Family A DNA Polymerase Phylogeny Uncovers Diversity and Replication Gene Organization in the Virioplankton

Shotgun metagenomics, which allows for broad sampling of viral diversity, has uncovered genes that are widely distributed among virioplankton populations and show linkages to important biological features of unknown viruses. Over 25% of known dsDNA phage carry the DNA polymerase I (polA) gene, making it one of the most widely distributed phage genes. Because of its pivotal role in DNA replication, this enzyme is linked to phage lifecycle characteristics. Previous research has suggested that a single amino acid substitution might be predictive of viral lifestyle. In this study Chesapeake Bay virioplankton were sampled by shotgun metagenomic sequencing (using long and short read technologies). More polA sequences were predicted from this single viral metagenome (virome) than from 86 globally distributed virome libraries (ca. 2,100, and 1,200, respectively). The PolA peptides predicted from the Chesapeake Bay virome clustered with 69% of PolA peptides from global viromes; thus, remarkably the Chesapeake Bay virome captured the majority of known PolA peptide diversity in viruses. This deeply sequenced virome also expanded the diversity of PolA sequences, increasing the number of PolA clusters by 44%. Contigs containing polA sequences were also used to examine relationships between phylogenetic clades of PolA and other genes within unknown viral populations. Phylogenic analysis revealed five distinct groups of phages distinguished by the amino acids at their 762 (Escherichia coli IAI39 numbering) positions and replication genes. DNA polymerase I sequences from Tyr762 and Phe762 groups were most often neighbored by ring-shaped superfamily IV helicases and ribonucleotide reductases (RNRs). The Leu762 groups had non-ring shaped helicases from superfamily II and were further distinguished by an additional helicase gene from superfamily I and the lack of any identifiable RNR genes. Moreover, we found that the inclusion of ribonucleotide reductase associated with PolA helped to further differentiate phage diversity, chiefly within lytic podovirus populations. Altogether, these data show that DNA Polymerase I is a useful marker for observing the diversity and composition of the virioplankton and may be a driving factor in the divergence of phage replication components

RefSeq database growth influences the accuracy of k-mer-based lowest common ancestor species identification

Abstract In order to determine the role of the database in taxonomic sequence classification, we examine the influence of the database over time on k-mer-based lowest common ancestor taxonomic classification. We present three major findings: the number of new species added to the NCBI RefSeq database greatly outpaces the number of new genera; as a result, more reads are classified with newer database versions, but fewer are classified at the species level; and Bayesian-based re-estimation mitigates this effect but struggles with novel genomes. These results suggest a need for new classification approaches specially adapted for large databases

Agricultural Freshwater Pond Supports Diverse and Dynamic Bacterial and Viral Populations

Agricultural ponds have a great potential as a means of capture and storage of water for irrigation. However, pond topography (small size, shallow depth) leaves them susceptible to environmental, agricultural, and anthropogenic exposures that may influence microbial dynamics. Therefore, the aim of this project was to characterize the bacterial and viral communities of pond water in the Mid-Atlantic United States with a focus on the late season (October–December), where decreasing temperature and nutrient levels can affect the composition of microbial communities. Ten liters of freshwater from an agricultural pond were sampled monthly, and filtered sequentially through 1 and 0.2 μm filter membranes. Total DNA was then extracted from each filter, and the bacterial communities were characterized using 16S rRNA gene sequencing. The remaining filtrate was chemically concentrated for viruses, DNA-extracted, and shotgun sequenced. Bacterial community profiling showed significant fluctuations over the sampling period, corresponding to changes in the condition of the pond freshwater (e.g., pH, nutrient load). In addition, there were significant differences in the alpha-diversity and core bacterial operational taxonomic units (OTUs) between water fractions filtered through different pore sizes. The viral fraction was dominated by tailed bacteriophage of the order Caudovirales, largely those of the Siphoviridae family. Moreover, while present, genes involved in virulence/antimicrobial resistance were not enriched within the viral fraction during the study period. Instead, the viral functional profile was dominated by phage associated proteins, as well as those related to nucleotide production. Overall, these data suggest that agricultural pond water harbors a diverse core of bacterial and bacteriophage species whose abundance and composition are influenced by environmental variables characteristic of pond topology and the late season

Seasonal dynamics in taxonomy and function within bacterial and viral metagenomic assemblages recovered from a freshwater agricultural pond

