Biology subjects

Polson, S. W.

Publications and source records attributed to Polson, S. W..

At least 19 recordsLinked to original sources

bioRxiv2026

Single amino acid substitution in DNA Polymerase I dramatically alters infection dynamics of bacteriophage T7

Viruses constitute a significant proportion of Earths genetic diversity, yet most remain uncharacterized beyond their sequences in viral metagenomes. Linking viral genotypes to phenotypes--especially enzyme function to phage infection dynamics--is challenging due to the lack of cultured virus-host systems. DNA polymerase I (PolA), essential for genome replication in [~]25% of dsDNA phages, provides an opportunity to explore these connections. In phage T7, residue 526 is critical for nucleotide incorporation, with previous in vitro evidence indicating impacts on enzyme efficiency and fidelity. Previous analyses identified three substitutions at this position (Tyr/Y, Phe/F, Leu/L) linked with deeply rooted viral PolA clades. Mutation impacts at residue 526 were tested in vitro and in vivo. The Y526F protein exhibited a 50% reduction in specific activity, and when introduced via High Complexity Golden Gate Assembly into T7 demonstrated a 53% decrease in burst size and significantly longer latent period compared to wild type. The Y526L protein exhibited a 97% decrease in activity, and the Y526L phage was incapable of completing its lifecycle. These findings confirm historical biochemical data, provide in vivo context for these mutations in the T7-E. coli system, and offer experimental support for genotype-to-phenotype associations in viral PolA, informing viral metagenomics studies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/726624v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@14395e5org.highwire.dtl.DTLVardef@261504org.highwire.dtl.DTLVardef@2dc1e4org.highwire.dtl.DTLVardef@147a7f_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Keown, R. (2026) https://BioRender.com/mhrmup3

microbiology↗

bioRxiv2026

Ribonucleotide reductases recapitulate biogeographic patterns within virioplankton according to ocean biogeochemistry

Many of the important biogeochemical implications of marine viruses focus on the activity of lytic viruses. It is generally thought that lytic viral activity contributes to maintaining bioavailable nutrient pools and controlling host community composition through specific infection and lysis processes. Among lytic viruses, genome replication is under stringent selection pressure as advantages in replication speed and fidelity can influence viral fitness. Deoxyribonucleotides (dNTPs) are the precursor substrate for DNA synthesis and ribonucleotide reductase (RNR) is the only known enzyme capable of reducing ribonucleotides into dNTPs. Thus, for a virus, encoding an RNR gene provides control over dNTP production. Overall, distributional patterns of the RNR-encoding virioplankton community mirrored that seen for total virioplankton throughout the global ocean. A majority of RNR-encoding virioplankton ([~]66%) carried the Class II enzyme and most of these carried the monomeric (NrdJm) gene. This was significant as NrdJm utilizes triphosphate ribonucleotides as opposed to the diphosphate ribonucleotides used by Class I and Class II dimeric RNRs. The distribution and frequency of Class I RNRs followed the concentration of required iron and manganese co-factors. Oceanic virioplankton encompassed a high diversity of Class I and II RNRs with the discovery of new phylogenetic clades not previously observed among viruses or within cellular life. Representing [~]10% of all virioplankton populations, RNR-encoding viral populations demonstrated surprising fidelity with the major biogeographical features defining oceanic ecosystems.

microbiology↗

bioRxiv2025

Multi-site Assessment of Methods for Cell Preservation Upstream of Single Cell RNA Sequencing

