Massively parallel single-cell sequencing of diverse microbial populations.

Single-cell genetic heterogeneity is ubiquitous in microbial populations and an important aspect of microbial biology; however, we lack a broadly applicable and accessible method to study this heterogeneity in microbial populations. Here, we show a simple, robust and generalizable method for high-throughput single-cell sequencing of target genetic loci in diverse microbes using simple droplet microfluidics devices (droplet targeted amplicon sequencing; DoTA-seq). DoTA-seq serves as a platform to perform diverse assays for single-cell genetic analysis of microbial populations. Using DoTA-seq, we demonstrate the ability to simultaneously track the prevalence and taxonomic associations of >10 antibiotic-resistance genes and plasmids within human and mouse gut microbial communities. This workflow is a powerful and accessible platform for high-throughput single-cell sequencing of diverse microbial populations.

Clades of huge phages from across Earth's ecosystems.

Bacteriophages typically have small genomes1 and depend on their bacterial hosts for replication2. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is-to our knowledge-the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR-Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR-Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR-Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth's ecosystems.

Efficient plasmid transfer via natural competence in a microbial co-culture.

The molecular and ecological factors shaping horizontal gene transfer (HGT) via natural transformation in microbial communities are largely unknown, which is critical for understanding the emergence of antibiotic-resistant pathogens. We investigate key factors shaping HGT in a microbial co-culture by quantifying extracellular DNA release, species growth, and HGT efficiency over time. In the co-culture, plasmid release and HGT efficiency are significantly enhanced than in the respective monocultures. The donor is a key determinant of HGT efficiency as plasmids induce the SOS response, enter a multimerized state, and are released in high concentrations, enabling efficient HGT. However, HGT is reduced in response to high donor lysis rates. HGT is independent of the donor viability state as both live and dead cells transfer the plasmid with high efficiency. In sum, plasmid HGT via natural transformation depends on the interplay of plasmid properties, donor stress responses and lysis rates, and interspecies interactions.

vRhyme enables binning of viral genomes from metagenomes.

Genome binning has been essential for characterization of bacteria, archaea, and even eukaryotes from metagenomes. Yet, few approaches exist for viruses. We developed vRhyme, a fast and precise software for construction of viral metagenome-assembled genomes (vMAGs). vRhyme utilizes single- or multi-sample coverage effect size comparisons between scaffolds and employs supervised machine learning to identify nucleotide feature similarities, which are compiled into iterations of weighted networks and refined bins. To refine bins, vRhyme utilizes unique features of viral genomes, namely a protein redundancy scoring mechanism based on the observation that viruses seldom encode redundant genes. Using simulated viromes, we displayed superior performance of vRhyme compared to available binning tools in constructing more complete and uncontaminated vMAGs. When applied to 10,601 viral scaffolds from human skin, vRhyme advanced our understanding of resident viruses, highlighted by identification of a Herelleviridae vMAG comprised of 22 scaffolds, and another vMAG encoding a nitrate reductase metabolic gene, representing near-complete genomes post-binning. vRhyme will enable a convention of binning uncultivated viral genomes and has the potential to transform metagenome-based viral ecology.

Accurate and complete genomes from metagenomes.

Genomes are an integral component of the biological information about an organism; thus, the more complete the genome, the more informative it is. Historically, bacterial and archaeal genomes were reconstructed from pure (monoclonal) cultures, and the first reported sequences were manually curated to completion. However, the bottleneck imposed by the requirement for isolates precluded genomic insights for the vast majority of microbial life. Shotgun sequencing of microbial communities, referred to initially as community genomics and subsequently as genome-resolved metagenomics, can circumvent this limitation by obtaining metagenome-assembled genomes (MAGs); but gaps, local assembly errors, chimeras, and contamination by fragments from other genomes limit the value of these genomes. Here, we discuss genome curation to improve and, in some cases, achieve complete (circularized, no gaps) MAGs (CMAGs). To date, few CMAGs have been generated, although notably some are from very complex systems such as soil and sediment. Through analysis of about 7000 published complete bacterial isolate genomes, we verify the value of cumulative GC skew in combination with other metrics to establish bacterial genome sequence accuracy. The analysis of cumulative GC skew identified potential misassemblies in some reference genomes of isolated bacteria and the repeat sequences that likely gave rise to them. We discuss methods that could be implemented in bioinformatic approaches for curation to ensure that metabolic and evolutionary analyses can be based on very high-quality genomes.

