Intraspecific variation in antibiotic resistance potential within E. coli.

Intraspecific genomic diversity brings the potential for an unreported and diverse reservoir of cryptic antibiotic resistance genes in pathogens, as cryptic resistance can occur without major mutations and horizontal transmission. Here, we predicted the differences in the types of antibiotics and genes that induce cryptic and latent resistance between micro-diverse Escherichia coli strains. For example, we hypothesize that known resistance genes will be the culprit of latent resistance within clinical strains. We used a modified functional metagenomics method to induce expression in eight E. coli strains. We found a total of 66 individual genes conferring phenotypic resistance to 11 out of 16 antibiotics. A total of 14 known antibiotic resistance genes comprised 21% of total identified genes, whereas the majority (52 genes) were unclassified cryptic resistance genes. Between the eight strains, 1.2% of core orthologous genes were positive (conferred resistance in at least one strain). Sixty-four percent of positive orthologous genes conferred resistance to only one strain, demonstrating high intraspecific variability of latent resistance genes. Cryptic resistance genes comprised most resistance genes among laboratory and clinical strains as well as natural, semisynthetic, and synthetic antibiotics. Known antibiotic resistance genes primarily conferred resistance to multiple antibiotics from varying origins and within multiple strains. Hence, it is uncommon for E. coli to develop cross-cryptic resistance to antibiotics from multiple origins or within multiple strains. We have uncovered prospective and previously unknown resistance genes as well as antibiotics that have the potential to trigger latent antibiotic resistance in E. coli strains from varying origins.IMPORTANCEIntraspecific genomic diversity may be a driving force in the emergence of adaptive antibiotic resistance. Adaptive antibiotic resistance enables sensitive bacterial cells to acquire temporary antibiotic resistance, creating an optimal window for the development of permanent mutational resistance. In this study, we investigate cryptic resistance, an adaptive resistance mechanism, and unveil novel (cryptic) antibiotic resistance genes that confer resistance when amplified within eight E. coli strains derived from clinical and laboratory origins. We identify the potential of cryptic resistance genes to confer cross-resistance to antibiotics from varying origins and within multiple strains. We discern antibiotic characteristics that promote latent resistance in multiple strains, considering intraspecific diversity. This study may help detect novel resistance genes and functional genes that could become responsible for cryptic resistance among diverse strains and antibiotics, thus also identifying potential novel antibiotic targets and mechanisms.

Proteome trait regulation of marine Synechococcus elemental stoichiometry under global change.

Recent studies have demonstrated regional differences in marine ecosystem C:N:P with implications for carbon and nutrient cycles. Due to strong co-variance, temperature and nutrient stress explain variability in C:N:P equally well. A reductionistic approach can link changes in individual environmental drivers with changes in biochemical traits and cell C:N:P. Thus, we quantified effects of temperature and nutrient stress on Synechococcus chemistry using laboratory chemostats, chemical analyses, and data-independent acquisition mass spectrometry proteomics. Nutrient supply accounted for most C:N:Pcell variability and induced tradeoffs between nutrient acquisition and ribosomal proteins. High temperature prompted heat-shock, whereas thermal effects via the "translation-compensation hypothesis" were only seen under P-stress. A Nonparametric Bayesian Local Clustering algorithm suggested that changes in lipopolysaccharides, peptidoglycans, and C-rich compatible solutes may also contribute to C:N:P regulation. Physiological responses match field-based trends in ecosystem stoichiometry and suggest a hierarchical environmental regulation of current and future ocean C:N:P.

Intraspecific trait variation modulates the temperature effect on elemental quotas and stoichiometry in marine Synechococcus.

Diverse phytoplankton modulate the coupling between the ocean carbon and nutrient cycles through life-history traits such as cell size, elemental quotas, and ratios. Biodiversity is mostly considered at broad functional levels, but major phytoplankton lineages are themselves highly diverse. As an example, Synechococcus is found in nearly all ocean regions, and we demonstrate contains extensive intraspecific variation. Here, we grew four closely related Synechococcus isolates in serially transferred cultures across a range of temperatures (16-25°C) to quantify for the relative role of intraspecific trait variation vs. environmental change. We report differences in cell size (p<0.01) as a function of strain and clade (p<0.01). The carbon (QC), nitrogen (QN), and phosphorus (QP) cell quotas all increased with cell size. Furthermore, cell size has an inverse relationship to growth rate. Within our experimental design, temperature alone had a weak physiological effect on cell quota and elemental ratios. Instead, we find systemic intraspecific variance of C:N:P, with cell size and N:P having an inverse relationship. Our results suggest a key role for intraspecific life history traits in determining elemental quotas and stoichiometry. Thus, the extensive biodiversity harbored within many lineages may modulate the impact of environmental change on ocean biogeochemical cycles.