Single cell RNA sequencing (scRNA-seq) is a revolutionary technique to identify cell types and their molecular phenotype in heterogeneous biological specimens. ScRNA-seq typically requires fresh, high quality single cell suspensions that are processed immediately to preserve their molecular profiles. This presents a challenge for samples with long preparation times and prevents collection at remote sites lacking the required instrumentation for sample processing. Recently, several commercial assays have been released that enable sample preservation at the time of collection either via fixation or cryopreservation, allowing for sample processing to occur months after the initial collection. The Association of Biomolecular Research Facilities (ABRF) DNA Sequencing (DSRG) and Genomics Bioinformatics (GBiRG) Research Groups have undertaken a cross-platform, multi-site study to assess the performance and reproducibility of three platforms: a) 10x Genomics FLEX, b) Parse Bioscience Evercode WT v2 and c) Honeycomb Bio HIVE. Total leukocytes were isolated from a single healthy individual using the EasySep RBC depletion reagent. Cells were then characterized by collecting a 21-color flow cytometry dataset for reference and the remaining material was used for scRNA-seq procedures where different sites then processed either the fixed or cryopreserved cells for each method. We evaluated performance of each method across traditional scRNA-seq quality control metrics and analysis applications, including gene/transcript detection sensitivity, cell type discovery and annotation, and differential expression. We demonstrate that data from the methods tested can be effectively integrated and produce concordant results with regard to cell type annotation and relative abundance, though we observe platform-specific differences in the expression of a subset of genes. Preservation-based methods also show better retention of fragile granulocyte populations compared with fresh samples processed using the 10x 3 workflow. The improvements to preservation methods are changing the way research is conducted and our thorough investigation into the performance of each method provides a valuable resource to help scientists determine the most appropriate single cell preservation workflow given their sample collection logistics and laboratory infrastructure constraints.

genomics↗

bioRxiv2025

Comparative Analysis Reveals Host Species-Dependent Diversity Among 16 Virulent Bacteriophages Isolated Against Soybean Bradyrhizobium spp.

Phages play a role in shaping ecosystems by controlling host abundance via cell lysis, driving host evolution via horizontal gene transfer, and promoting nutrient cycling. The genus Bradyrhizobium includes bacteria able to symbiotically nodulate the roots of soybean (Glycine max), providing the plant with a direct source of biologically fixed nitrogen. Optimizing this symbiosis can minimize the use of nitrogen fertilizers and make soybean production more sustainable. Phages targeting Bradyrhizobium may modify their hosts genotype, alter phenotypic traits such as symbiotic effectiveness, and mediate competition among strains for nodulation sites. Sixteen phages were isolated against B. elkanii strains USDA94 and USDA31, and B. diazoefficiens strain USDA110. Comparative analyses revealed host species-dependent diversity in morphology, host range, and genome composition, leading to the identification of three previously undescribed phage species. Remarkably, all B. elkanii phages shared a siphophage morphology and formed a single species with >97% nucleotide identity, even when isolated from farms separated by up to [~]70 km, suggesting genomic stability across geographic scales. In contrast, phages isolated against B. diazoefficiens displayed podophage-like morphology, greater genetic diversity, and divided into two distinct species. Although no phages were recovered against B. japonicum strains or native Delaware Bradyrhizobium isolates tested, some Delaware isolates showed susceptibility during the host range assay. The phage genomes demonstrated features predicting phenotypes. Terminase genes predicted headful packaging among the phages which is critical for generalized transduction. The B. elkanii phages all carried tmRNA genes capable of recruiting stalled ribosomes and both phage groups carried DNA polymerase A indicating greater control of phage genome replication. State-of-the-art structural annotation revealed a tail fiber gene within a phage genome having the highest proportion (80.77%) of unknown genes. Together this work expands the limited knowledge available on soybean Bradyrhizobium phage ecology and genomics.

