS: For Escherichia coli in solution culture, soil microbial communities, and respiring tissues of plants, the amount of H2 18 O produced was strongly correlated with that of 18 O2 consumed during incubations. Measurements of 18 O in DNA, microbial biomass, and CO2 showed that metabolic water was an important substrate in biosynthesis reactions. CONCLUSIONS: Any organism with aerobic respiration is amenable to labeling with 18 O2 , and the method described here enables a new approach to investigate questions regarding plant and microbial physiology. In plants, 18 O introduced as metabolic water could be tracked as it moves between living cells and exchanges with external water. For probing soil microbial physiology, the method described here has the advantage over the application of exogenous H2 18 O of not increasing the soil moisture, a disturbance that can affect microbial metabolism.

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.

Phylogenetic organization in the assimilation of chemically distinct substrates by soil bacteria.

Soils are among the most biodiverse habitats on earth and while the species composition of microbial communities can influence decomposition rates and pathways, the functional significance of many microbial species and phylogenetic groups remains unknown. If bacteria exhibit phylogenetic organization in their function, this could enable ecologically meaningful classification of bacterial clades. Here, we show non-random phylogenetic organization in the rates of relative carbon assimilation for both rapidly mineralized substrates (amino acids and glucose) assimilated by many microbial taxa and slowly mineralized substrates (lipids and cellulose) assimilated by relatively few microbial taxa. When mapped onto bacterial phylogeny using ancestral character estimation this phylogenetic organization enabled the identification of clades involved in the decomposition of specific soil organic matter substrates. Phylogenetic organization in substrate assimilation could provide a basis for predicting the functional attributes of uncharacterized microbial taxa and understanding the significance of microbial community composition for soil organic matter decomposition.

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.

Limits to the three domains of life: lessons from community assembly along an Antarctic salinity gradient.

Extremophiles exist among all three domains of life; however, physiological mechanisms for surviving harsh environmental conditions differ among Bacteria, Archaea and Eukarya. Consequently, we expect that domain-specific variation of diversity and community assembly patterns exist along environmental gradients in extreme environments. We investigated inter-domain community compositional differences along a high-elevation salinity gradient in the McMurdo Dry Valleys, Antarctica. Conductivity for 24 soil samples collected along the gradient ranged widely from 50 to 8355 µS cm-1. Taxonomic richness varied among domains, with a total of 359 bacterial, 2 archaeal, 56 fungal, and 69 non-fungal eukaryotic operational taxonomic units (OTUs). Richness for bacteria, archaea, fungi, and non-fungal eukaryotes declined with increasing conductivity (all P < 0.05). Principal coordinate ordination analysis (PCoA) revealed significant (ANOSIM R = 0.97) groupings of low/high salinity bacterial OTUs, while OTUs from other domains were not significantly clustered. Bacterial beta diversity was unimodally distributed along the gradient and had a nested structure driven by species losses, whereas in fungi and non-fungal eukaryotes beta diversity declined monotonically without strong evidence of nestedness. Thus, while increased salinity acts as a stressor in all domains, the mechanisms driving community assembly along the gradient differ substantially between the domains.

Invasive and native grasses exert negative plant-soil feedbacks on the woody shrub Artemisia tridentata.

Displacement of diverse native plant communities by low-diversity invasive communities is a global problem. In the western United States, the displacement of sagebrush-dominated communities by cheatgrass has increased since the 1920s. Restoration outcomes are poor, potentially due to soil alteration by cheatgrass. We explored the poorly understood role of plant-soil feedbacks in the dominance of cheatgrass in a greenhouse study where uninvaded sagebrush soils were conditioned with either cheatgrass, a native bunchgrass or sagebrush. Sagebrush seedlings were grown in the soils that remained following the removal of conditioning plants. We expected cheatgrass to strongly suppress sagebrush due to a change in belowground microbial communities, conspecifics to have neutral effects and the native bunchgrass to have intermediate effects as it coevolved with sagebrush but belongs to a different functional group. We assessed the effects of conditioning on sagebrush growth, tissue nutrients, and carbon allocation. We also characterized the abundance, diversity and community composition of root microbial associates. Cheatgrass strongly suppressed sagebrush growth at high and low conditioning densities, the native bunchgrass showed suppression at high conditioning densities only and conspecific effects were neutral. Tissue nutrients, amount of root colonization by soil fungi or root microbial community composition were not associated with these plant-soil feedbacks. Although we did not identify the precise mechanism, our results provide key evidence that rapid soil alteration by cheatgrass results in negative plant-soil feedbacks on sagebrush growth. These feedbacks likely contribute to cheatgrass dominance and the poor success of sagebrush restoration.

