Biology subjects

Nielsen, T. N.

Publications and source records attributed to Nielsen, T. N..

11 recordsLinked to original sources

bioRxiv2026

A complete-genome view of phylum Omnitrophota and a multi-order capacity for very long proteins

Phylum Omnitrophota (formerly candidate division OP3) is represented in public databases almost entirely by metagenome-assembled genomes; GTDB R232 contains two complete Omnitrophota assemblies. We present 176 complete and 53 high-quality Omnitrophota genomes from Oxford Nanopore metagenomes of Fennoscandian deep groundwater and the Baltic Sea water column, an 88-fold expansion of the complete Omnitrophota count. The 229 genomes resolve to 202 distinct species at 95 % ANI; 162 of these have no conspecific match in the 714 NCBI HQ Omnitrophota MAGs. Phylogenomically, 171 of the 176 complete genomes fall in class Gorgyraeia -- which contains the cultured episymbiont Velamenicoccus archaeovorus -- and 5 in Omnitrophia, with multiple Gorgyraeia orders and families represented. Phylum Omnitrophota hosts many very long proteins, with the longest in our corpus reaching 147,155 AA; the long end of the length distribution is concentrated on Gorgyraeia contigs across multiple Gorgyraeia orders. 24-28 % of GTDB-Tk-classified Omnitrophota contigs in the deep-groundwater and Baltic samples host at least one [≥] 10-kAA protein. Across the 916-protein long-protein domain-architecture catalog, 94 % carry transmembrane helices or a signal peptide; the four complete-genome proteins above 100,000 amino acids are all in inner-membrane-anchored architectures, the 147,155-AA protein with 147 TM helices. The 176 complete genomes share a uniform metabolic profile across the dominant orders: intact bacterial peptidoglycan biosynthesis alongside fragmentary TCA, incomplete electron transport, absent aerobic terminal oxidase, and partial cofactor and amino-acid biosynthesis. The profile matches the cultured V. archaeovorus phenotype and is consistent with a host-dependent episymbiotic lifestyle. Hypervariable-region calling across the 229 chromosomes returns 1,909 candidate loci, distributed across 223 of them; ribosomal-protein and EF-Tu / EF-G content sits inside called HVRs on 150 of those 223 (67 %), recovering across the collection the housekeeping-cargo integrations documented in Nielsen (2026b). All genomes, the OrthoFinder supermatrix and its ML tree, the 916-protein giant-protein domain-architecture catalog, and per-step scripts are released as a community resource at Zenodo (DOI [TBD]).

microbiology↗

bioRxiv2026

Cohort-HMM marker recruitment with per-OG orthology QC for phylogenomic supermatrices

OrthoFinders all-vs-all DIAMOND step systematically misses single-copy orthogroups (SC OGs) at deep taxonomic divergence: a marker recovered cleanly within a tightly defined cohort is dropped when the same marker is searched against phylum-broad metagenome-assembled genome (MAG) sets, because pairwise sequence similarity falls below DIAMONDs detection threshold even when the underlying ortholog is present. The result is biased dropout -- supermatrices that retain genomes near the cohort but lose genomes from the deeper, more diverged corners of the same phylum. We describe a two-stage cohort-HMM recruitment pipeline (per-OG profile HMMs built from cohort alignments, then hmmsearch against the broader proteome set) followed by an independent per-OG gene-tree QC step that classifies each recruited hit relative to the cohorts most recent common ancestor (MRCA) descendant set, with a per-MAG paralog-rate filter applied before supermatrix concatenation. We characterize the pipeline across three taxonomic ranks. At phylum scale (Omnitrophota, 97 cohort OGs, 714 NCBI MAGs), the recruitment recovers MAGs that the OrthoFinder-only supermatrix would otherwise drop, and the QC identifies 2 deep-peripheral MAGs -- divergent genomes whose per-OG tips repeatedly place outside the cohort MRCA descendant set despite being orthologs -- that the per-MAG filter removes. At family scale (Pelagibacteraceae, 146 cohort OGs, 366 NCBI MAGs) and at genus scale (Actinomarina, 289 cohort OGs, 23 NCBI MAGs), the per-tip paralog-candidate rate drops to 0.0 %. The pipeline addresses two independent failure modes. Cohort paralog density breaks strict-SC OG discovery at the cohort step (the family-rank case, where every candidate marker has at least one cohort species carrying multiple copies; the relaxed cohort criterion supplies the marker set and HMM recruitment disambiguates which copy each NCBI MAG contributes). DIAMOND-reach attrition breaks OG assignment for the most divergent NCBI MAGs (the phylum-rank case, where pairwise similarities fall below DIAMONDs detection threshold; HMM recruitment recovers the dropouts and the per-OG QC step filters residual paralog candidates). At genus rank both modes are inactive and OrthoFinder suffices directly; HMM recruitment runs but finds no new orthologs. Code and per-case data products are released as a community resource at Zenodo (DOI 10.5281/zenodo.20422348).