microbiology↗

bioRxiv2025

EXTENDING PROTEIN LANGUAGE MODELS TO A VIRAL GENOMIC SCALE USING BIOLOGICALLY INDUCED SPARSE ATTENTION

The transformer architecture in deep learning has revolutionized protein sequence analysis. Recent advancements in protein language models have paved the way for significant progress across various domains, including protein function and structure prediction, multiple sequence alignments and mutation effect prediction. A protein language model is commonly trained on individual proteins, ignoring the interdependencies between sequences within a genome. However, biological understanding reveals that protein-protein interactions span entire genomic regions, underscoring the limitations of focusing solely on individual proteins. To address these limitations, we propose a novel approach that extends the context size of transformer models across the entire viral genome. By training on large genomic fragments, our method captures long-range interprotein interactions and encodes protein sequences with integrated information from distant proteins within the same genome, offering substantial benefits in various tasks. Viruses, with their densely packed genomes, minimal intergenic regions, and protein annotation challenges, are ideal candidates for genome-wide learning. We introduce a long-context protein language model, trained on entire viral genomes, leveraging a sparse attention mechanism based on protein-protein interactions. Our semi-supervised approach supports long sequences of up to 61,000 amino acids (aa). Our evaluations demonstrate that the resulting embeddings significantly surpass those generated by single-protein models and outperform alternative large-context architectures that rely on static masking or non-transformer frameworks.

bioinformatics↗

bioRxiv2025

Fine-Tuning Protein Language Models Unlocks the Potential of Underrepresented Viral Proteomes

Protein language models (pLMs) have revolutionized computational biology by generating rich protein vector representations--or embeddings--enabling major advancements in de novo protein design, structure prediction, variant effect analysis, and evolutionary studies. Despite these breakthroughs, current pLMs often exhibit biases against proteins from underrepresented species, with viral proteins being particularly affected--frequently referred to as the "dark matter" of the biological world due to their vast diversity and ubiquity, yet sparse representation in training datasets. Here, we show that fine-tuning pre-trained pLMs on viral protein sequences, using diverse learning frameworks and parameter-efficient strategies, significantly enhances representation quality and improves performance on downstream tasks. To support further research, we provide source code for fine-tuning pLMs and benchmarking embedding quality. By enabling more accurate modeling of viral proteins, our approach advances tools for understanding viral biology, combating emerging infectious diseases, and driving biotechnological innovation.

bioinformatics↗

bioRxiv2024

Genomic diversity and global distribution of four new prasinoviruses from the tropical North Pacific

Viruses that infect phytoplankton are an integral part of marine ecosystems, but the vast majority of viral diversity remains uncultivated. Here we introduce four near-complete genomic assemblies of viruses that infect the widespread marine picoeukaryote Micromonas commoda, doubling the number of published genomes of Micromonas dsDNA viruses. All host and virus isolates were obtained from tropical waters of the North Pacific, a first for viruses infecting green algae in the order Mamiellales. Genome length of the new isolates ranges from 205-212 kb, and phylogenetic analysis shows that all four are members of the genus Prasinovirus. Three of the viruses form a clade that is adjacent to previously sequenced Micromonas viruses, while the fourth virus is relatively divergent from previously sequenced prasinoviruses. We identified 61 putative genes not previously found in prasinoviruses, including a phosphate transporter from a family novel to viruses, and a potential apoptosis inhibitor novel to marine viruses. Forty-eight genes in the new viruses are also found in host genome(s) and may have been acquired through horizontal gene transfer. By analyzing the coding sequences of all published prasinoviruses we found that [~]25% of prasinovirus gene content is significantly correlated with host genus identity (i.e., Micromonas, Ostreococcus, or Bathycoccus), and the functions of these genes suggest that much of the viral life cycle is differentially adapted to the three host genera. Mapping of metagenomic reads from global survey data indicates that one of the new isolates, McV-SA1, is relatively common in multiple ocean basins. Importance statementThe genomes analyzed here represent the first viruses from the tropical North Pacific that infect the abundant phytoplankton order Mamiellales. Comparing isolates from the same location demonstrates high genomic diversity among viruses that co-occur and presumably compete for hosts. Comparing all published prasinovirus genomes highlights gene functions that are likely associated with adaptation to different host genera. Metagenomic data indicates these viruses are globally distributed, and one of the novel isolates may be among the most abundant marine viruses.

ecology↗

bioRxiv2024

An integrative proteotranscriptomics approach reveals new ADAM9 substrates and downstream pathways