Life history strategies of soil bacterial communities across global terrestrial biomes.

The life history strategies of soil microbes determine their metabolic potential and their response to environmental changes. Yet these strategies remain poorly understood. Here we use shotgun metagenomes from terrestrial biomes to characterize overarching covariations of the genomic traits that capture dominant life history strategies in bacterial communities. The emerging patterns show a triangle of life history strategies shaped by two trait dimensions, supporting previous theoretical and isolate-based studies. The first dimension ranges from streamlined genomes with simple metabolisms to larger genomes and expanded metabolic capacities. As metabolic capacities expand, bacterial communities increasingly differentiate along a second dimension that reflects a trade-off between increasing capacities for environmental responsiveness or for nutrient recycling. Random forest analyses show that soil pH, C:N ratio and precipitation patterns together drive the dominant life history strategy of soil bacterial communities and their biogeographic distribution. Our findings provide a trait-based framework to compare life history strategies of soil bacteria.

Global scale phylogeography of functional traits and microdiversity in Prochlorococcus.

Prochlorococcus is the most numerically abundant photosynthetic organism in the surface ocean. The Prochlorococcus high-light and warm-water adapted ecotype (HLII) is comprised of extensive microdiversity, but specific functional differences between microdiverse sub-clades remain elusive. Here we characterized both functional and phylogenetic diversity within the HLII ecotype using Bio-GO-SHIP metagenomes. We found widespread variation in gene frequency connected to local environmental conditions. Metagenome-assembled marker genes and genomes revealed a globally distributed novel HLII haplotype defined by adaptation to chronically low P conditions (HLII-P). Environmental correlation analysis revealed different factors were driving gene abundances verses phylogenetic differences. An analysis of cultured HLII genomes and metagenome-assembled genomes revealed a subclade within HLII, which corresponded to the novel HLII-P haplotype. This work represents the first global assessment of the HLII ecotype's phylogeography and corresponding functional differences. These findings together expand our understanding of how microdiversity structures functional differences and reveals the importance of nutrients as drivers of microdiversity in Prochlorococcus.

Investigating the eco-evolutionary response of microbiomes to environmental change.

Microorganisms are the primary engines of biogeochemical processes and foundational to the provisioning of ecosystem services to human society. Free-living microbial communities (microbiomes) and their functioning are now known to be highly sensitive to environmental change. Given microorganisms' capacity for rapid evolution, evolutionary processes could play a role in this response. Currently, however, few models of biogeochemical processes explicitly consider how microbial evolution will affect biogeochemical responses to environmental change. Here, we propose a conceptual framework for explicitly integrating evolution into microbiome-functioning relationships. We consider how microbiomes respond simultaneously to environmental change via four interrelated processes that affect overall microbiome functioning (physiological acclimation, demography, dispersal and evolution). Recent evidence in both the laboratory and the field suggests that ecological and evolutionary dynamics occur simultaneously within microbiomes; however, the implications for biogeochemistry under environmental change will depend on the timescales over which these processes contribute to a microbiome's response. Over the long term, evolution may play an increasingly important role for microbially driven biogeochemical responses to environmental change, particularly to conditions without recent historical precedent.

Global scale phylogeography of functional traits and microdiversity in Prochlorococcus.

Prochlorococcus is the most numerically abundant photosynthetic organism in the surface ocean. The Prochlorococcus high-light and warm-water adapted ecotype (HLII) is comprised of extensive microdiversity, but specific functional differences between microdiverse sub-clades remain elusive. Here we characterized both functional and phylogenetic diversity within the HLII ecotype using Bio-GO-SHIP metagenomes. We found widespread variation in gene frequency connected to local environmental conditions. Metagenomically assembled marker genes and genomes revealed a globally distributed novel HLII haplotype defined by adaptation to chronically low P conditions (HLII-P). Environmental correlation analysis revealed different factors were driving gene abundances verses phylogenetic differences. An analysis of cultured HLII genomes and metagenomically assembled genomes revealed a subclade within HLII, which corresponded to the novel HLII-P haplotype. This work represents the first global assessment of the HLII ecotype’s phylogeography and corresponding functional differences. These findings together expand our understanding of how microdiversity structures functional differences and reveals the importance of nutrients as drivers of microdiversity in Prochlorococcus .