bioinformatics↗

bioRxiv2026

Omnitrophota dominate per-genome cross-replicon lateralgene transfer in a Fennoscandian deep-groundwatermetagenome

In a single Oxford Nanopore long-read metagenome from a Fennoscandian deep-groundwater borehole (KR0015B, Aspo Hard Rock Laboratory, Sweden), 791 protein clusters span at least one chromosomal contig and at least one co-sampled circular mobile element -- the cross-replicon LGT-candidate cohort. The 199 participating chromosomes are dominated by three small-genome / symbiont-associated lineages -- Patescibacteriota, Omnitrophota, and Nanobdellota -- but the per-chromosome participation rate tells a different story: Omnitrophota chromosomes participate at an order-of-magnitude higher rate (mean 56 cross-replicon clusters per genome), while Patescibacteriota and Nanobdellota dominate by compositional abundance only. Two large divergent circular mobile elements (233-kbp u20424375 and 123-kbp u29249220) -- each lineage-restricted within a single Omnitrophota genus, with sparse cross-phylum reach (only 12 of their combined 289 cross-replicon clusters involve a non-Omnitrophota partner) -- together account for 37% of the cohort and lack canonical plasmid or phage signatures. The 233-kbp element carries a Mu-class DDE transposase, is found integrated in one host chromosome at 99.3% nucleotide identity over 87% of element length, and carries an essentially complete bacterial big-operon r-protein cluster (31 r-protein KOs) as cargo with no rRNA genes -- a cargo profile with no published precedent in the mobile-element literature. Seven cross-replicon clusters span both domains; per-cluster phylogenies confirm gene-tree topologies that violate the species-tree expectation in 6 of 10 callable smoking-gun trees. We release the cross-replicon cluster table, integrated mobile-partner classifications, and chromosome taxonomy as a community resource. A parallel cross-chromosome catalog without the mobile-partner requirement contains 957 clusters, 95% of which carry no co-sampled circular plasmid or virus partner -- a chromosome-only LGT footprint that bounds the MGE-coupled cohort and is consistent with vehicle-free / direct-contact transfer in lineages whose close-contact symbiotic biology is well-documented.

microbiology↗

bioRxiv2026

A complete-genome view of phylum Nanobdellota and recurrent Form III RuBisCO transfer between archaea and Patescibacteriota

The archaeal phylum Nanobdellota (formerly Nanoarchaeota) was previously represented by four complete genomes. We present 208 complete Nanobdellota genomes from Oxford Nanopore metagenomes of the Baltic Sea water column and Fennoscandian groundwater (69-201 m below sea level), rotated to the ORC1/Cdc6 replication origin -- a 52-fold expansion of complete-genome representation. Across the ar53 supermatrix and a Nanobdellota-tuned 71-marker supermatrix on 1,239 taxa, the named GTDB orders within Nanobdellota are recovered as monophyletic clades, including the three orders that dominate our environmental sampling: Woesearchaeales, Pacearchaeales, and the GTDB placeholder order SCGC-AAA011-G17. This is consistent with the existing GTDB R232 order-level circumscription. We retire the SCGC-AAA011-G17 placeholder name, replacing it with a complete-genome-anchored SeqCode nomenclatural chain (Maxwellarchaeales ord. nov., Maxwellarchaeaceae fam. nov., Maxwellarchaeum gen. nov., and Maxwellarchaeum balticum sp. nov.) without altering the order-level circumscription. Pacearchaeales and Maxwellarchaeales retain no central or energy metabolism beyond Form III RuBisCO, PEP synthase, and ferredoxin; Woesearchaeales retains partial glycolysis and a V/A-type ATPase. A 4,262-tip phylogeny of rbcL (the RuBisCO large-subunit gene) identifies nine candidate archaea-to-Patescibacteriota Form III RuBisCO transfer events -- including one to a Baltic Minisyncoccia -- versus two reciprocal candidates, consistent with archaea-to-CPR being the more frequently identified direction in our data. All 256 Nanobdellota genomes (208 complete + 48 high-quality non-circular), the ar71 marker set with its 1,239-taxon ML tree, 154 Nanobdellota-trained HMMs for KEGG-ortholog detection in DPANN proteomes (94 ROBUST), and the 4,262-tip rbcL reference tree are released as a community resource, alongside the full analysis archive -- alignments, intermediate trees, structural predictions, and per-step scripts -- at Zenodo (DOI 10.5281/zenodo.20174424; see Using the resource).