The disintegrin metalloprotease ADAM9 is a cell-surface protease that can shed the ectodomain of membrane protein substrates. Dysregulated ADAM9 activity has been implicated in several diseases such as solid tumors, autoimmunity, inflammatory diseases, and COVID-19. Despite its importance, the substrates and targets of ADAM9 in normal and pathological processes are poorly understood. Here, we developed an integrative proteotranscriptomics approach to systematically identify the transcriptional and post-transcriptional targets of ADAM9 in HCT116 cells, which have a stable diploid karyotype suitable for omics analyses. Using this approach, we uncovered major signaling pathways downstream of ADAM9, including the oncogenic mTOR pathway and the tumor suppressor FOXO pathway. We also identified several direct and indirect substrates for ADAM9, which may mediate the pathophysiological roles of this protease. This study provides new mechanistic insights into the function of ADAM9 as well as a method that can be applied to other membrane proteases.

biochemistry↗

bioRxiv2023

B12-dependent virioplankton demonstrate interseasonal dynamics and associate with a diversity of pelagic bacterioplankton

Through infection and lysis of their coexisting bacterial hosts, viruses impact the biogeochemical cycles sustaining globally significant pelagic oceanic ecosystems. Currently, little is known of the ecological interactions between lytic viruses and their bacterial hosts underlying these biogeochemical impacts at ecosystem scales. This study focused on populations of lytic viruses carrying the B12- dependent Class II monomeric ribonucleotide reductase (RNR) gene, ribonucleotide triphosphate reductase (RTPR), documenting seasonal changes in pelagic virioplankton and bacterioplankton using amplicon sequences of RTPR and the 16S rRNA gene, respectively. Amplicon sequence libraries were analyzed using compositional data analysis tools that account for the compositional nature of these data. Both virio- and bacterioplankton communities responded to environmental changes typically seen across seasonal cycles as well as shorter term upwelling-downwelling events. Defining RTPR-carrying viral populations according to major phylogenetic clades proved a more robust means of exploring virioplankton ecology than operational taxonomic units defined by percent sequence homology. Virioplankton RTPR populations showed positive associations with a broad phylogenetic diversity of bacterioplankton including dominant taxa within pelagic oceanic ecosystems such as Prochlorococcus and SAR11. Temporal changes in RTPR-virioplankton, occurring as both free viruses and within infected cells, indicated possible viral-host pairs undergoing sustained infection and lysis cycles throughout the seasonal study. Phylogenetic relationships inferred from RTPR sequences mirrored ecological patterns in virio- and bacterioplankton populations demonstrating possible genome to phenome associations for an essential viral replication gene.

ecology↗

bioRxiv2023

Gallionellaceae pangenomic analysis reveals insight into phylogeny, metabolic flexibility, and iron oxidation mechanisms