Basin-scale biogeography of Prochlorococcus and SAR11 ecotype replication.

Establishing links between microbial diversity and environmental processes requires resolving the high degree of functional variation among closely related lineages or ecotypes. Here, we implement and validate an improved metagenomic approach that estimates the spatial biogeography and environmental regulation of ecotype-specific replication patterns (RObs) across ocean regions. A total of 719 metagenomes were analyzed from meridional Bio-GO-SHIP sections in the Atlantic and Indian Ocean. Accounting for sequencing bias and anchoring replication estimates in genome structure were critical for identifying physiologically relevant biological signals. For example, ecotypes within the dominant marine cyanobacteria Prochlorococcus exhibited distinct diel cycles in RObs that peaked between 19:00-22:00. Additionally, both Prochlorococcus ecotypes and ecotypes within the highly abundant heterotroph Pelagibacter (SAR11) demonstrated systematic biogeographies in RObs that differed from spatial patterns in relative abundance. Finally, RObs was significantly regulated by nutrient stress and temperature, and explained by differences in the genomic potential for nutrient transport, energy production, cell wall structure, and replication. Our results suggest that our new approach to estimating replication is reflective of gross population growth. Moreover, this work reveals that the interaction between adaptation and environmental change drives systematic variability in replication patterns across ocean basins that is ecotype-specific, adding an activity-based dimension to our understanding of microbial niche space.

Global patterns and predictors of C:N:P in marine ecosystems.

Oceanic nutrient cycles are coupled, yet carbon-nitrogen-phosphorus (C:N:P) stoichiometry in marine ecosystems is variable through space and time, with no clear consensus on the controls on variability. Here, we analyze hydrographic, plankton genomic diversity, and particulate organic matter data from 1970 stations sampled during a global ocean observation program (Bio-GO-SHIP) to investigate the biogeography of surface ocean particulate organic matter stoichiometry. We find latitudinal variability in C:N:P stoichiometry, with surface temperature and macronutrient availability as strong predictors of stoichiometry at high latitudes. Genomic observations indicated community nutrient stress and suggested that nutrient supply rate and nitrogen-versus-phosphorus stress are predictive of hemispheric and regional variations in stoichiometry. Our data-derived statistical model suggests that C:P and N:P ratios will increase at high latitudes in the future, however, changes at low latitudes are uncertain. Our findings suggest systematic regulation of elemental stoichiometry among ocean ecosystems, but that future changes remain highly uncertain.

Global Ocean Particulate Organic Phosphorus, Carbon, Oxygen for Respiration, and Nitrogen (GO-POPCORN).

Concentrations and elemental stoichiometry of suspended particulate organic carbon, nitrogen, phosphorus, and oxygen demand for respiration (C:N:P:-O2) play a vital role in characterizing and quantifying marine elemental cycles. Here, we present Version 2 of the Global Ocean Particulate Organic Phosphorus, Carbon, Oxygen for Respiration, and Nitrogen (GO-POPCORN) dataset. Version 1 is a previously published dataset of particulate organic matter from 70 different studies between 1971 and 2010, while Version 2 is comprised of data collected from recent cruises between 2011 and 2020. The combined GO-POPCORN dataset contains 2673 paired surface POC/N/P measurements from 70°S to 73°N across all major ocean basins at high spatial resolution. Version 2 also includes 965 measurements of oxygen demand for organic carbon respiration. This new dataset can help validate and calibrate the next generation of global ocean biogeochemical models with flexible elemental stoichiometry. We expect that incorporating variable C:N:P:-O2 into models will help improve our estimates of key ocean biogeochemical fluxes such as carbon export, nitrogen fixation, and organic matter remineralization.

Gene Amplification Uncovers Large Previously Unrecognized Cryptic Antibiotic Resistance Potential in E. coli.