microbiology↗

bioRxiv2026

Actinomarina, resolved

Actinomarina (Ca. Actinomarina minuta) is among the smallest known free-living bacteria and among the most abundant photoheterotrophs in ocean surface waters, yet not a single complete genome has been published for any member of the order Actinomarinales. Here we present 84 complete Actinomarina genomes assembled from Oxford Nanopore metagenomes of the San Francisco Estuary (SFE), together with 29 additional high-quality ([≥]95% complete, <5% contamination, single contig) non-circular assemblies. These genomes define 9 species at 95% ANI, three of which are novel, and represent, to our knowledge, the first complete genomes for the entire Actinomarinales order. An expanded phylogeny incorporating 23 high-quality NCBI genomes and a Casp-actino5 outgroup confirms species boundaries and places NCBI genomes among SFE clades. The pangenome of 9,278 clusters is approaching closure (decay ratio 0.55), with a core genome of 227 single-copy genes (2.4%). Singletons (3,858 clusters, 42%) are concentrated in a tRNA-bounded hypervariable region (HVR) at 85-90% of the chromosome from dnaA -- a similar replicative distance to the HVR recently described in Pelagibacter (7-15% from dnaA; Lui & Nielsen, in preparation) but on the opposite replichore. The HVR is flanked by tRNA genes at both boundaries, and in 32 of 84 genomes, the selenocysteine tRNA (selC) is physically located inside the HVR. All 84 genomes encode selenocysteine tRNA (selC) and the selenocysteine biosynthesis genes selA and selD; the elongation factor selB is present but divergent. This has not been previously reported for Actinomarina. Deep learning selenoprotein prediction (deep-Sep) identifies [~]5 selenoproteins per genome in 69 families, including a selenoprotein form of selD itself. Retention of dedicated Sec biosynthetic machinery serving multiple targets in a genome of [~]1.1 Mbp implies that the catalytic advantage of selenocysteine over cysteine is large enough to justify the cost. KofamScan pathway reconstruction reveals the most extensive set of auxotrophies yet documented from complete genomes in a free-living marine bacterium: arginine, histidine, tryptophan, and thiamine biosynthesis are universally absent, biotin biosynthesis is non-functional (final step absent), and only 5 of 8 TCA cycle steps are retained at the standard detection threshold. Gene order is extensively rearranged between species: no gene adjacency is universal across all 84 genomes. NCBI currently lists 396 Actinomarina genomes, none complete; of the 39 that pass quality thresholds, 41% are misclassified by NCBI, belonging to other genera by GTDB-Tk reclassification.

microbiology↗

bioRxiv2026

Pelagibacter, resolved

Pelagibacter, the largest genus within the SAR11 clade, is the most abundant bacterium in the ocean, yet the vast majority of its species-level diversity remains uncharacterized at the genomic level. Here we present 135 complete Pelagibacter genomes -- the largest such collection assembled to date -- comprising 75 from Oxford Nanopore metagenomes of the San Francisco Estuary (SFE), 31 from a deeply sequenced station within the same transect, and 29 from public databases. These genomes define 52 species at 95% ANI, of which 44 (85%) are taxonomically novel. An expanded phylogeny incorporating 89 additional high-quality NCBI genomes confirms that our collection captures the phylogenetic backbone of the genus, with genomes from Hawaii, Namibia, and the Sargasso Sea nesting within SFE clades. The pangenome is open (14,862 singletons, 62%), driven by two distinct mechanisms. First, a universal hypervariable region (HVR) at a conserved chromosomal position (7-15% from dnaA) is present in all 135 genomes, anchored by tRNA genes at both boundaries (Phe/His and Arg). The HVR carries genome-specific surface polysaccharide biosynthesis genes with a GC age gradient -- highest GC at the tRNA boundaries, lowest in the center -- consistent with a two-ended phage insertion model. Only this HVR is positionally conserved across the genus; the three other hypervariable regions previously described in a single reference genome are not. Second, scattered genomic islands throughout the chromosome contribute the remaining singleton content, including chimeric islands with genes from four bacterial phyla. Biosynthetic pathway reconstruction reveals auxotrophies that are phylogenetically structured, not uniform: biotin, reduced sulfur, and glycine are genus-wide dependencies, while isoleucine, pantothenate, histidine, and glyoxylate cycle capacity vary across lineages with significant phylogenetic clustering. Structural annotation with ESMFold and Foldseek resolved 3,125 hypothetical proteins; 1,222 remain uncharacterized by any method, including a 47-amino-acid protein conserved in two-thirds of all genomes within a fixed operonic context -- independently predicted by two gene callers yet matching nothing in any database. A controlled depth comparison at one station demonstrates that standard metagenome sequencing systematically underestimates Pelagibacter diversity, with three species recovered only at elevated depth and the species count at that station more than doubling (9 vs 4).