The iron-oxidizing Gallionellaceae drive a wide variety of biogeochemical cycles through their metabolisms and biominerals. To better understand the environmental impacts of Gallionellaceae, we need to improve our knowledge of their diversity and metabolisms, especially any novel iron oxidation mechanisms. Here, we used a pangenomic analysis of 103 genomes to resolve Gallionellaceae phylogeny and explore the range of genomic potential. Using a concatenated ribosomal protein tree and key gene patterns, we determined Gallionellaceae has four genera, divided into two groups-iron-oxidizing bacteria (FeOB) Gallionella, Sideroxydans, and Ferriphaselus with known iron oxidases (Cyc2, MtoA) and nitrite-oxidizing bacteria (NOB) Candidatus Nitrotoga with nitrite oxidase (Nxr). The FeOB and NOB have similar electron transport chains, including genes for reverse electron transport and carbon fixation. Auxiliary energy metabolisms including S oxidation, denitrification, and organotrophy were scattered throughout the Gallionellaceae FeOB. Within FeOB, we found genes that may represent adaptations for iron oxidation, including a variety of extracellular electron uptake (EEU) mechanisms. FeOB genomes encoded more predicted c-type cytochromes overall, notably more multiheme c-type cytochromes (MHCs) with >10 CXXCH motifs. These include homologs of several predicted outer membrane porin-MHC complexes, including MtoAB and Uet. MHCs are known to efficiently conduct electrons across longer distances and function across a wide range of redox potentials that overlap with mineral redox potentials, which can help expand the range of usable iron substrates. Overall, the results of pangenome analyses suggest that the Gallionellaceae genera Gallionella, Sideroxydans, and Ferriphaselus are primarily iron oxidizers, capable of oxidizing dissolved Fe2+ as well as a range of solid iron or other mineral substrates. ImportanceNeutrophilic iron-oxidizing bacteria (FeOB) produce copious iron (oxyhydr)oxides that can profoundly influence biogeochemical cycles, notably the fate of carbon and many metals. To fully understand environmental microbial iron oxidation, we need a thorough accounting of iron oxidation mechanisms. In this study we show the Gallionellaceae FeOB have both known iron oxidases as well as uncharacterized multiheme cytochromes (MHCs). MHCs are predicted to transfer electrons from extracellular substrates and likely confer metabolic capabilities that help Gallionellaceae occupy a range of different iron- and mineral-rich niches. Gallionellaceae appear to specialize in iron oxidation, so it makes sense that they would have multiple mechanisms to oxidize various forms of iron, given the many iron minerals on Earth, as well as the physiological and kinetic challenges faced by FeOB. The multiple iron/mineral oxidation mechanisms may help drive the widespread ecological success of Gallionellaceae.

microbiology↗

bioRxiv2022

Metagenomic analysis of concrete bridge reveals a microbial community dominated by halophilic Bacteria and Archaea

Concrete hosts a small but diverse microbiome that changes over time. Shotgun metagenomic sequencing would enable assessment of both diversity and function of the microbial community in concrete, but because the biomass in concrete is so low, this analysis is highly affected by laboratory contamination. Here, we demonstrate improved DNA extraction from concrete, and show that this method provides DNA of sufficient quality and quantity to do shotgun metagenomic sequencing. DNA was extracted from a sample of concrete obtained from a road bridge and sequenced with Illumina MiSeq. This microbial community was dominated by halophilic Bacteria and Archaea, with enriched functional pathways related to osmotic stress responses. Prior work found that halophilic bacteria were relatively rare in younger concrete samples, which had abundant oligotrophic taxa. These results suggest that as concrete ages and weathers, salt and osmotic stresses become more important selective pressures, and suggest that long-term persistence and performance of microbes for biorepair or biosensing applications might improve if halophilic strains were used.

microbiology↗

bioRxiv2022

Spontaneously produced lysogenic phages are an important component of the soybean Bradyrhizobium mobilome

The ability to nodulate and fix atmospheric nitrogen in soybean root nodules makes soybean Bradyrhizobium spp. (SB) critical in supplying humanitys nutritional needs. The intricacies of SB-plant interactions have been studied extensively; however, bradyrhizobial ecology as influenced by phages has received somewhat less attention even though these interactions may significantly impact soybean yield. In batch culture four SB strains, S06B (B. japonicum, S06B-Bj), S10J (B. japonicum, S10J-Bj), USDA 122 (B. diazoefficiens, USDA 122-Bd), and USDA 76T (B. elkanii, USDA 76-Be), spontaneously (without apparent exogenous chemical or physical induction) produced phages throughout the growth cycle; for three strains, phage concentrations exceeded cell numbers by ca. 3-fold after 48 h incubation. Observed spontaneously produced phages (SPP) were tailed. Phage terminase large-subunit protein phylogeny revealed possible differences in phage packaging and replication mechanisms. Bioinformatic analyses predicted multiple prophage regions within each SB genome preventing accurate identification of SPP genomes. A DNA sequencing approach was developed that accurately delineated the boundaries of four SPP genomes within three of the SB chromosomes. Read mapping suggested that the SPP are capable of transduction. In addition to the phages, bacterial strains S06B-Bj and USDA 76-Be were rich in mobile elements consisting of insertion sequences (IS) and large, conjugable, broad host range plasmids. The prevalence of SPP along with IS and plasmids indicate that horizontal gene transfer likely plays an outsized role in SB ecology and may subsequently impact soybean productivity. ImportancePrevious studies have shown that IS and plasmids mediate horizontal gene transfer (HGT) of symbiotic nodulation (nod) genes in SB; however, these events require close cell to cell contact which could be limited in soil environments. Bacteriophage assisted gene transduction through spontaneously produced prophages could provide stable means of HGT not limited by the constraints of proximal cell to cell contact. Phage mediated HGT events could be important in SB population ecology with concomitant impacts on soybean agriculture.

