Biology subjects

Banfield, J. F.

Publications and source records attributed to Banfield, J. F..

At least 19 recordsLinked to original sources

bioRxiv2026

Genetic exchange drives relatedness of large linear and circular extrachromosomal elements of archaea

Archaeal extra-chromosomal elements (ECEs) play key roles in archaeal host biology and evolution, yet little is known about their diversity and interactions with each other. Here, we report 42 complete 66 - 165 kbp genomes: 23 "Klingons", 8 "Romulans" and "Romulan-like elements", and 11 "Ferengi", three new families of archaeal ECEs from diverse terrestrial environments. Methylation patterns, phylogenetics and gene sharing analyses suggest that Klingons and Romulans associate with Candidatus Methanoperedens and Ferengi are likely hosted by Candidatus Bathyarchaeaota. Like known archaeal viruses and plasmids, these ECEs both have circular genomes and replicate bi-directionally, unlike the linear Borg and mini-Borg ECEs that also replicate in Methanoperedens. The new ECE genomes all encode genes for methylation, glycosylation, protein degradation, and novel TnpB-like enzymes, which may interact with novel CRISPR-like arrays. All genomes also encode proteins with strong structural similarity to archaeal viral capsid proteins, and some are predicted to multimerize into pentameric or hexameric subunit capsid-like arrays and thus may encapsulate themselves. The three new ECE families share gene content with each other and with Borgs and mini-Borgs, often concentrated in specific genomic regions. Discordance among single gene and genomic phylogenies suggests that apparent relatedness of the five distinct ECE types stems from extensive horizontal gene transfer, rather than evolution from a common ancestor. The three new families of ECEs broaden our understanding of the diversity of archaeal ECEs and highlight the role of horizontal gene transfer in modulating the archaeal and archaeal ECE gene pools.

genomics↗

bioRxiv2026

Microbial inoculation accelerates post-fire soil recovery in a mixed conifer forest

High-severity wildfires of increasing size and frequency result in release of carbon dioxide and loss of timber resources, reduction in biodiversity, loss of soil, diminished water quality, and reduced recreational opportunities. Forest recovery strongly depends on the reestablishment of soil microbial communities, motivating research on how restoration of soil microbiomes in burned forests can be accelerated. Here, we used a high intensity burn pile experiment to test the effectiveness of post-fire native soil amendment. This design enabled us to sequentially and simultaneously sample unburned, burned, and inoculated burned soils while holding post-fire abiotic factors constant. All conditions were sampled at six time points across an annual hydrological cycle and analyzed using 16S and ITS rRNA amplicon sequencing, genome resolved metagenomics, metatranscriptomics, and soil chemistry. Fire sharply reduced bacterial and fungal diversity and eliminated ectomycorrhizal and ericoid symbiotic fungi. Inoculating the burned soil with native microbes accelerated recovery of microbial diversity and of functions associated with nutrient cycling, especially nitrogen transformations. Despite introducing the full diversity of soil microbes from adjacent unburned forest, only a small subset of adapted organisms were engrafted. Native soil inoculation stimulated reestablishment of mycorrhizal fungi, including genera that form essential symbioses with conifers, although this response was not persistent over the full year. Nonetheless, reestablishment of mycorrhizal fungi for even a window of time may facilitate early forest regrowth. We conclude that, by microbial inoculation, recovery that would otherwise rely on dispersal from distant sites is accelerated, potentially enhancing reforestation efforts.

microbiology↗

bioRxiv2026

Establishment of microbial strains from the river, groundwater, and soil in the hyporheic zone is limited despite connectivity