microbiology↗

bioRxiv2026

General-purpose embeddings for long-read metagenomic sequences via β-VAE on multi-scale k-mer frequencies

Long-read metagenomics routinely produces millions of assembled contigs, creating a need for methods that organize sequences into biologically meaningful groups across samples and environments. We present a general-purpose compositional embedding for metagenomic sequences based on a {beta}-variational autoencoder ({beta}-VAE) trained on multi-scale k-mer frequencies (1-mers through 6-mers; 2,772 features with centered log-ratio transformation). The embedding compresses each contig into a 384-dimensional vector that preserves local compositional similarity, enabling similarity search and graph-based clustering from sequence composition alone. Through systematic comparison of fifteen models trained on up to 17.4 million contigs (525.5 Gbp) from brackish, terrestrial, and reference genome sources, we find that a small set of curated prokaryotic reference genomes (656,000 contigs) outperforms ten-fold larger domain-specific training sets, and that neither reconstruction loss nor Spearman correlation reliably predicts downstream clustering quality. On nearest-neighbor graphs, flow-based clustering (MCL) markedly outperforms modularity-based methods (Leiden), yielding 12,123 clusters from 154,041 contigs ([≥] 100 kbp) with 99.2% phylum-level purity confirmed by independent marker gene phylogenetics. Multi-method taxonomic annotation achieves 87% coverage and reveals that 16.4% of contigs are eukaryotic--the single largest component invisible to standard prokaryotic annotation tools. The embedding provides a sample-independent coordinate system for organizing metagenomic sequence space at scale.

bioinformatics↗

bioRxiv2024

Genomic and environmental controls on Castellaniella biogeography in an anthropogenically disturbed subsurface

Castellaniella species have been isolated from a variety of mixed-waste environments including the nitrate and multiple metal contaminated subsurface at the Oak Ridge Reservation (ORR). Previous studies examining microbial community composition and nitrate removal at ORR during biostimulation efforts reported increased abundances of members of the Castellaniella genus concurrent to increased denitrification rates. Thus, we asked how genomic and abiotic factors control the Castellaniella biogeography at the site to understand how these factors may influence nitrate transformation in an anthropogenically impacted setting. ORR Castellaniella strains showed a higher degree of genetic diversification than those originating from non-ORR sites, which we attribute to the multitude of extreme stressors faced in the ORR subsurface. We report the isolation and characterization of several Castellaniella strains from the ORR subsurface. Five of these isolates match at 100% identity (at the 16S rRNA gene V4 region) to two Castellaniella amplicon sequence variants (ASVs), ASV1 and ASV2, that have persisted in the ORR subsurface for at least two decades. However, ASV2 has consistently higher relative abundance in samples taken from the site and was also the dominant blooming denitrifier population during a prior biostimulation effort. We found that the ASV2 representative strain has greater resistance to mixed metal stress than the ASV1 representative strains. We attribute this resistance, in part, to the large number of unique heavy metal resistance genes identified on a genomic island in the ASV2 representative genome. Additionally, we suggest that the relatively lower fitness of ASV1 may be connected to the loss of the nitrous oxide reductase (nos) operon (and associated nitrous oxide reductase activity) due to the insertion at this genomic locus of a mobile genetic element carrying copper resistance genes. This study demonstrates the value of integrating genomic, environmental, and phenotypic data to characterize the biogeography of key microorganisms in contaminated sites.

microbiology↗

bioRxiv2023

Mixed Waste Contamination Selects for a Mobile Genetic Element Population Enriched in Multiple Heavy Metal Resistance Genes