The activation of unrecognized antibiotic resistance genes in the bacterial cell can give rise to antibiotic resistance without the need for major mutations or horizontal gene transfer. We hypothesize that bacteria harbor an extensive array of diverse cryptic genes that can be activated in response to antibiotics via adaptive resistance. To test this hypothesis, we developed a plasmid assay to randomly manipulate gene copy numbers in Escherichia coli cells and identify genes that conferred resistance when amplified. We then tested for cryptic resistance to 18 antibiotics and identified genes conferring resistance. E. coli could become resistant to 50% of the antibiotics tested, including chloramphenicol, d-cycloserine, polymyxin B, and 6 beta-lactam antibiotics, following this manipulation. Known antibiotic resistance genes comprised 13% of the total identified genes, where 87% were unclassified (cryptic) antibiotic resistance genes. These unclassified genes encoded cell membrane proteins, stress response/DNA repair proteins, transporters, and miscellaneous or hypothetical proteins. Stress response/DNA repair genes have a broad antibiotic resistance potential, as this gene class, in aggregate, conferred cryptic resistance to nearly all resistance-positive antibiotics. We found that antibiotics that are hydrophilic, those that are amphipathic, and those that inhibit the cytoplasmic membrane or cell wall biosynthesis were more likely to induce cryptic resistance in E. coli. This study reveals a diversity of cryptic genes that confer an antibiotic resistance phenotype when present in high copy number. Thus, our assay can identify potential novel resistance genes while also describing which antibiotics are prone to induce cryptic antibiotic resistance in E. coli. IMPORTANCE Predicting where new antibiotic resistance genes will rise is a challenge and is especially important when new antibiotics are developed. Adaptive resistance allows sensitive bacterial cells to become transiently resistant to antibiotics. This provides an opportune time for cells to develop more efficient resistance mechanisms, such as tolerance and permanent resistance to higher antibiotic concentrations. The biochemical diversity harbored within bacterial genomes may lead to the presence of genes that could confer resistance when timely activated. Therefore, it is crucial to understand adaptive resistance to identify potential resistance genes and prolong antibiotics. Here, we investigate cryptic resistance, an adaptive resistance mechanism, and identify unknown (cryptic) antibiotic resistance genes that confer resistance when amplified in a laboratory strain of E. coli. We also pinpoint antibiotic characteristics that are likely to induce cryptic resistance. This study may help detect novel antibiotic resistance genes and provide the foundation to help develop more effective antibiotics.

Exploring Trait Trade-Offs for Fungal Decomposers in a Southern California Grassland.

Fungi are important decomposers in terrestrial ecosystems, so their responses to climate change might influence carbon (C) and nitrogen (N) dynamics. We investigated whether growth and activity of fungi under drought conditions were structured by trade-offs among traits in 15 fungal isolates from a Mediterranean Southern California grassland. We inoculated fungi onto sterilized litter that was incubated at three moisture levels (4, 27, and 50% water holding capacity, WHC). For each isolate, we characterized traits that described three potential lifestyles within the newly proposed "YAS" framework: growth yield, resource acquisition, and stress tolerance. Specifically, we measured fungal hyphal length per unit litter decomposition for growth yield; the potential activities of the extracellular enzymes cellobiohydrolase (CBH), ß -glucosidase (BG), ß -xylosidase (BX), and N-acetyl- ß - D -glucosaminidase (NAG) for resource acquisition; and ability to grow in drought vs. higher moisture levels for drought stress tolerance. Although, we had hypothesized that evolutionary and physiological trade-offs would elicit negative relationships among traits, we found no supporting evidence for this hypothesis. Across isolates, growth yield, drought stress tolerance, and extracellular enzyme activities were not significantly related to each other. Thus, it is possible that drought-induced shifts in fungal community composition may not necessarily lead to changes in fungal biomass or decomposer ability in this arid grassland.

Metagenomic analysis reveals global-scale patterns of ocean nutrient limitation.

Nutrient supply regulates the activity of phytoplankton, but the global biogeography of nutrient limitation and co-limitation is poorly understood. Prochlorococcus adapt to local environments by gene gains and losses, and we used genomic changes as an indicator of adaptation to nutrient stress. We collected metagenomes from all major ocean regions as part of the Global Ocean Ship-based Hydrographic Investigations Program (Bio-GO-SHIP) and quantified shifts in genes involved in nitrogen, phosphorus, and iron assimilation. We found regional transitions in stress type and severity as well as widespread co-stress. Prochlorococcus stress genes, bottle experiments, and Earth system model predictions were correlated. We propose that the biogeography of multinutrient stress is stoichiometrically linked by controls on nitrogen fixation. Our omics-based description of phytoplankton resource use provides a nuanced and highly resolved description of nutrient stress in the global ocean.

High spatial resolution global ocean metagenomes from Bio-GO-SHIP repeat hydrography transects.