BackgroundPonds are important freshwater habitats that support both human and environmental activities. However, relative to their larger counterparts (e.g. rivers, lakes), ponds are understudied, especially with regard to their microbial communities. Our study aimed to fill this knowledge gap by using culture-independent, high-throughput sequencing to assess the dynamics, taxonomy, and functionality of bacterial and viral communities in a freshwater agricultural pond.ResultsWater samples (n = 14) were collected from a Mid-Atlantic agricultural pond between June 2017 and May 2018 and filtered sequentially through 1 and 0.2 μm filter membranes. Total DNA was then extracted from each filter, pooled, and subjected to 16S rRNA gene and shotgun sequencing on the Illumina HiSeq 2500 platform. Additionally, on eight occasions water filtrates were processed for viral metagenomes (viromes) using chemical concentration and then shotgun sequenced. A ubiquitous freshwater phylum, Proteobacteria was abundant at all sampling dates throughout the year. However, environmental characteristics appeared to drive the structure of the community. For instance, the abundance of Cyanobacteria (e.g. Nostoc) increased with rising water temperatures, while a storm event appeared to trigger an increase in overall bacterial diversity, as well as the relative abundance of Bacteroidetes. This event was also associated with an increase in the number of antibiotic resistance genes. The viral fractions were dominated by dsDNA of the order Caudovirales, namely Siphoviridae and Myovirdae.ConclusionsOverall, this study provides one of the largest datasets on pond water microbial ecology to date, revealing seasonal trends in the microbial taxonomic composition and functional potential

CRISPR Spacers Indicate Preferential Matching of Specific Virioplankton Genes

The CRISPR-Cas system is one means by which bacterial and archaeal populations defend against viral infection which causes 20 to 50% of cell mortality in the ocean. We tested the hypothesis that certain viral genes are preferentially targeted for the initial attack of the CRISPR-Cas system on a viral genome. Using CASC, a pipeline for CRISPR spacer discovery, and metagenome data from oceanic microbes and viruses, we found a clear subset of viral genes with high match frequencies to CRISPR spacers. Moreover, we observed a many-to-many relationship of spacers and viral genes. These high-match viral genes were involved in nucleotide metabolism, DNA methylation, and viral structure. It is possible that CRISPR spacer matching is an evolutionary algorithm pointing to those viral genes most important to sustaining infection and lysis. Studying these genes may advance the understanding of virus-host interactions in nature and provide new technologies for leveraging CRISPR-Cas systems in beneficial microbes.Viral infection exerts selection pressure on marine microbes, as virus-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 clustered regularly interspaced short palindromic repeat (CRISPR)-associated (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, no viral genes should be preferentially matched by CRISPR spacers. However, if there are differences in effectiveness, 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 virioplankton 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, 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 virus-host interactions and provide new technologies for optimizing CRISPR defense in beneficial microbes

Zero-valent iron sand filtration reduces concentrations of virus-like particles and modifies virome community composition in reclaimed water used for agricultural irrigation

Abstract Objective Zero-valent iron sand filtration can remove multiple contaminants, including some types of pathogenic bacteria, from contaminated water. However, its efficacy at removing complex viral populations, such as those found in reclaimed water used for agricultural irrigation, has not been fully evaluated. Therefore, this study utilized metagenomic sequencing and epifluorescent microscopy to enumerate and characterize viral populations found in reclaimed water and zero-valent iron-sand filtered reclaimed water sampled three times during a larger greenhouse study. Results Zero-valent iron-sand filtered reclaimed water samples had significantly less virus-like particles than reclaimed water samples at all collection dates, with the reclaimed water averaging between 108 and 109 and the zero-valent iron-sand filtered reclaimed water averaging between 106 and 107 virus-like particles per mL. In addition, for both sample types, viral metagenomes (viromes) were dominated by bacteriophages of the order Caudovirales, largely Siphoviridae, and genes related to DNA metabolism. However, the proportion of sequences homologous to bacteria, as well as the abundance of genes possibly originating from a bacterial host, was higher in the viromes of zero-valent iron-sand filtered reclaimed water samples. Overall, zero-valent iron-sand filtered reclaimed water had a lower total concentration of virus-like particles and a different virome community composition compared to unfiltered reclaimed water

Current progress and future opportunities in applications of bioinformatics for biodefense and pathogen detection: report from the Winter Mid-Atlantic Microbiome Meet-up, College Park, MD, January 10, 2018

Abstract The Mid-Atlantic Microbiome Meet-up (M3) organization brings together academic, government, and industry groups to share ideas and develop best practices for microbiome research. In January of 2018, M3 held its fourth meeting, which focused on recent advances in biodefense, specifically those relating to infectious disease, and the use of metagenomic methods for pathogen detection. Presentations highlighted the utility of next-generation sequencing technologies for identifying and tracking microbial community members across space and time. However, they also stressed the current limitations of genomic approaches for biodefense, including insufficient sensitivity to detect low-abundance pathogens and the inability to quantify viable organisms. Participants discussed ways in which the community can improve software usability and shared new computational tools for metagenomic processing, assembly, annotation, and visualization. Looking to the future, they identified the need for better bioinformatics toolkits for longitudinal analyses, improved sample processing approaches for characterizing viruses and fungi, and more consistent maintenance of database resources. Finally, they addressed the necessity of improving data standards to incentivize data sharing. Here, we summarize the presentations and discussions from the meeting, identifying the areas where microbiome analyses have improved our ability to detect and manage biological threats and infectious disease, as well as gaps of knowledge in the field that require future funding and focus