microbiology↗

bioRxiv2021

Unraveling Fe(II)-oxidizing mechanisms in a facultative Fe(II) oxidizer Sideroxydans lithotrophicus ES-1 via culturing, transcriptomics, and RT-qPCR

Sideroxydans lithotrophicus ES-1 grows autotrophically either by Fe(II) oxidation or thiosulfate oxidation, in contrast to most other neutrophilic Fe(II)-oxidizing bacteria (FeOB) isolates. This provides a unique opportunity to explore the physiology of a facultative FeOB and constrain the genes specific to Fe(II) oxidation. We compared the growth of S. lithotrophicus ES-1 on Fe(II), thiosulfate, and both substrates together. While initial growth rates were similar, thiosulfate-grown cultures had higher yield with or without Fe(II) present, which may give ES-1 an advantage over obligate FeOB. To investigate the Fe(II) and S oxidation pathways, we conducted transcriptomics experiments, validated with RT-qPCR. We explored the long-term gene expression response at different growth phases (over days-week) and expression changes during a short-term switch from thiosulfate to Fe(II) (90 min). The dsr and sox sulfur oxidation genes were upregulated in thiosulfate cultures. The Fe(II) oxidase gene cyc2 was among the top expressed genes during both Fe(II) and thiosulfate oxidation, and addition of Fe(II) to thiosulfate-grown cells caused an increase in cyc2 expression. These results support the role of Cyc2 as the Fe(II) oxidase and suggest that ES-1 maintains readiness to oxidize Fe(II) even in the absence of Fe(II). We used gene expression profiles to further constrain the ES-1 Fe(II) oxidation pathway. Notably, among the most highly upregulated genes during Fe(II) oxidation were genes for alternative complex III, reverse electron transport and carbon fixation. This implies a direct connection between Fe(II) oxidation and carbon fixation, suggesting that CO2 is an important electron sink for Fe(II) oxidation. ImportanceNeutrophilic FeOB are increasingly observed in various environments, but knowledge of their ecophysiology and Fe(II) oxidation mechanisms is still relatively limited. Sideroxydans are widely observed in aquifers, wetlands, and sediments, and genome analysis suggests metabolic flexibility contributes to their success. The type strain ES-1 is unusual amongst neutrophilic FeOB isolates as it can grow on either Fe(II) or a non-Fe(II) substrate, thiosulfate. Almost all our knowledge of neutrophilic Fe(II) oxidation pathways comes from genome analyses, with some work on metatranscriptomes. This study used culture-based experiments to test the genes specific to Fe(II) oxidation in a facultative FeOB and refine our model of the Fe(II) oxidation pathway. We gained insight into how facultative FeOB like ES-1 connect Fe, S, and C biogeochemical cycling in the environment, and suggest a multi-gene indicator would improve understanding of Fe(II) oxidation activity in environments with facultative FeOB.

microbiology↗

bioRxiv2021

Deletion of Fibroblast growth factor 9 globally and in skeletal muscle results in enlarged tuberosities at sites of deltoid tendon attachments

