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DOE OSTI2023

Structures, activity and mechanism of the Type IIS restriction endonuclease PaqCI

Abstract Type IIS restriction endonucleases contain separate DNA recognition and catalytic domains and cleave their substrates at well-defined distances outside their target sequences. They are employed in biotechnology for a variety of purposes, including the creation of gene-targeting zinc finger and TAL effector nucleases and DNA synthesis applications such as Golden Gate assembly. The most thoroughly studied Type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or similar enzymes forms a DNA-bound reaction complex have not been described at atomic resolution. Here we describe biochemical analyses of DNA cleavage by the Type IIS PaqCI restriction endonuclease and a series of molecular structures in the presence and absence of multiple bound DNA targets. The enzyme displays a similar tetrameric organization of target recognition domains in the absence or presence of bound substrate, with a significant repositioning of endonuclease domains in a trapped DNA-bound complex that is poised to deliver the first of a series of double-strand breaks. PaqCI and FokI share similar structural mechanisms of DNA cleavage, but considerable differences in their domain organization and quaternary architecture, facilitating comparisons between distinct Type IIS enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis for cloverleaf RNA-initiated viral genome replication

The genomes of positive-strand RNA viruses serve as a template for both protein translation and genome replication. In enteroviruses, a cloverleaf RNA structure at the 5' end of the genome functions as a switch to transition from viral translation to replication by interacting with host poly(C)-binding protein 2 (PCBP2) and the viral 3CD pro protein. We determined the structures of cloverleaf RNA from coxsackievirus and poliovirus. Cloverleaf RNA folds into an H-type four-way junction and is stabilized by a unique adenosine-cytidine-uridine (A•C-U) base triple involving the conserved pyrimidine mismatch region. The two PCBP2 binding sites are spatially proximal and are located on the opposite end from the 3CD pro binding site on cloverleaf. We determined that the A•C-U base triple restricts the flexibility of the cloverleaf stem–loops resulting in partial occlusion of the PCBP2 binding site, and elimination of the A•C-U base triple increases the binding affinity of PCBP2 to the cloverleaf RNA. Based on the cloverleaf structures and biophysical assays, we propose a new mechanistic model by which enteroviruses use the cloverleaf structure as a molecular switch to transition from viral protein translation to genome replication.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Novobiocin blocks nucleic acid binding to Polθ and inhibits stimulation of its ATPase activity

Polymerase theta (Polθ) acts in DNA replication and repair, and its inhibition is synthetic lethal in BRCA1 and BRCA2-deficient tumor cells. Novobiocin (NVB) is a first-in-class inhibitor of the Polθ ATPase activity, and it is currently being tested in clinical trials as an anti-cancer drug. Here, we investigated the molecular mechanism of NVB-mediated Polθ inhibition. Using hydrogen deuterium exchange-mass spectrometry (HX-MS), biophysical, biochemical, computational and cellular assays, we found NVB is a non-competitive inhibitor of ATP hydrolysis. NVB sugar group deletion resulted in decreased potency and reduced HX-MS interactions, supporting a specific NVB binding orientation. Collective results revealed that NVB binds to an allosteric site to block DNA binding, both in vitro and in cells. Comparisons of The Cancer Genome Atlas (TCGA) tumors and matched controls implied that POLQ upregulation in tumors stems from its role in replication stress responses to increased cell proliferation: this can now be tested in fifteen tumor types by NVB blocking ssDNA-stimulation of ATPase activity, required for Polθ function at replication forks and DNA damage sites. Structural and functional insights provided in this study suggest a path for developing NVB derivatives with improved potency for Polθ inhibition by targeting ssDNA binding with entropically constrained small molecules.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes

Abstract CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

TroR is the primary regulator of the iron homeostasis transcription network in the halophilic archaeon Haloferax volcanii

Abstract Maintaining the intracellular iron concentration within the homeostatic range is vital to meet cellular metabolic needs and reduce oxidative stress. Previous research revealed that the haloarchaeon Halobacterium salinarum encodes four diphtheria toxin repressor (DtxR) family transcription factors (TFs) that together regulate the iron response through an interconnected transcriptional regulatory network (TRN). However, the conservation of the TRN and the metal specificity of DtxR TFs remained poorly understood. Here we identified and characterized the TRN of Haloferax volcanii for comparison. Genetic analysis demonstrated that Hfx. volcanii relies on three DtxR transcriptional regulators (Idr, SirR, and TroR), with TroR as the primary regulator of iron homeostasis. Bioinformatics and molecular approaches revealed that TroR binds a conserved cis-regulatory motif located ∼100 nt upstream of the start codon of iron-related target genes. Transcriptomics analysis demonstrated that, under conditions of iron sufficiency, TroR repressed iron uptake and induced iron storage mechanisms. TroR repressed the expression of one other DtxR TF, Idr. This reduced DtxR TRN complexity relative to that of Hbt. salinarum appeared correlated with natural variations in iron availability. Based on these data, we hypothesize that variable environmental conditions such as iron availability appear to select for increasing TRN complexity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Site-specific acetylation of polynucleotide kinase 3'-phosphatase regulates its distinct role in DNA repair pathways