Microbial diversity across ecosystems may be impacted by dispersal pathways and selection due to physiochemical conditions, and so it is hard to predict the extent of strain sharing across heterogeneous environmental compartments. Evaluation of patterns of strain-level overlap requires genome-level comparisons using extensive datasets collected over large spatial scales. Here, we sampled microbiomes beneath (hyporheic zone) and within the East River (Colorado, USA) and found little or no strain overlap at sites along the river corridor, despite connection by river flow, suggesting selection due to the local environment. Comparisons involving microbiomes from hillslopes and riparian zone soil yielded essentially no strain-level overlap with the microbiomes of river and hyporheic zones. We also sampled a nearby groundwater well and found near-perfect genotypic overlap with strains in one hyporheic zone location that exhibited active groundwater upwelling. Given an absence of direct connectivity between these two locations via discrete hydrologic flow paths, we conclude that groundwater strains are widely dispersed in the aquifer. As hyporheic zone microbiomes in zones with low river flow share no strains with groundwater microbiomes, we infer that strains introduced by groundwater mixing in the hyporheic zone are transitory. We conclude that despite evidence for mixing of river-and-hyporheic zone water, and river-and-groundwater on short time scales, establishment of transported strains in the hyporheic zone is minimal.

microbiology↗

bioRxiv2026

Microbial autotrophy is widespread across soils and most prevalent in deep and saturated environments

Genes for CO2 fixation occur in soil microorganisms, but little is known about the pathways that are most common across ecosystem types, the organisms with these genes, where different CO2 fixation pathways are most prevalent, and the energy sources that support autotrophy across ecosystems. Here, we investigated microbial capacity for autotrophy in soils using 853 metagenomes and 201 metatranscriptomes from a wide range of terrestrial ecosystems (agricultural soils, wetlands, weathering rock). Autotrophy-associated RuBisCO (Form I and II) is widely encoded across all soils and occurs in bacteria from numerous lineages (38 phyla). RuBisCO Form IE is consistently more phylogenetically diverse in soils than in marine ecosystems, suggesting that it may have evolved to function in soil-like environments. A newly discovered deeply branching Form I RuBisCO, Form I, supports the hypothesis that Form I RuBisCO originated in anaerobic environments. Further, saturated soils harbor more, and more distinct, autotrophic microbes, many of which may use the Calvin-Benson-Bassham cycle or Wood-Ljungdahl pathway for CO2 fixation. Overall, the results indicate that autotrophy is a particularly important metabolism in deep, saturated soils and weathering rock.

microbiology↗

bioRxiv2026

Metagenomic strain-resolved DNA modification patterns link extrachromosomal genetic elements to host strains

DNA modification is central to microbial defense against extrachromosomal genetic elements (ECEs), consequently ECEs tend to adopt their hosts modification patterns. Shared ECE-host modification patterns enable linking ECEs to their hosts, but modification detection tools are designed for single genomes and are ineffective at metagenome scale. Here, we present MODIFI, software for detecting DNA modifications in metagenomes. MODIFI assumes that each k-mer in a metagenome is mostly unmodified and calculates background signal levels for that k-mer from PacBio HiFi reads, eliminating the need for matched control experiments. MODIFI ECE-host linkages were validated using >1,000 isolate and mock microbiome datasets. Illustrating the approach, we identified 315 strain-resolved, non-redundant ECE-host linkages in environmental and human metagenomes. In infant gut microbiomes, a chromosomal inversion in Enterococcus faecalis alters host and associated plasmid methylation motifs simultaneously. Overall, MODIFI solves a major bottleneck in DNA modification analysis and provides a foundational tool for understanding microbial epigenomics.

microbiology↗

bioRxiv2026

A Novel Pilus System in Candidate Phyla Radiation Bacteria

The Candidate Phyla Radiation (CPR) represents a bacterial superphylum estimated to include between 15-50% of all bacterial species, yet CPR bacteria remain challenging to culture and have been primarily identified through metagenomic approaches. Candidatus Mycosynbacter amalyticus is a parasitic CPR bacterium that specifically targets Gordonia amarae, an actinobacterium with a hydrophobic, mycolic acid-rich cell envelope. Previous cryo-electron tomography indicated that Ca. M. amalyticus assembles thin extracellular filaments that are important for host interaction, yet their molecular identity remains unknown. Here, we applied single-particle cryo-electron microscopy to determine high-resolution structures of these filaments (2.8 and 3.6 [A]), enabling the unambiguous identification of two previously uncharacterized pilins from the experimental density maps. These pilins, designated PamA and PamB, assemble into unique helical filaments distinct from all previously characterized filaments in both domain architecture and assembly mechanism. Despite low sequence identity, both PamA and PamB share conserved structural principles, including Ig-like folds and donor-strand exchange-mediated assembly. Phylogenetic analysis indicates that Pam pilins are exclusive to CPR bacteria, with homologues distributed predominantly across the classes Saccharimonadia and Microgenomatia. Analysis of the conserved pam operon identifies putative chaperones (PamC and PamD) and assembly factors structurally homologous to chaperone-usher pili components, suggesting an analogous but distinct assembly pathway. These findings expand the known diversity of bacterial pilus systems and demonstrate the power of structural approaches for characterizing uncharacterized proteins encoded within CPR genomes.