Detailed descriptions of microbial communities have lagged far behind physical and chemical measurements in the marine environment. Here, we present 971 globally distributed surface ocean metagenomes collected at high spatio-temporal resolution. Our low-cost metagenomic sequencing protocol produced 3.65 terabases of data, where the median number of base pairs per sample was 3.41 billion. The median distance between sampling stations was 26 km. The metagenomic libraries described here were collected as a part of a biological initiative for the Global Ocean Ship-based Hydrographic Investigations Program, or "Bio-GO-SHIP." One of the primary aims of GO-SHIP is to produce high spatial and vertical resolution measurements of key state variables to directly quantify climate change impacts on ocean environments. By similarly collecting marine metagenomes at high spatiotemporal resolution, we expect that this dataset will help answer questions about the link between microbial communities and biogeochemical fluxes in a changing ocean.

Persistent El Niño driven shifts in marine cyanobacteria populations.

In the California Current Ecosystem, El Niño acts as a natural phenomenon that is partially representative of climate change impacts on marine bacteria at timescales relevant to microbial communities. Between 2014-2016, the North Pacific warm anomaly (a.k.a., the "blob") and an El Niño event resulted in prolonged ocean warming in the Southern California Bight (SCB). To determine whether this "marine heatwave" resulted in shifts in microbial populations, we sequenced the rpoC1 gene from the biogeochemically important picocyanobacteria Prochlorococcus and Synechococcus at 434 time points from 2009-2018 in the MICRO time series at Newport Beach, CA. Across the time series, we observed an increase in the abundance of Prochlorococcus relative to Synechococcus as well as elevated frequencies of ecotypes commonly associated with low-nutrient and high-temperature conditions. The relationships between environmental and ecotype trends appeared to operate on differing temporal scales. In contrast to ecotype trends, most microdiverse populations were static and possibly reflect local habitat conditions. The only exceptions were microdiversity from Prochlorococcous HLI and Synechococcus Clade II that shifted in response to the 2015 El Niño event. Overall, Prochlorococcus and Synechococcus populations did not return to their pre-heatwave composition by the end of this study. This research demonstrates that extended warming in the SCB can result in persistent changes in key microbial populations.

Latitudinal gradient in the respiration quotient and the implications for ocean oxygen availability.

Climate-driven depletion of ocean oxygen strongly impacts the global cycles of carbon and nutrients as well as the survival of many animal species. One of the main uncertainties in predicting changes to marine oxygen levels is the regulation of the biological respiration demand associated with the biological pump. Derived from the Redfield ratio, the molar ratio of oxygen to organic carbon consumed during respiration (i.e., the respiration quotient, [Formula: see text]) is consistently assumed constant but rarely, if ever, measured. Using a prognostic Earth system model, we show that a 0.1 increase in the respiration quotient from 1.0 leads to a 2.3% decline in global oxygen, a large expansion of low-oxygen zones, additional water column denitrification of 38 Tg N/y, and the loss of fixed nitrogen and carbon production in the ocean. We then present direct chemical measurements of [Formula: see text] using a Pacific Ocean meridional transect crossing all major surface biome types. The observed [Formula: see text] has a positive correlation with temperature, and regional mean values differ significantly from Redfield proportions. Finally, an independent global inverse model analysis constrained with nutrients, oxygen, and carbon concentrations supports a positive temperature dependence of [Formula: see text] in exported organic matter. We provide evidence against the common assumption of a static biological link between the respiration of organic carbon and the consumption of oxygen. Furthermore, the model simulations suggest that a changing respiration quotient will impact multiple biogeochemical cycles and that future warming can lead to more intense deoxygenation than previously anticipated.

Linking regional shifts in microbial genome adaptation with surface ocean biogeochemistry.

Linking 'omics measurements with biogeochemical cycles is a widespread challenge in microbial community ecology. Here, we propose applying genomic adaptation as 'biosensors' for microbial investments to overcome nutrient stress. We then integrate this genomic information with a trait-based model to predict regional shifts in the elemental composition of marine plankton communities. We evaluated this approach using metagenomic and particulate organic matter samples from the Atlantic, Indian and Pacific Oceans. We find that our genome-based trait model significantly improves our prediction of particulate C : P (carbon : phosphorus) across ocean regions. Furthermore, we detect previously unrecognized ocean areas of iron, nitrogen and phosphorus stress. In many ecosystems, it can be very challenging to quantify microbial stress. Thus, a carefully calibrated genomic approach could become a widespread tool for understanding microbial responses to environmental changes and the biogeochemical outcomes. This article is part of the theme issue 'Conceptual challenges in microbial community ecology'.