Mammalian polynucleotide kinase 3'-phosphatase (PNKP), a DNA end-processing enzyme with 3'-phosphatase and 5'-kinase activities, is involved in multiple DNA repair pathways, including base excision (BER), single-strand break (SSBR), and double-strand break repair (DSBR). However, little is known as to how PNKP functions in such diverse repair processes. Here we report that PNKP is acetylated at K142 (AcK142) by p300 constitutively but at K226 (AcK226) by CBP, only after DSB induction. Co-immunoprecipitation analysis using AcK142 or AcK226 PNKP-specific antibodies showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP with DSBR proteins. Despite the modest effect of acetylation on PNKP’s enzymatic activity in vitro, cells expressing non-acetylable PNKP (K142R or K226R) accumulated DNA damage in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This is consistent with the reported degradation of CBP, but not p300, in HD cells. Moreover, transcribed genomes of HD cells progressively accumulated DSBs. Chromatin-immunoprecipitation analysis demonstrated the association of Ac-PNKP with the transcribed genes, consistent with PNKP’s role in transcription-coupled repair. Thus, our findings demonstrate that acetylation at two lysine residues, located in different domains of PNKP, regulates its distinct role in BER/SSBR versus DSBR.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structure of native four-repeat satellite III sequence with non-canonical base interactions

Abstract Tandem-repetitive DNA (where two or more DNA bases are repeated numerous times) can adopt non-canonical secondary structures. Many of these structures are implicated in important biological processes. Human Satellite III (HSat3) is enriched for tandem repeats of the sequence ATGGA and is located in pericentromeric heterochromatin in many human chromosomes. Here, we investigate the secondary structure of the four-repeat HSat3 sequence 5′-ATGGA ATGGA ATGGA ATGGA-3′ using X-ray crystallography, NMR, and biophysical methods. Circular dichroism spectroscopy, thermal stability, native PAGE, and analytical ultracentrifugation indicate that this sequence folds into a monomolecular hairpin with non-canonical base pairing and B-DNA characteristics at concentrations below 0.9 mM. NMR studies at 0.05–0.5 mM indicate that the hairpin is likely folded-over into a compact structure with high dynamics. Crystallographic studies at 2.5 mM reveal an antiparallel self-complementary duplex with the same base pairing as in the hairpin, extended into an infinite polymer. The non-canonical base pairing includes a G–G intercalation sandwiched by sheared A–G base pairs, leading to a cross-strand four guanine stack, so called guanine zipper. The guanine zippers are spaced throughout the structure by A–T/T–A base pairs. Our findings lend further insight into recurring structural motifs associated with the HSat3 and their potential biological functions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Engineering an Escherichia coli strain for production of long single-stranded DNA

Long single-stranded DNA (ssDNA) is a versatile molecular reagent with applications including RNA-guided genome engineering and DNA nanotechnology, yet its production is typically resource-intensive. We introduce a novel method utilizing an engineered Escherichia coli ‘helper’ strain and phagemid system that simplifies long ssDNA generation to a straightforward transformation and purification procedure. Our method obviates the need for helper plasmids and their associated contamination by integrating M13mp18 genes directly into the E. coli chromosome. We achieved ssDNA lengths ranging from 504 to 20 724 nt with titers up to 250 μg/l following alkaline lysis purification. The efficacy of our system was confirmed through its application in primary T-cell genome modifications and DNA origami folding. The reliability, scalability and ease of our approach promise to unlock new experimental applications requiring large quantities of long ssDNA.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB

Mitomycin C (MMC) repair factor A (mrfA) and factor B (mrfB), encode a conserved helicase and exonuclease that repair DNA damage in the soil-dwelling bacterium Bacillus subtilis. Here we have focused on the characterization of MrfB, a DEDDh exonuclease in the DnaQ superfamily. We solved the structure of the exonuclease core of MrfB to a resolution of 2.1 Å, in what appears to be an inactive state. In this conformation, a predicted α-helix containing the catalytic DEDDh residue Asp172 adopts a random coil, which moves Asp172 away from the active site and results in the occupancy of only one of the two catalytic Mg 2+ ions. We propose that MrfB resides in this inactive state until it interacts with DNA to become activated. By comparing our structure to an AlphaFold prediction as well as other DnaQ-family structures, we located residues hypothesized to be important for exonuclease function. Using exonuclease assays we show that MrfB is a Mg 2+ -dependent 3'–5' DNA exonuclease. We show that Leu113 aids in coordinating the 3' end of the DNA substrate, and that a basic loop is important for substrate binding. This work provides insight into the function of a recently discovered bacterial exonuclease important for the repair of MMC-induced DNA adducts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Symmetry breaking of fluorophore binding to a G-quadruplex generates an RNA aptamer with picomolar K D

Fluorogenic RNA aptamer tags with high affinity enable RNA purification and imaging. The G-quadruplex (G4) based Mango (M) series of aptamers were selected to bind a thiazole orange based (TO1-Biotin) ligand. Using a chemical biology and reselection approach, we have produced a MII.2 aptamer–ligand complex with a remarkable set of properties: Its unprecedented K D of 45 pM, formaldehyde resistance (8% v/v), temperature stability and ligand photo-recycling properties are all unusual to find simultaneously within a small RNA tag. Crystal structures demonstrate how MII.2, which differs from MII by a single A23U mutation, and modification of the TO1-Biotin ligand to TO1-6A-Biotin achieves these results. MII binds TO1-Biotin heterogeneously via a G4 surface that is surrounded by a stadium of five adenosines. Breaking this pseudo-rotational symmetry results in a highly cooperative and homogeneous ligand binding pocket: A22 of the G4 stadium stacks on the G4 binding surface while the TO1-6A-Biotin ligand completely fills the remaining three quadrants of the G4 ligand binding face. Similar optimization attempts with MIII.1, which already binds TO1-Biotin in a homogeneous manner, did not produce such marked improvements. We use the novel features of the MII.2 complex to demonstrate a powerful optically-based RNA purification system.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Contrasting transcriptional responses to Fusarium virguliforme colonization in symptomatic and asymptomatic hosts

The broad host range of Fusarium virguliforme represents a unique comparative system to identify and define differentially induced responses between an asymptomatic monocot host, maize (Zea mays), and a symptomatic eudicot host, soybean (Glycine max). Using a temporal, comparative transcriptome-based approach, we observed that early gene expression profiles of root tissue from infected maize suggest that pathogen tolerance coincides with the rapid induction of senescence dampening transcriptional regulators, including ANACs (Arabidopsis thaliana NAM/ATAF/CUC protein) and Ethylene-Responsive Factors. In contrast, the expression of senescence-associated processes in soybean was coincident with the appearance of disease symptom development, suggesting pathogen-induced senescence as a key pathway driving pathogen susceptibility in soybean. Based on the analyses described herein, we posit that root senescence is a primary contributing factor underlying colonization and disease progression in symptomatic versus asymptomatic host–fungal interactions. This process also supports the lifestyle and virulence of F. virguliforme during biotrophy to necrotrophy transitions. Further support for this hypothesis lies in comprehensive co-expression and comparative transcriptome analyses, and in total, supports the emerging concept of necrotrophy-activated senescence. We propose that F. virguliforme conditions an environment within symptomatic hosts, which favors susceptibility through transcriptomic reprogramming, and as described herein, the induction of pathways associated with senescence during the necrotrophic stage of fungal development.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

Chlamydomonas as a model for reactive oxygen species signaling and thiol redox regulation in the green lineage

While the maintenance of redox balance is important for all organisms, the energy balance in the chloroplast is particularly tightly linked to the overall cellular redox status in photosynthetic organisms. Excess light can cause overreduction of the photosynthetic electron transport chain, which, when unchecked, results in the generation of reactive oxygen species (ROS) that can oxidize cellular components including nucleic acids, proteins, and lipids (Li et al., 2009). A photosynthetic cell possesses multiple layers of defense against oxidative stress, such as antioxidant enzymes (Foyer and Noctor, 2005) and alternative electron transfer pathways (Alric and Johnson, 2017). At the same time, ROS can function as important signals that mediate responses to biotic and abiotic stresses (Apel and Hirt, 2004). Reversibly oxidizable small molecules and proteins can serve as ROS sinks and signals and are re-reduced by multiple mechanisms. For example, the ascorbate–glutathione (GSH) cycle is central in controlling ROS levels and regenerating/reducing oxidized cellular redox agents (Figure 1A; reviewed by Foyer and Noctor, 2011). The functions of many proteins can be regulated by the redox state of cysteine residues, such as the formation or reduction of disulfides by thioredoxins (TRX) (Buchanan, 1991) and glutaredoxins (Michelet et al., 2013b; Figure 1, B and C).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The extracellular vesicle proteomes of Sorghum bicolor and Arabidopsis thaliana are partially conserved