New CRISPR-Cas systems from uncultivated microbes.

CRISPR-Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped. Metagenomics, the sequencing of DNA extracted directly from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms. Here, using genome-resolved metagenomics, we identify a number of CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life, to our knowledge. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two previously unknown systems, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.

Asgard archaea illuminate the origin of eukaryotic cellular complexity.

The origin and cellular complexity of eukaryotes represent a major enigma in biology. Current data support scenarios in which an archaeal host cell and an alphaproteobacterial (mitochondrial) endosymbiont merged together, resulting in the first eukaryotic cell. The host cell is related to Lokiarchaeota, an archaeal phylum with many eukaryotic features. The emergence of the structural complexity that characterizes eukaryotic cells remains unclear. Here we describe the 'Asgard' superphylum, a group of uncultivated archaea that, as well as Lokiarchaeota, includes Thor-, Odin- and Heimdallarchaeota. Asgard archaea affiliate with eukaryotes in phylogenomic analyses, and their genomes are enriched for proteins formerly considered specific to eukaryotes. Notably, thorarchaeal genomes encode several homologues of eukaryotic membrane-trafficking machinery components, including Sec23/24 and TRAPP domains. Furthermore, we identify thorarchaeal proteins with similar features to eukaryotic coat proteins involved in vesicle biogenesis. Our results expand the known repertoire of 'eukaryote-specific' proteins in Archaea, indicating that the archaeal host cell already contained many key components that govern eukaryotic cellular complexity.

Rice Paddy Nitrospirae Carry and Express Genes Related to Sulfate Respiration: Proposal of the New Genus "Candidatus Sulfobium".

Nitrospirae spp. distantly related to thermophilic, sulfate-reducing Thermodesulfovibrio species are regularly observed in environmental surveys of anoxic marine and freshwater habitats. Here we present a metaproteogenomic analysis of Nitrospirae bacterium Nbg-4 as a representative of this clade. Its genome was assembled from replicated metagenomes of rice paddy soil that was used to grow rice in the presence and absence of gypsum (CaSO4·2H2O). Nbg-4 encoded the full pathway of dissimilatory sulfate reduction and showed expression of this pathway in gypsum-amended anoxic bulk soil as revealed by parallel metaproteomics. In addition, Nbg-4 encoded the full pathway of dissimilatory nitrate reduction to ammonia (DNRA), with expression of its first step being detected in bulk soil without gypsum amendment. The relative abundances of Nbg-4 were similar under both treatments, indicating that Nbg-4 maintained stable populations while shifting its energy metabolism. Whether Nbg-4 is a strict sulfate reducer or can couple sulfur oxidation to DNRA by operating the pathway of dissimilatory sulfate reduction in reverse could not be resolved. Further genome reconstruction revealed the potential to utilize butyrate, formate, H2, or acetate as an electron donor; the Wood-Ljungdahl pathway was expressed under both treatments. Comparison to publicly available Nitrospirae genome bins revealed the pathway for dissimilatory sulfate reduction also in related Nitrospirae recovered from groundwater. Subsequent phylogenomics showed that such microorganisms form a novel genus within the Nitrospirae, with Nbg-4 as a representative species. Based on the widespread occurrence of this novel genus, we propose for Nbg-4 the name "Candidatus Sulfobium mesophilum," gen. nov., sp. nov.IMPORTANCE Rice paddies are indispensable for the food supply but are a major source of the greenhouse gas methane. If it were not counterbalanced by cryptic sulfur cycling, methane emission from rice paddy fields would be even higher. However, the microorganisms involved in this sulfur cycling are little understood. By using an environmental systems biology approach with Italian rice paddy soil, we could retrieve the population genome of a novel member of the phylum Nitrospirae This microorganism encoded the full pathway of dissimilatory sulfate reduction and expressed it in anoxic paddy soil under sulfate-enriched conditions. Phylogenomics and comparison to the results of environmental surveys showed that such microorganisms are actually widespread in freshwater and marine environments. At the same time, they represent an undiscovered genus within the little-explored phylum Nitrospirae Our results will be important for the design of enrichment strategies and postgenomic studies to further understanding of the contribution of these novel Nitrospirae spp. to the global sulfur cycle.