microbiology↗

bioRxiv2026

Jumbo circular extrachromosomal elements of methane-oxidizing archaea with variably extensive metabolic and defense gene repertoires

Archaeal extrachromosomal elements (ECEs) are arguably the least well understood of all genetic elements, and few have > 200 kbp ("jumbo") genomes. Here, we report circular, jumbo ECEs with genomes of up to 535 kbp in length that associate with anaerobic methane-oxidizing Methanoperedens archaea. Notably, a 409-kbp genome related to jumbo ECEs is integrated into a subset of the [~]4.2 Mbp Methanoperedens chromosomes at the tRNA-Asp genes. This represents the largest integrative element in Archaea and supports the jumbo ECE - host association. Multiple genome alignment and phylogenetic analyses suggest that the large sizes were developed by extensive DNA acquisition from Methanoperedens. The newly identified ECEs encode, and in some cases express, metabolic genes such as tetrahydromethanopterin S-methyltransferase exclusively involved in methane oxidation, and genes for nitrogen and sulfur compound transformations. Also encoded are defense systems, some of which are absent in hosts, such as hybrid Type I/Type III-A CRISPR-Cas systems. In contrast to viruses and plasmids, they have host-like replication machinery and occur at stable copy ratios of 1.44 {+/-} 0.24 : 1 to the host. Overall, our results reveal a spectrum of jumbo ECEs of Methanoperedens, ranging from plasmid-like to minichromosome-like.

microbiology↗

bioRxiv2025

Structure and evolution-guided design of minimal RNA-guided nucleases

The design of RNA-guided nucleases with properties not limited by evolution can expand programmable genome editing capabilities. However, generating diverse multi-domain proteins with robust enzymatic properties remains challenging. Here we use an artificial intelligence-driven strategy that couples structure-guided inverse protein folding with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease. High-throughput functional screening of AI-generated variants yielded editors that retained or exceeded wild-type activity in bacterial, plant and human cells. Cryo-EM-based structure determination of the most divergent active variant revealed new stabilizing contacts in the RNA/DNA interfaces across conformational states, demonstrating the design potential of this approach. Together these results establish a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space. One-sentence abstractAn evolution- and structure-conditioned model enables design of active RNA-guided nucleases with new nucleic acid contacts resolved by cryo-EM.

synthetic biology↗

bioRxiv2025

Engineered orthogonal translation systems from metagenomic libraries expand the genetic code

Genetic code expansion with non-canonical amino acids (ncAAs) opens new opportunities for the function and design of proteins by broadening their chemical repertoire. Unfortunately, ncAA incorporation is limited both by a small collection of orthogonal aminoacyl-tRNA synthetases (aaRSs) and tRNAs and by low-throughput methods to discover them. Here, we report the discovery, characterization, and engineering of a UGA suppressing orthogonal translation system mined from metagenomic data. We developed an integrated computational and experimental pipeline to profile the orthogonality of >200 tRNAs, test >1,250 combinations of aaRS:tRNA pairs, and identify the AP1 TrpRS:tRNATrpUCA as an orthogonal pair that natively encodes tryptophan at the UGA codon. We demonstrate that the AP1 TrpRS:tRNATrpUCA is highly active in cell-free and cellular contexts. We then use Ochre, a genomically recoded Escherichia coli strain that lacks UAG and UGA codons, to engineer an AP1 TrpRS variant capable of 5-hydroxytryptophan incorporation at an open UGA codon. We anticipate that our strategy of integrating metagenomic bioprospecting with cell-free screening and cell-based engineering will accelerate the discovery and optimization of orthogonal translation systems for genetic code expansion.