Abstract Plant extracellular vesicles (EVs) are membrane-bound organelles involved mainly in intercellular communications and defense responses against pathogens. Recent studies have demonstrated the presence of proteins, nucleic acids including small RNAs, and lipids along with other metabolites in plant EVs. Here, we describe the isolation and characterization of EVs from sorghum (Sorghum bicolor). Nanoparticle tracking analysis, dynamic light scattering, and cryo-electron tomography showed the presence of a heterogeneous population of EVs isolated from the apoplastic wash of sorghum leaves. Cryo-electron microscopy revealed that EVs had a median size of 110 nm and distinct populations of vesicles with single or multiple lipid bilayers and low or high amounts of contents. The heterogeneity was further supported by data showing that only a subset of EVs that were stained with a membrane dye, Potomac Gold, were also stained with the membrane-permeant esterase-dependent dye, calcein acetoxymethyl ester. Proteomic analysis identified 437 proteins that were enriched in multiple EV isolations, with the majority of these also found in the EV proteome of Arabidopsis (Arabidopsis thaliana). These data suggest a partial conservation of EV contents and function between the monocot, sorghum, and a distantly related eudicot, Arabidopsis.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Disentangling Sources of Gene Tree Discordance in Phylogenomic Data Sets: Testing Ancient Hybridizations in Amaranthaceae s.l

Gene tree discordance in large genomic data sets can be caused by evolutionary processes such as incomplete lineage sorting and hybridization, as well as model violation, and errors in data processing, orthology inference, and gene tree estimation. Species tree methods that identify and accommodate all sources of conflict are not available, but a combination of multiple approaches can help tease apart alternative sources of conflict. Here, using a phylotranscriptomic analysis in combination with reference genomes, we test a hypothesis of ancient hybridization events within the plant family Amaranthaceae s.l. that was previously supported by morphological, ecological, and Sanger-based molecular data. The data set included seven genomes and 88 transcriptomes, 17 generated for this study. We examined gene-tree discordance using coalescent-based species trees and network inference, gene tree discordance analyses, site pattern tests of introgression, topology tests, synteny analyses, and simulations. We found that a combination of processes might have generated the high levels of gene tree discordance in the backbone of Amaranthaceae s.l. Furthermore, we found evidence that three consecutive short internal branches produce anomalous trees contributing to the discordance. Overall, our results suggest that Amaranthaceae s.l. might be a product of an ancient and rapid lineage diversification, and remains, and probably will remain, unresolved. This work highlights the potential problems of identifiability associated with the sources of gene tree discordance including, in particular, phylogenetic network methods. Our results also demonstrate the importance of thoroughly testing for multiple sources of conflict in phylogenomic analyses, especially in the context of ancient, rapid radiations. We provide several recommendations for exploring conflicting signals in such situations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Intra-canopy leaf trait variation facilitates high leaf area index and compensatory growth in a clonal woody encroaching shrub

Leaf trait variation enables plants to utilize large gradients of light availability that exist across canopies of high leaf area index (LAI), allowing for greater net carbon gain while reducing light availability for understory competitors. While these canopy dynamics are well understood in forest ecosystems, studies of canopy structure of woody shrubs in grasslands are lacking. To evaluate the investment strategy used by these shrubs, we investigated the vertical distribution of leaf traits and physiology across canopies of Cornus drummondii, the predominant woody encroaching shrub in the Kansas tallgrass prairie. We also examined the impact of disturbance by browsing and grazing on these factors. Our results reveal that leaf mass per area (LMA) and leaf nitrogen per area (Na) varied approximately threefold across canopies of C. drummondii, resulting in major differences in the physiological functioning of leaves. High LMA leaves had high photosynthetic capacity, while low LMA leaves had a novel strategy for maintaining light compensation points below ambient light levels. The vertical allocation of leaf traits in C. drummondii canopies was also modified in response to browsing, which increased light availability at deeper canopy depths. As a result, LMA and Na increased at lower canopy depths, leading to a greater photosynthetic capacity deeper in browsed canopies compared to control canopies. This response, along with increased light availability, facilitated greater photosynthesis and resource-use efficiency deeper in browsed canopies compared to control canopies. Furthermore, our results illustrate how C. drummondii facilitates high LAI canopies and a compensatory growth response to browsing—both of which are key factors contributing to the success of C. drummondii and other species responsible for grassland woody encroachment.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