Genomic resolution of a cold subsurface aquifer community provides metabolic insights for novel microbes adapted to high CO2 concentrations.

As in many deep underground environments, the microbial communities in subsurface high-CO2 ecosystems remain relatively unexplored. Recent investigations based on single-gene assays revealed a remarkable variety of organisms from little studied phyla in Crystal Geyser (Utah, USA), a site where deeply sourced CO2 -saturated fluids are erupted at the surface. To provide genomic resolution of the metabolisms of these organisms, we used a novel metagenomic approach to recover 227 high-quality genomes from 150 microbial species affiliated with 46 different phylum-level lineages. Bacteria from two novel phylum-level lineages have the capacity for CO2 fixation. Analyses of carbon fixation pathways in all studied organisms revealed that the Wood-Ljungdahl pathway and the Calvin-Benson-Bassham Cycle occurred with the highest frequency, whereas the reverse TCA cycle was little used. We infer that this, and selection for form II RuBisCOs, are adaptions to high CO2 -concentrations. However, many autotrophs can also grow mixotrophically, a strategy that confers metabolic versatility. The assignment of 156 hydrogenases to 90 different organisms suggests that H2 is an important inter-species energy currency even under gaseous CO2 -saturation. Overall, metabolic analyses at the organism level provided insight into the biochemical cycles that support subsurface life under the extreme condition of CO2 saturation.

Genome-Resolved Meta-Omics Ties Microbial Dynamics to Process Performance in Biotechnology for Thiocyanate Degradation.

Remediation of industrial wastewater is important for preventing environmental contamination and enabling water reuse. Biological treatment for one industrial contaminant, thiocyanate (SCN-), relies upon microbial hydrolysis, but this process is sensitive to high loadings. To examine the activity and stability of a microbial community over increasing SCN- loadings, we established and operated a continuous-flow bioreactor fed increasing loadings of SCN-. A second reactor was fed ammonium sulfate to mimic breakdown products of SCN-. Biomass was sampled from both reactors for metagenomics and metaproteomics, yielding a set of genomes for 144 bacteria and one rotifer that constituted the abundant community in both reactors. We analyzed the metabolic potential and temporal dynamics of these organisms across the increasing loadings. In the SCN- reactor, Thiobacillus strains capable of SCN- degradation were highly abundant, whereas the ammonium sulfate reactor contained nitrifiers and heterotrophs capable of nitrate reduction. Key organisms in the SCN- reactor expressed proteins involved in SCN- degradation, sulfur oxidation, carbon fixation, and nitrogen removal. Lower performance at higher loadings was linked to changes in microbial community composition. This work provides an example of how meta-omics can increase our understanding of industrial wastewater treatment and inform iterative process design and development.

Functional metagenomic selection of ribulose 1, 5-bisphosphate carboxylase/oxygenase from uncultivated bacteria.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a critical yet severely inefficient enzyme that catalyses the fixation of virtually all of the carbon found on Earth. Here, we report a functional metagenomic selection that recovers physiologically active RubisCO molecules directly from uncultivated and largely unknown members of natural microbial communities. Selection is based on CO2 -dependent growth in a host strain capable of expressing environmental deoxyribonucleic acid (DNA), precluding the need for pure cultures or screening of recombinant clones for enzymatic activity. Seventeen functional RubisCO-encoded sequences were selected using DNA extracted from soil and river autotrophic enrichments, a photosynthetic biofilm and a subsurface groundwater aquifer. Notably, three related form II RubisCOs were recovered which share high sequence similarity with metagenomic scaffolds from uncultivated members of the Gallionellaceae family. One of the Gallionellaceae RubisCOs was purified and shown to possess CO2 /O2 specificity typical of form II enzymes. X-ray crystallography determined that this enzyme is a hexamer, only the second form II multimer ever solved and the first RubisCO structure obtained from an uncultivated bacterium. Functional metagenomic selection leverages natural biological diversity and billions of years of evolution inherent in environmental communities, providing a new window into the discovery of CO2 -fixing enzymes not previously characterized.