Mobile genetic elements (MGEs) like plasmids, viruses, and transposable elements can provide fitness benefits to their hosts for survival in the presence of environmental stressors. Heavy metal resistance genes (HMRGs) are frequently observed on MGEs, suggesting that MGEs may be an important driver of adaptive evolution in environments contaminated with heavy metals. Here, we report the meta-mobilome of the heavy metal contaminated regions of the Oak Ridge Reservation (ORR) subsurface. This meta-mobilome was compared to one derived from samples collected from unimpacted regions of the ORR subsurface. We assembled 1,615 unique circularized DNA elements that we propose to be MGEs. The circular elements from the highly contaminated subsurface were enriched in HMRG clusters relative to those from the nearby unimpacted regions. Additionally, we found that these HMRGs were associated with Gamma and Betaproteobacteria hosts in the contaminated subsurface and potentially facilitate the persistence and dominance of these taxa in this region. Finally, the HMRGs were associated with conjugative elements, suggesting their potential for future lateral transfer. We demonstrate how our understanding of MGE ecology, evolution, and function can be enhanced through the genomic context provided by completed MGE assemblies.

microbiology↗

bioRxiv2023

Decomposing a San Francisco Estuary microbiome using long read metagenomics reveals species and species- and strain-level dominance from picoeukaryotes to viruses

Although long read sequencing has enabled obtaining high-quality and complete prokaryotic genomes from metagenomes, many challenges still remain to completely decompose a metagenome into its constituent genomes. These challenges include obtaining enough biomass, high-molecular weight DNA extraction, determining the appropriate depth of sequencing, and bioinformatics challenges to separate closely related genomes. This study focuses on decomposing an estuarine water metagenome from USGS Station 36 in the South San Francisco Bay into its constituent genomes and counting the number of organisms present. To achieve this, we developed a new bead-based DNA extraction method, a novel bin refinement method, and sequenced the sample with 150 Gbases of nanopore sequencing. With our results, we were able to estimate that there are [~]500 bacteria and archaeal species in our sample, obtain 68 high-quality bins (>90% complete, <5% contamination, [≤]5 contigs, no contigs shorter than 100 Kbases, and all ribosomal and necessary tRNA genes). Since we pre-filtered the sample at 11m and then collected directly on to a 0.1m filter, we also obtained many contigs of picoeukaryotes, environmental DNA of larger eukaryotes such as mammals, complete mitochondrial and chloroplast genomes, and detected [~]40,000 viral populations. This deep analysis of the taxonomy of the sample down to the strain and individual contig level allowed us to find that among picoeukaryotes, prokaryotes, and viruses there are likely only a few strains that comprise most of the species abundances. These results also indicate that to truly decompose a metagenome into its constituent genomes, we likely need 1Tbase of sequencing. If you are reading this preprint, know that this is the paper we wanted to write, but it will likely be shortened for submission to a journal.

microbiology↗

bioRxiv2020

A method for achieving complete microbial genomes and better quality bins from metagenomics data

Metagenomics facilitates the study of the genetic information from uncultured microbes and complex microbial communities. Assembling complete microbial genomes (i.e., circular with no misassemblies) from metagenomics data is difficult because most samples have high organismal complexity and strain diversity. Less than 100 circularized bacterial and archaeal genomes have been assembled from metagenomics data despite the thousands of datasets that are available. Circularized genomes are important for (1) building a reference collection as scaffolds for future assemblies, (2) providing complete gene content of a genome, (3) confirming little or no contamination of a genome, (4) studying the genomic context and synteny of genes, and (5) linking protein coding genes to ribosomal RNA genes to aid metabolic inference in 16S rRNA gene sequencing studies. We developed a method to achieve circularized genomes using iterative assembly, binning, and read mapping. In addition, this method exposes potential misassemblies from k-mer based assemblies. We chose species of the Candidate Phyla Radiation (CPR) to focus our initial efforts because they have small genomes and are only known to have one ribosomal RNA operon. We present 34 circular CPR genomes, one circular Margulisbacteria genome, and two circular megaphage genomes from 19 public and published datasets. We demonstrate findings that would likely be difficult without circularizing genomes, including that ribosomal genes are likely not operonic in the majority of CPR, and that some CPR harbor diverged forms of RNase P RNA. Code and a tutorial for this method is available at https://github.com/lmlui/Jorg.

bioinformatics↗