synthetic biology↗

bioRxiv2025

Reduced legacy precipitation decreases microbial community growth efficiency and alters soil organic carbon in a California grassland

Changes in global patterns can leave a lasting legacy in semi-arid grasslands by reshaping microbial growth dynamics and carbon cycling during the first wet-up in the autumn--a period known for intense microbial activity and significant carbon emissions. To study the lasting impacts of decreased winter rain, we implemented two precipitation regimes (100% vs. 50% mean annual precipitation) in California Mediterranean-climate grassland field plots. After the dry season, soils were rewetted in the laboratory with H218O, and sampled at 0 h, 3 h, 24 h, 48 h, 72 h, and 168 h post rewet. We quantified CO2 efflux; measured microbial growth and mortality via quantitative 18O stable isotope probing and 16S rRNA gene amplicon sequencing; and characterized the soil organic carbon chemical composition, metagenomes, and metatranscriptomes. We found that reduced winter precipitation imposed a strong legacy effect on microbial turnover; despite maintaining similar respiration rates, microbial growth declined by [~]1 order of magnitude, yielding decreased community growth efficiency (CGE = gross community growth/net respiration), and microbial mortality declined by [~]2 orders of magnitude. Soil organic carbon also shifted from lipid-like, amino-sugar-like, and protein-like compounds (indicative of microbial necromass) to more oxidized lignin-like and tannin-like compounds (indicative of decomposing plant-derived compounds). Meta-omics revealed distinct metabolic strategies linked to CGE. At high-CGE, microbes appeared to consume more energetically favorable N-rich necromass (released via high microbial turnover), this allowed for increased amino acids and peptidoglycan biosynthesis and greater aromatic compound degradation, fueling further energy production and growth efficiency. At low-CGE, communities had elevated carbohydrate metabolism and lipid turnover, consistent with increased investment in plant detritus degradation and membrane repair and maintenance rather than growth. Together, our findings demonstrate that reduced winter rainfall decreases microbial turnover following rewetting. Persistent decreases in CGE due to reduced winter rainfall result in consistent carbon loss as CO2, which, if sustained over multiple years, could ultimately lead to a net decline in total soil organic carbon.

microbiology↗

bioRxiv2025

Phage-based microbiome manipulation elucidates interactions within community context

Testing microbial interaction hypotheses remains a major challenge in microbiome research. Bacteriophages have the potential to probe organism-organism interactions within complex microbiomes, yet are rarely used for this purpose. Here, we isolated nine narrow host range phages that replicate in several ecologically important Variovorax species. As Variovorax CL14 degrades the plant root-stunting hormone auxin, we used phages to eliminate V. CL14 from a rhizosphere consortium and reestablished the stunted root phenotype. We used three of the phages to deplete another Variovorax, SCN45, from complex communities to test correlation network-based hypotheses of thiamine interdependencies. Genome-resolved metagenomics revealed that specific taxa decreased in relative abundance following Variovorax depletion and could be rescued by thiamine supplementation. Thus, we confirmed thiamine production as the mechanistic basis for interdependence. These experiments lay the foundation for research that employs wildtype and engineered phages to test interaction hypotheses and for targeted microbiome manipulation.

microbiology↗

bioRxiv2025

Assemblies of long-read metagenomes suffer from diverse errors

Genomes from metagenomes have revolutionised our understanding of microbial diversity, ecology, and evolution, propelling advances in basic science, biomedicine, and biotechnology. Assembly algorithms that take advantage of increasingly available long-read sequencing technologies bring the recovery of complete genomes directly from metagenomes within reach. However, assessing the accuracy of the assembled long reads, especially from complex environments that often include poorly studied organisms, poses remarkable challenges. Here we show that erroneous reporting is pervasive among long-read assemblers and can take many forms, including multi-domain chimeras, prematurely circularized sequences, haplotyping errors, excessive repeats, and phantom sequences. Our study highlights the need for rigorous evaluation of the algorithms while they are in development, and options for users who may opt for more accurate reads than shorter runtimes.