At least it is a dry cold: the global distribution of freeze–thaw and drought stress and the traits that may impart poly-tolerance in conifers

Abstract Conifers inhabit some of the most challenging landscapes where multiple abiotic stressors (e.g., aridity, freezing temperatures) often co-occur. Physiological tolerance to multiple stressors (‘poly-tolerance’) is thought to be rare because exposure to one stress generally limits responses to another through functional trade-offs. However, the capacity to exhibit poly-tolerance may be greater when combined abiotic stressors have similar physiological impacts, such as the disruption of hydraulic function imposed by drought or freezing. Here, we reviewed empirical data in light of theoretical expectations for conifer adaptations to drought and freeze–thaw cycles with particular attention to hydraulic traits of the stem and leaf. Additionally, we examined the commonality and spatial distribution of poly-stress along indices of these combined stressors. We found that locations with the highest values of our poly-stress index (PSi) are characterized by moderate drought and moderate freeze–thaw, and most of the global conifer distribution occupies areas of moderate poly-stress. Among traits examined, we found diverse responses to the stressors. Turgor loss point did not correlate with freeze–thaw or drought stress individually, but did with the PSi, albeit inverse to what was hypothesized. Leaf mass per area was more strongly linked with drought stress than the poly-stress and not at all with freeze–thaw stress. In stems, the water potential causing 50% loss of hydraulic conductivity became more negative with increasing drought stress and poly-stress but did not correlate with freeze–thaw stress. For these traits, we identified a striking lack of coverage for substantial portions of species ranges, particularly at the upper boundaries of their respective PSis, demonstrating a critical gap in our understanding of trait prevalence and plasticity along these stress gradients. Future research should investigate traits that confer tolerance to both freeze–thaw and drought stress in a wide range of species across broad geographic scales.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Brilliant angle-independent structural colours preserved in weevil scales from the Swiss Pleistocene

Extant weevils exhibit a remarkable colour palette that ranges from muted monochromatic tones to rainbow-like iridescence, with the most vibrant colours produced by three-dimensional photonic nanostructures housed within cuticular scales. Although the optical properties of these nanostructures are well understood, their evolutionary history is not fully resolved, in part due to a poor knowledge of their fossil record. Here, we report three-dimensional photonic nanostructures preserved in brightly coloured scales of two weevils, belonging to the genus Phyllobius or Polydrusus , from the Pleistocene (16–10 ka) of Switzerland. The scales display vibrant blue, green and yellow hues that resemble those of extant Phyllobius/Polydrusus . Scanning electron microscopy and small-angle X-ray scattering analyses reveal that the subfossil scales possess a single-diamond photonic crystal nanostructure. In extant Phyllobius/Polydrusus , the near-angle-independent blue and green hues function primarily in crypsis. The preservation of far-field, angle-independent structural colours in the Swiss subfossil weevils and their likely function in substrate matching confirm the importance of investigating fossil and subfossil photonic nanostructures to understand the evolutionary origins and diversification of colours and associated behaviours (e.g. crypsis) in insects.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Phylogenetic conservation of soil bacterial responses to simulated global changes.

Soil bacterial communities are altered by anthropogenic drivers such as climate change-related warming and fertilization. However, we lack a predictive understanding of how bacterial communities respond to such global changes. Here, we tested whether phylogenetic information might be more predictive of the response of bacterial taxa to some forms of global change than others. We analysed the composition of soil bacterial communities from perturbation experiments that simulated warming, drought, elevated CO2 concentration and phosphorus (P) addition. Bacterial responses were phylogenetically conserved to all perturbations. The phylogenetic depth of these responses varied minimally among the types of perturbations and was similar when merging data across locations, implying that the context of particular locations did not affect the phylogenetic pattern of response. We further identified taxonomic groups that responded consistently to each type of perturbation. These patterns revealed that, at the level of family and above, most groups responded consistently to only one or two types of perturbations, suggesting that traits with different patterns of phylogenetic conservation underlie the responses to different perturbations. We conclude that a phylogenetic approach may be useful in predicting how soil bacterial communities respond to a variety of global changes. This article is part of the theme issue 'Conceptual challenges in microbial community ecology'.

54 ENVIRONMENTAL SCIENCES↗