Evidence for hydrogen oxidation and metabolic plasticity in widespread deep-sea sulfur-oxidizing bacteria.

Hydrothermal vents are a well-known source of energy that powers chemosynthesis in the deep sea. Recent work suggests that microbial chemosynthesis is also surprisingly pervasive throughout the dark oceans, serving as a significant CO(2) sink even at sites far removed from vents. Ammonia and sulfur have been identified as potential electron donors for this chemosynthesis, but they do not fully account for measured rates of dark primary production in the pelagic water column. Here we use metagenomic and metatranscriptomic analyses to show that deep-sea populations of the SUP05 group of uncultured sulfur-oxidizing Gammaproteobacteria, which are abundant in widespread and diverse marine environments, contain and highly express genes encoding group 1 Ni, Fe hydrogenase enzymes for H(2) oxidation. Reconstruction of near-complete genomes of two cooccurring SUP05 populations in hydrothermal plumes and deep waters of the Gulf of California enabled detailed population-specific metatranscriptomic analyses, revealing dynamic patterns of gene content and transcript abundance. SUP05 transcripts for genes involved in H(2) and sulfur oxidation are most abundant in hydrothermal plumes where these electron donors are enriched. In contrast, a second hydrogenase has more abundant transcripts in background deep-sea samples. Coupled with results from a bioenergetic model that suggest that H(2) oxidation can contribute significantly to the SUP05 energy budget, these findings reveal the potential importance of H(2) as a key energy source in the deep ocean. This study also highlights the genomic plasticity of SUP05, which enables this widely distributed group to optimize its energy metabolism (electron donor and acceptor) to local geochemical conditions.

Genomic and transcriptomic analyses of the facultative methanotroph Methylocystis sp. strain SB2 grown on methane or ethanol.

A minority of methanotrophs are able to utilize multicarbon compounds as growth substrates in addition to methane. The pathways utilized by these microorganisms for assimilation of multicarbon compounds, however, have not been explicitly examined. Here, we report the draft genome of the facultative methanotroph Methylocystis sp. strain SB2 and perform a detailed transcriptomic analysis of cultures grown with either methane or ethanol. Evidence for use of the canonical methane oxidation pathway and the serine cycle for carbon assimilation from methane was obtained, as well as for operation of the complete tricarboxylic acid (TCA) cycle and the ethylmalonyl-coenzyme A (EMC) pathway. Experiments with Methylocystis sp. strain SB2 grown on methane revealed that genes responsible for the first step of methane oxidation, the conversion of methane to methanol, were expressed at a significantly higher level than those for downstream oxidative transformations, suggesting that this step may be rate limiting for growth of this strain with methane. Further, transcriptomic analyses of Methylocystis sp. strain SB2 grown with ethanol compared to methane revealed that on ethanol (i) expression of the pathway of methane oxidation and the serine cycle was significantly reduced, (ii) expression of the TCA cycle dramatically increased, and (iii) expression of the EMC pathway was similar. Based on these data, it appears that Methylocystis sp. strain SB2 converts ethanol to acetyl-coenzyme A, which is then funneled into the TCA cycle for energy generation or incorporated into biomass via the EMC pathway. This suggests that some methanotrophs have greater metabolic flexibility than previously thought and that operation of multiple pathways in these microorganisms is highly controlled and integrated.

Protein Set Transformer: A protein-based genome language model to power high diversity viromics.

Exponential increases in microbial and viral genomic data demand transformational advances in scalable, generalizable frameworks for their interpretation. Standard homology-based functional analyses are hindered by the rapid divergence of microbial and especially viral genomes and proteins that significantly decreases the volume of usable data. Here, we present Protein Set Transformer (PST), a protein-based genome language model that models genomes as sets of proteins without considering sparsely available functional labels. Trained on >100k viruses, PST outperformed other homology- and language model-based approaches for relating viral genomes based on shared protein content. Further, PST demonstrated protein structural and functional awareness by clustering capsid-fold-containing proteins with known capsid proteins and uniquely clustering late gene proteins within related viruses. Our data establish PST as a valuable method for diverse viral genomics, ecology, and evolutionary applications. We posit that the PST framework can be a foundation model for microbial genomics when trained on suitable data.

Prophage-DB: A comprehensive database to explore diversity, distribution, and ecology of prophages.