bioinformatics↗

bioRxiv2025

Overturning circulation structures the microbial functional landscape of the South Pacific

Global overturning circulation partitions the deep ocean into regions with unique physicochemical characteristics, but the extent to which these water masses represent distinct ecosystems remains unknown. Here, we integrate extensive genomic information with hydrography and water mass age to delineate microbial taxonomic and functional boundaries across the South Pacific. Prokaryotic richness steeply increases with depth in the surface ocean, forming a "phylocline", below which richness is consistently high, dipping slightly in highly aged water. Reconstructed genomes self-organize into six spatially-distinct taxonomic cohorts and ten functionally-distinct biomes that are primarily structured by wind-driven circulation at the surface and density-driven circulation at depth. Overall, water physicochemistry, modulated at depth by water age, drives microbial diversity and functional potential in the pelagic ocean.

microbiology↗

bioRxiv2024

The active subset of grassland soil microbiomes changes with soil depth, water availability and prominently features predatory bacteria and episymbionts

Mediterranean grasslands, vital natural and agricultural ecosystems, experience seasonal variation in water content that likely affect microbial activity. We used metagenomics-informed stable isotope probing to investigate how the activities of microorganisms in Angelo Reserve (2160 mm rainfall) and Hopland (956 mm rainfall) soil change over depth and the seasons. At both sites, we find that the relative abundances of organisms in shallow soil changes relatively little but the most abundant organisms vary greatly with soil depth. Notably the highest levels of isotope incorporation, indicative of growth, occurs in deep soils. The active part of the 0-10 cm soil community varies over time, especially in Hopland soils during the fall rewetting. We defined a large, novel clade of Actinomycetota with notable capacity for thiosulfate oxidation whose representatives are prevalent and active in deep soils (>20 cm) across both ecosystems. Active Saccharibacteria unexpectedly encode nucleotide synthesis genes that enabled isotope incorporation while growing in shallow Angelo soils over all time periods. In contrast to predicted episymbiotic lifestyles of Saccharibacteria, other highly active bacteria are predicted predators. Obligately predatory Pseudobdellovibrio are active in intermediate depth Hopland soils whereas bacteria of the order Haliangiales are active in shallow Angelo soils. Supporting predatory lifestyles of Haliangiales, we used in silico structure prediction to assemble a large protein complex that we identify as a contractile injection system. Overall, the results indicate the potential for active carbon turnover in deep grassland soil and strong seasonal changes in the active members of microbial communities, despite relatively minor shifts in community composition.

bioinformatics↗

bioRxiv2024

Coordinated organic and inorganic nitrogen transformations fuel soil microbial blooms and increase nitrogen retention during snowmelt

Snowmelt in high-elevation watersheds triggers a microbial bloom and crash that affects nitrogen (N) export. Predicting watershed N dynamics as snowpack declines is a challenge because the mechanisms that underlie this microbial bloom and crash are uncertain. Using a multi-omic approach, we show that the dynamic molecular properties of dissolved organic N, plus high gene expression for peptidases that recycle microbial biomass, suggested that microbial turnover provided N for biosynthesis during the microbial bloom. Amino acid fermentation by Bradyrhizobia produced organic acids that also fueled denitrification and dissimilatory nitrate reduction to ammonia (DNRA) during snowmelt. Nitrification in spring was driven by Spring-Adapted Nitrososphaerales, which utilized ammonium derived from osmolyte degradation by Winter-Adapted Solirubrobacteraceae. High DNRA gene expression after snowmelt suggested significant nitrate retention, increasing watershed N retention potential. However, declining snowfall may compromise microbial regulation of soil N retention, with implications for watershed N export.