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.

Microbial rRNA Synthesis and Growth Compared through Quantitative Stable Isotope Probing with H218O.

Growing bacteria have a high concentration of ribosomes to ensure sufficient protein synthesis, which is necessary for genome replication and cellular division. To elucidate whether metabolic activity of soil microorganisms is coupled with growth, we investigated the relationship between rRNA and DNA synthesis in a soil bacterial community using quantitative stable isotope probing (qSIP) with H218O. Most soil bacterial taxa were metabolically active and grew, and there was no significant difference between the isotopic composition of DNA and RNA extracted from soil incubated with H218O. The positive correlation between 18O content of DNA and rRNA of taxa, with a slope statistically indistinguishable from 1 (slope = 0.96; 95% confidence interval [CI], 0.90 to 1.02), indicated that few taxa made new rRNA without synthesizing new DNA. There was no correlation between rRNA-to-DNA ratios obtained from sequencing libraries and the atom percent excess (APE) 18O values of DNA or rRNA, suggesting that the ratio of rRNA to DNA is a poor indicator of microbial growth or rRNA synthesis. Our results support the notion that metabolic activity is strongly coupled to cellular division and suggest that nondividing taxa do not dominate soil metabolic activity.IMPORTANCE Using quantitative stable isotope probing of microbial RNA and DNA with H218O, we show that most soil taxa are metabolically active and grow because their nucleic acids are significantly labeled with 18O. A majority of the populations that make new rRNA also grow, which argues against the common paradigm that most soil taxa are dormant. Additionally, our results indicate that relative sequence abundance-based RNA-to-DNA ratios, which are frequently used for identifying active microbial populations in the environment, underestimate the number of metabolically active taxa within soil microbial communities.

The Influence of Time and Plant Species on the Composition of the Decomposing Bacterial Community in a Stream Ecosystem.

Foliar chemistry influences leaf decomposition, but little is known about how litter chemistry affects the assemblage of bacterial communities during decomposition. Here we examined relationships between initial litter chemistry and the composition of the bacterial community in a stream ecosystem. We incubated replicated genotypes of Populus fremontii and P. angustifolia leaf litter that differ in percent tannin and lignin, then followed changes in bacterial community composition during 28 days of decomposition using 16S rRNA gene-based pyrosequencing. Using a nested experimental design, the majority of variation in bacterial community composition was explained by time (i.e., harvest day) (R(2) = 0.50). Plant species, nested within harvest date, explained a significant but smaller proportion of the variation (R(2) = 0.03). Significant differences in community composition between leaf species were apparent at day 14, but no significant differences existed among genotypes. Foliar chemistry correlated significantly with community composition at day 14 (r = 0.46) indicating that leaf litter with more similar phytochemistry harbor bacterial communities that are alike. Bacteroidetes and ß-proteobacteria dominated the bacterial assemblage on decomposing leaves, and Verrucomicrobia and α- and δ-proteobacteria became more abundant over time. After 14 days, bacterial diversity diverged significantly between leaf litter types with fast-decomposing P. fremontii hosting greater richness than slowly decomposing P. angustifolia; however, differences were no longer present after 28 days in the stream. Leaf litter tannin, lignin, and lignin: N ratios all correlated negatively with diversity. This work shows that the bacterial community on decomposing leaves in streams changes rapidly over time, influenced by leaf species via differences in genotype-level foliar chemistry.