Background: Viruses that infect prokaryotes (phages) constitute the most abundant group of biological agents, playing pivotal roles in microbial systems. They are known to impact microbial community dynamics, microbial ecology, and evolution. Efforts to document the diversity, host range, infection dynamics, and effects of bacteriophage infection on host cell metabolism are extremely underexplored. Phages are classified as virulent or temperate based on their life cycles. Temperate phages adopt the lysogenic mode of infection, where the genome integrates into the host cell genome forming a prophage. Prophages enable viral genome replication without host cell lysis, and often contribute novel and beneficial traits to the host genome. Current phage research predominantly focuses on lytic phages, leaving a significant gap in knowledge regarding prophages, including their biology, diversity, and ecological roles. Results: Here we develop and describe Prophage-DB, a database of prophages, their proteins, and associated metadata that will serve as a resource for viral genomics and microbial ecology. To create the database, we identified and characterized prophages from genomes in three of the largest publicly available databases. We applied several state-of-the-art tools in our pipeline to annotate these viruses, cluster and taxonomically classify them, and detect their respective auxiliary metabolic genes. In total, we identify and characterize over 350,000 prophages and 35,000 auxiliary metabolic genes. Our prophage database is highly representative based on statistical results and contains prophages from a diverse set of archaeal and bacterial hosts which show a wide environmental distribution. Conclusion: Prophages are particularly overlooked in viral ecology and merit increased attention due to their vital implications for microbiomes and their hosts. Here, we created Prophage-DB to advance our comprehension of prophages in microbiomes through a comprehensive characterization of prophages in publicly available genomes. We propose that Prophage-DB will serve as a valuable resource for advancing phage research, offering insights into viral taxonomy, host relationships, auxiliary metabolic genes, and environmental distribution.

Discarded diversity: Novel megaphages, auxiliary metabolic genes, and virally encoded CRISPR-Cas systems in landfills.

Background: Viruses are the most abundant microbial entity on the planet, impacting microbial community structure and ecosystem services. Despite outnumbering Bacteria and Archaea by an order of magnitude, viruses have been comparatively underrepresented in reference databases. Metagenomic examinations have illustrated that viruses of Bacteria and Archaea have been specifically understudied in engineered environments. Here we employed metagenomic and computational biology methods to examine the diversity, host interactions, and genetic systems of viruses predicted from 27 samples taken from three municipal landfills across North America. Results: We identified numerous viruses that are not represented in reference databases, including the third largest bacteriophage genome identified to date (~678 kbp), and note a cosmopolitan diversity of viruses in landfills that are distinct from viromes in other systems. Host-virus interactions were examined via host CRISPR spacer to viral protospacer mapping which captured hyper-targeted viral populations and six viral populations predicted to infect across multiple phyla. Virally-encoded auxiliary metabolic genes (AMGs) were identified with the potential to augment hosts' methane, sulfur, and contaminant degradation metabolisms, including AMGs not previously reported in literature. CRISPR arrays and CRISPR-Cas systems were identified from predicted viral genomes, including the two largest bacteriophage genomes to contain these genetic features. Some virally encoded Cas effector proteins appear distinct relative to previously reported Cas systems and are interesting targets for potential genome editing tools. Conclusions: Our observations indicate landfills, as heterogeneous contaminated sites with unique selective pressures, are key locations for diverse viruses and atypical virus-host dynamics.

Evolutionary history and origins of Dsr-mediated sulfur oxidation.