microbiology↗

bioRxiv2024

Structure of an Archaeal Ribosome with a Divergent Active Site

The ribosome is the universal translator of the genetic code and is shared across all life. Despite divergence in ribosome structure over the course of evolution, the peptidyl transferase center (PTC), the catalytic site of the ribosome, has been thought to be nearly universally conserved. Here, we identify clades of archaea that have highly divergent ribosomal RNA sequences in the PTC. To understand how these PTC sequences fold, we determined cryo-EM structures of the Pyrobaculum calidifontis ribosome. We find that sequence variation leads to the rearrangement of key PTC base triples and differences between archaeal and bacterial ribosomal proteins also enable sequence variation in archaeal PTCs. Finally, we identify a novel archaeal ribosome hibernation factor that differs from known bacterial and eukaryotic hibernation factors and is found in multiple archaeal phyla. Overall, this work identifies factors that regulate ribosome function in archaea and reveals a larger diversity of the most ancient sequences in the ribosome.

evolutionary biology↗

bioRxiv2024

Reduced methane emissions in transgenic rice genotypes are associated with altered rhizosphere microbial hydrogen cycling

Rice paddies contribute substantially to atmospheric methane (CH4) and these emissions are expected to increase as the need to feed the human population grows. Here, we show that two independent rice genotypes overexpressing genes for PLANT PEPTIDES CONTAINING SULFATED TYROSINE (PSY) reduced cumulative CH4 emissions by 38% (PSY1) and 58% (PSY2) over the growth period compared with controls. Genome-resolved metatranscriptomic data from rhizosphere soils reveal lower ratios of gene activities for CH4 production versus consumption, decrease in activity of H2-producing genes, and increase in bacterial H2 oxidation pathways in the PSY genotypes. Metabolic modeling using metagenomic and metabolomic data predicts elevated levels of H2 oxidation and suppressed H2 production in the PSY rhizosphere. The H2-oxidizing bacteria have more genes for utilization of gluconeogenic acids than H2-producing counterparts, and their activities were likely stimulated by the observed enrichment of gluconeogenic acids (mostly amino acids) in PSY root exudates. Together these results suggest that decreased CH4 emission is due to the reduction of H2 available for hydrogenotrophic methanogenesis. The combination of rice phenotypic characterization, microbiome multi-omic analysis, and metabolic modeling described here provides a powerful strategy to discover the mechanisms by which specific plant genotypes can alter biogeochemical cycles to reduce CH4 emissions.

microbiology↗

bioRxiv2024

Characterization of sediment and granite hosted deep underground research laboratories reveals diverse microbiome functions, limited temporal variation and substantial genomic conservation

Underground research laboratories (URLs) provide a window on the deep biosphere and enable investigation of potential microbial impacts on nuclear waste, CO2 and H2 stored in the subsurface. We carried out the first multi-year study of groundwater microbiomes sampled from defined intervals between 140 and 400 m below the surface of the Horonobe and Mizunami URLs, Japan. We reconstructed draft genomes for >90% of all organisms detected over a four year period. The Horonobe and Mizunami microbiomes are dissimilar, likely because the Mizunami URL is hosted in granitic rock and the Horonobe URL in sedimentary rock. Despite this, hydrogen metabolism, rubisco-based CO2 fixation, reduction of nitrogen compounds and sulfate reduction are well represented functions in microbiomes from both URLs, although methane metabolism is more prevalent at the organic- and CO2-rich Horonobe URL. High fluid flow zones and proximity to subsurface tunnels select for candidate phyla radiation bacteria in the Mizunami URL. We detected near-identical genotypes for approximately one third of all genomically defined organisms at multiple depths within the Horonobe URL. This cannot be explained by inactivity, as in situ growth was detected for some bacteria, albeit at slow rates. Given the current low hydraulic conductivity and groundwater compositional heterogeneity, ongoing inter-site strain dispersal seems unlikely. Alternatively, the Horonobe URL microbiome homogeneity may be explained by higher groundwater mobility during the last glacial period. Genotypically-defined species closely related to those detected in the URLs were identified in three other subsurface environments in the USA. Thus, dispersal rates between widely separated underground sites may be fast enough relative to mutation rates to have precluded substantial divergence in species composition. Species overlaps between subsurface locations on different continents constrain expectations regarding the scale of global subsurface biodiversity. Overall, microbiome and geochemical stability over the study period has important implications for underground storage applications.

microbiology↗