Quantitative microbial ecology through stable isotope probing.

Bacteria grow and transform elements at different rates, and as yet, quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification to stable isotope probing (SIP) that enables the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers to be determined. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon-specific density curves are produced for labeled and nonlabeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxon's density shift relative to that taxon's density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative stable isotope probing (qSIP). We demonstrated qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variations in (18)O and (13)C composition after exposure to [(18)O]water or [(13)C]glucose. The addition of glucose increased the assimilation of (18)O into DNA from [(18)O]water. However, the increase in (18)O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because the addition of glucose indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing.

Growth and death of bacteria and fungi underlie rainfall-induced carbon dioxide pulses from seasonally dried soil.

The rapid increase in microbial 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 x 10(8) bacterial 16S rRNA gene copies x [g dry mass](-1) x h(-1) and 4.3 x 10(7) fungal ITS copies x [g dry mass](-1) x 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 wet-up, 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.

Minor changes in soil bacterial and fungal community composition occur in response to monsoon precipitation in a semiarid grassland.

Arizona and New Mexico receive half of their annual precipitation during the summer monsoon season, making this large-scale rain event critical for ecosystem productivity. We used the monsoon rains to explore the responses of soil bacterial and fungal communities to natural moisture pulses in a semiarid grassland. Through 454 pyrosequencing of the 16S rRNA gene and ITS region, we phylogenetically characterized these communities at 22 time points during a summer season. Relative humidity increased before the rains arrived, creating conditions in soil that allowed for the growth of microorganisms. During the course of the study, the relative abundances of most bacterial phyla showed little variation, though some bacterial populations responded immediately to an increase in soil moisture once the monsoon rains arrived. The Firmicutes phylum experienced over a sixfold increase in relative abundance with increasing water availability. Conversely, Actinobacteria, the dominant taxa at our site, were negatively affected by the increase in water availability. No relationship was found between bacterial diversity and soil water potential. Bacterial community structure was unrelated to all environmental variables that we measured, with the exception of a significant relationship with atmospheric relative humidity. Relative abundances of fungal phyla fluctuated more throughout the season than bacterial abundances did. Variation in fungal community structure was unrelated to soil water potential and to most environmental variables. However, ordination analysis showed a distinct fungal community structure late in the season, probably due to plant senescence.

Rapid growth rate responses of terrestrial bacteria to field warming on the Antarctic Peninsula.

Ice-free terrestrial environments of the western Antarctic Peninsula are expanding and subject to colonization by new microorganisms and plants, which control biogeochemical cycling. Measuring growth rates of microbial populations and ecosystem carbon flux is critical for understanding how terrestrial ecosystems in Antarctica will respond to future warming. We implemented a field warming experiment in early (bare soil; +2 °C) and late (peat moss-dominated; +1.2 °C) successional glacier forefield sites on the western Antarctica Peninsula. We used quantitative stable isotope probing with H218O using intact cores in situ to determine growth rate responses of bacterial taxa to short-term (1 month) warming. Warming increased the growth rates of bacterial communities at both sites, even doubling the number of taxa exhibiting significant growth at the early site. Growth responses varied among taxa. Despite that warming induced a similar response for bacterial relative growth rates overall, the warming effect on ecosystem carbon fluxes was stronger at the early successional site-likely driven by increased activity of autotrophs which switched the ecosystem from a carbon source to a carbon sink. At the late-successional site, warming caused a significant increase in growth rate of many Alphaproteobacteria, but a weaker and opposite gross ecosystem productivity response that decreased the carbon sink-indicating that the carbon flux rates were driven more strongly by the plant communities. Such changes to bacterial growth and ecosystem carbon cycling suggest that the terrestrial Antarctic Peninsula can respond fast to increases in temperature, which can have repercussions for long-term elemental cycling and carbon storage.

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.

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.

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.

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.