The growth of most bony tuberosities, like the deltoid tuberosity (DT), rely on the transmission of muscle forces at the tendon-bone attachment during skeletal growth. Tuberosities distribute muscle forces and provide mechanical leverage at attachment sites for joint stability and mobility. The genetic factors that regulate tuberosity growth remain largely unknown. In mouse embryos with global deletion of fibroblast growth factor 9 (Fgf9), the DT size is notably enlarged. In this study, we explored the tissue-specific regulation of DT size using both global and targeted deletion of Fgf9. We showed that cell hypertrophy and mineralization dynamics of the DT, as well as transcriptional signatures from skeletal muscle but not bone, were influenced by the global loss of Fgf9. Loss of Fgf9 during embryonic growth led to increased chondrocyte hypertrophy and reduced cell proliferation at the DT attachment site. This endured hypertrophy and limited proliferation may explain the abnormal mineralization patterns and locally dysregulated expression of markers of endochondral development in Fgf9null attachments. We then showed that targeted deletion of Fgf9 in skeletal muscle leads to postnatal enlargement of the DT. Taken together, we discovered that Fgf9 may play an influential role in muscle-bone crosstalk during embryonic and postnatal development.

developmental biology↗

bioRxiv2021

PASV: Automatic protein partitioning and validation using conserved residues

BackgroundIncreasingly, researchers use protein-coding genes from targeted PCR amplification or direct metagenomic sequencing in community and population ecology. Analysis of protein-coding genes presents different challenges from those encountered in traditional SSU rRNA studies. Most protein-coding sequences are annotated based on homology to other computationally-annotated sequences, which can lead to inaccurate annotations. Therefore, the results of sensitive homology searches must be validated to remove false-positives and assess functionality. Multiple lines of in silico evidence can be gathered by examining conserved domains and residues identified through biochemical investigations. However, manually validating sequences in this way can be time consuming and error prone, especially in large environmental studies. ResultsAn automated pipeline for protein active site validation (PASV) was developed to improve validation and partitioning accuracy for protein-coding sequences, combining multiple sequence alignment with expert domain knowledge. PASV was tested using commonly misannotated proteins: ribonucleotide reductase (RNR), alternative oxidase (AOX), and plastid terminal oxidase (PTOX). PASV partitioned 9,906 putative Class I alpha and Class II RNR sequences from bycatch in a global viral metagenomic investigation with >99% true positive and true negative rates. PASV predicted the class of 2,579 RNR sequences in >98% agreement with manual annotations. PASV correctly partitioned all 336 tested AOX and PTOX sequences. ConclusionsPASV provides an automated and accurate way to address post-homology search validation and partitioning of protein-coding marker genes. Source code is released under the MIT license and is found with documentation and usage examples on GitHub at https://github.com/mooreryan/pasv.

bioinformatics↗

bioRxiv2019

Validating the Cyc2 neutrophilic Fe oxidation pathway using meta-omics of Zetaproteobacteria iron mats at marine hydrothermal vents

Zetaproteobacteria create extensive iron (Fe) oxide mats at marine hydrothermal vents, making them an ideal model for microbial Fe oxidation at circumneutral pH. Comparison of neutrophilic Fe-oxidizer isolate genomes has revealed a hypothetical Fe oxidation pathway, featuring a homolog of the Fe oxidase Cyc2 from Acidithiobacillus ferrooxidans. However, Cyc2 function is not well verified in neutrophilic Fe-oxidizers, particularly in Fe-oxidizing environments. Toward this, we analyzed genomes and metatranscriptomes of Zetaproteobacteria, using 53 new high-quality metagenome assembled genomes reconstructed from Fe mats at Mid-Atlantic Ridge, Mariana Backarc, and Loihi Seamount (Hawaii) hydrothermal vents. Phylogenetic analysis demonstrated conservation of Cyc2 sequences among most neutrophilic Fe-oxidizers, suggesting a common function. We confirmed the widespread distribution of cyc2 and other model Fe oxidation pathway genes across all represented Zetaproteobacteria lineages. High expression of these genes was observed in diverse Zetaproteobacteria under multiple environmental conditions, and in incubations. The putative Fe oxidase gene, cyc2, was highly expressed in situ, often as the top expressed gene. The cyc2 gene showed increased expression in Fe(II)-amended incubations, with corresponding increases in carbon fixation and central metabolism gene expression. These results substantiate the Cyc2-based Fe oxidation pathway in neutrophiles and demonstrate its significance in marine Fe-mineralizing environments.