Microorganisms play vital roles in sulfur cycling through the oxidation of elemental sulfur and reduction of sulfite. These metabolisms are catalyzed by dissimilatory sulfite reductases (Dsr) functioning in either the reductive or reverse, oxidative direction. Dsr-mediated sulfite reduction is an ancient metabolism proposed to have fueled energy metabolism in some of Earth's earliest microorganisms, whereas sulfur oxidation is believed to have evolved later in association with the widespread availability of oxygen on Earth. Organisms are generally believed to carry out either the reductive or oxidative pathway, yet organisms from diverse phyla have been discovered with gene combinations that implicate them in both pathways. A comprehensive investigation into the metabolisms of these phyla regarding Dsr is currently lacking. Here, we selected one of these phyla, the metabolically versatile candidate phylum SAR324, to study the ecology and evolution of Dsr-mediated metabolism. We confirmed that diverse SAR324 encode genes associated with reductive Dsr, oxidative Dsr, or both. Comparative analyses with other Dsr-encoding bacterial and archaeal phyla revealed that organisms encoding both reductive and oxidative Dsr proteins are constrained to a few phyla. Further, DsrAB sequences from genomes belonging to these phyla are phylogenetically positioned at the interface between well-defined oxidative and reductive bacterial clades. The phylogenetic context and dsr gene content in these organisms points to an evolutionary transition event that ultimately gave way to oxidative Dsr-mediated metabolism. Together, this research suggests that SAR324 and other phyla with mixed dsr gene content are associated with the evolution and origins of Dsr-mediated sulfur oxidation.

Systematic genome-wide discovery of host factors governing bacteriophage infectivity.

Bacterial host factors regulate the infection cycle of bacteriophages. Except for some well-studied host factors (e.g., receptors or restriction-modification systems), the contribution of the rest of the host genome on phage infection remains poorly understood. We developed PHAGEPACK, a pooled assay that systematically and comprehensively measures each host-gene impact on phage fitness. PHAGEPACK combines CRISPR interference with phage packaging to link host perturbation to phage fitness during active infection. Using PHAGEPACK, we constructed a genome-wide map of genes impacting T7 phage fitness in permissive E. coli, revealing pathways previously unknown to affect phage packaging. When applied to the non-permissive E. coli O121, PHAGEPACK identified pathways leading to host resistance; their removal increased phage susceptibility up to a billion-fold. Bioinformatic analysis indicates phage genomes carry homologs or truncations of key host factors, potentially for fitness advantage. In summary, PHAGEPACK offers valuable insights into phage-host interactions, phage evolution, and bacterial resistance.

Shaping human gut community assembly and butyrate production by controlling the arginine dihydrolase pathway.

The arginine dihydrolase pathway (arc operon) present in a subset of diverse human gut species enables arginine catabolism. This specialized metabolic pathway can alter environmental pH and nitrogen availability, which in turn could shape gut microbiota inter-species interactions. By exploiting synthetic control of gene expression, we investigated the role of the arc operon in probiotic Escherichia coli Nissle 1917 on human gut community assembly and health-relevant metabolite profiles in vitro and in the murine gut. By stabilizing environmental pH, the arc operon reduced variability in community composition across different initial pH perturbations. The abundance of butyrate producing bacteria were altered in response to arc operon activity and butyrate production was enhanced in a physiologically relevant pH range. While the presence of the arc operon altered community dynamics, it did not impact production of short chain fatty acids. Dynamic computational modeling of pH-mediated interactions reveals the quantitative contribution of this mechanism to community assembly. In sum, our framework to quantify the contribution of molecular pathways and mechanism modalities on microbial community dynamics and functions could be applied more broadly.

Temperature-dependent trophic associations modulate soil bacterial communities along latitudinal gradients.

Understanding the environmental and biological mechanisms shaping latitudinal patterns in microbial diversity is challenging in the field of ecology. Although multiple hypotheses have been proposed to explain these patterns, a consensus has rarely been reached. Here, we conducted a large-scale field survey and microcosm experiments to investigate how environmental heterogeneity and putative trophic interactions (exerted by protist-bacteria associations and T4-like virus-bacteria associations) affect soil bacterial communities along a latitudinal gradient. We found that the microbial latitudinal diversity was kingdom dependent, showing decreasing, clumped, and increasing trends in bacteria, protists, and T4-like viruses, respectively. Climatic and edaphic drivers played predominant roles in structuring the bacterial communities; the intensity of the climatic effect increased sharply from 30°N to 32°N, whereas the intensity of the edaphic effect remained stable. Biotic associations were also essential in shaping the bacterial communities, with protist-bacteria associations showing a quadratic distribution, whereas virus-bacteria associations were significant only at high latitudes. The microcosm experiments further revealed that the temperature component, which is affiliated with climate conditions, is the primary regulator of trophic associations along the latitudinal gradient. Overall, our study highlights a previously underestimated mechanism of how the putative biotic interactions influence bacterial communities and their response to environmental gradients.