microbiology↗

bioRxiv2018

CRISPR spacers indicate preferential matching of specific virioplankton genes

Viral infection exerts selection pressure on marine microbes as viral-induced cell lysis causes 20 to 50% of cell mortality resulting in fluxes of biomass into oceanic dissolved organic matter. Archaeal and bacterial populations can defend against viral infection using the CRISPR-Cas system which relies on specific matching between a spacer sequence and a viral gene. If a CRISPR spacer match to any gene within a viral genome is equally effective in preventing lysis, then no viral genes should be preferentially matched by CRISPR spacers. However, if there are differences in effectiveness then certain viral genes may demonstrate a greater frequency of CRISPR spacer matches. Indeed, homology search analyses of bacterioplankton CRISPR spacer sequences against virioplankton sequences revealed preferential matching of replication proteins, nucleic acid binding proteins, and viral structural proteins. Positive selection pressure for effective viral defense is one parsimonious explanation for these observations. CRISPR spacers from virioplankton metagenomes preferentially matched methyltransferase and phage integrase genes within virioplankton sequences. These viriolankton CRISPR spacers may assist infected host cells in defending against competing phage. Analyses also revealed that half of the spacer-matched viral genes were unknown and that some genes matched several spacers and some spacers matched multiple genes, a many-to-many relationship. Thus, CRISPR spacer matching may be an evolutionary algorithm, agnostically identifying those genes under stringent selection pressure for sustaining viral infection and lysis. Investigating this subset of viral genes could reveal those genetic mechanisms essential to viral-host interactions and provide new technologies for optimizing CRISPR defense in beneficial microbes.

microbiology↗

bioRxiv2018

Reannotation of the ribonucleotide reductase in a cyanophage reveals life history strategies within the virioplankton

Ribonucleotide reductases (RNRs) are ancient enzymes that catalyze the reduction of ribonucleotides to deoxyribonucleotides. They are required for virtually all cellular life and are prominent within viral genomes. RNRs share a common ancestor and must generate a protein radical for direct ribonucleotide reduction. The mechanisms by which RNRs produce radicals are diverse and divide RNRs into three major classes and several subclasses. The diversity of radical generation methods means that cellular organisms and viruses typically contain the RNR best-suited to the environmental conditions surrounding DNA replication. However, such diversity has also fostered high rates of RNR misannotation within subject sequence databases. These misannotations have resulted in incorrect translative presumptions of RNR biochemistry and have diminished the utility of this marker gene for ecological studies of viruses. We discovered a misannotation of the RNR gene within the Prochlorococcus phage P-SSP7 genome, which caused a chain of misannotations within commonly observed RNR genes from marine virioplankton communities. These RNRs are found in marine cyanopodo- and cyanosiphoviruses and are currently misannotated as Class II RNRs, which are O2-independent and require cofactor B12. In fact, these cyanoviral RNRs are Class I enzymes that are O2-dependent and may require a di-metal cofactor made of Fe, Mn, or a combination of the two metals. The discovery of an overlooked Class I {beta} subunit in the P-SSP7 genome, together with phylogenetic analysis of the and {beta} subunits confirms that the RNR from P-SSP7 is a Class I RNR. Phylogenetic and conserved residue analyses also suggest that the P-SSP7 RNR may constitute a novel Class I subclass. The reannotation of the RNR clade represented by P-SSP7 means that most lytic cyanophage contain Class I RNRs, while their hosts, B12-producing Synechococcus and Prochlorococcus, contain Class II RNRs. By using a Class I RNR, cyanophage avoid a dependence on host-produced B12, a more effective strategy for a lytic virus. The discovery of a novel RNR {beta} subunit within cyanopodoviruses also implies that some unknown viral genes may be familiar cellular genes that are too divergent for homology-based annotation methods to identify.

bioinformatics↗