Ribonucleotide reductases reveal novel viral diversity and predict biological and ecological features of unknown marine viruses.

Virioplankton play a crucial role in aquatic ecosystems as top-down regulators of bacterial populations and agents of horizontal gene transfer and nutrient cycling. However, the biology and ecology of virioplankton populations in the environment remain poorly understood. Ribonucleotide reductases (RNRs) are ancient enzymes that reduce ribonucleotides to deoxyribonucleotides and thus prime DNA synthesis. Composed of three classes according to O2 reactivity, RNRs can be predictive of the physiological conditions surrounding DNA synthesis. RNRs are universal among cellular life, common within viral genomes and virioplankton shotgun metagenomes (viromes), and estimated to occur within >90% of the dsDNA virioplankton sampled in this study. RNRs occur across diverse viral groups, including all three morphological families of tailed phages, making these genes attractive for studies of viral diversity. Differing patterns in virioplankton diversity were clear from RNRs sampled across a broad oceanic transect. The most abundant RNRs belonged to novel lineages of podoviruses infecting α-proteobacteria, a bacterial class critical to oceanic carbon cycling. RNR class was predictive of phage morphology among cyanophages and RNR distribution frequencies among cyanophages were largely consistent with the predictions of the "kill the winner-cost of resistance" model. RNRs were also identified for the first time to our knowledge within ssDNA viromes. These data indicate that RNR polymorphism provides a means of connecting the biological and ecological features of virioplankton populations.

Bacteria exposed to antiviral drugs develop antibiotic cross-resistance and unique resistance profiles.

Antiviral drugs are used globally as treatment and prophylaxis for long-term and acute viral infections. Even though antivirals also have been shown to have off-target effects on bacterial growth, the potential contributions of antivirals to antimicrobial resistance remains unknown. Herein we explored the ability of different classes of antiviral drugs to induce antimicrobial resistance. Our results establish the previously unrecognized capacity of antivirals to broadly alter the phenotypic antimicrobial resistance profiles of both gram-negative and gram-positive bacteria Escherichia coli and Bacillus cereus. Bacteria exposed to antivirals including zidovudine, dolutegravir and raltegravir developed cross-resistance to commonly used antibiotics including trimethoprim, tetracycline, clarithromycin, erythromycin, and amoxicillin. Whole genome sequencing of antiviral-resistant E. coli isolates revealed numerous unique single base pair mutations, as well as multi-base pair insertions and deletions, in genes with known and suspected roles in antimicrobial resistance including those coding for multidrug efflux pumps, carbohydrate transport, and cellular metabolism. The observed phenotypic changes coupled with genotypic results indicate that bacteria exposed to antiviral drugs with antibacterial properties in vitro can develop multiple resistance mutations that confer cross-resistance to antibiotics. Our findings underscore the potential contribution of wide scale usage of antiviral drugs to the development and spread of antimicrobial resistance in humans and the environment.

Ubiquitous, B12-dependent virioplankton utilizing ribonucleotide-triphosphate reductase demonstrate interseasonal dynamics and associate with a diverse range of bacterial hosts in the pelagic ocean.

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 (Class II RTPR), documenting seasonal changes in pelagic virioplankton and bacterioplankton using amplicon sequences of Class II 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 Class II 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 Class II 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 Class II 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 Class II RTPR sequences mirrored ecological patterns in virio- and bacterioplankton populations demonstrating possible genome to phenome associations for an essential viral replication gene.

Influence of Simplified Microbial Community Biofilms on Bacterial Retention in Porous Media under Conditions of Stormwater Biofiltration.

Porous media filters are used widely to remove bacteria from contaminated water, such as stormwater runoff. Biofilms that colonize filter media during normal function can significantly alter performance, but it is not clear how characteristics of individual populations colonizing porous media combine to affect bacterial retention. We assess how four bacterial strains isolated from stormwater and a laboratory strain, Pseudomonas aeruginosa PAO1, alter Escherichia coli retention in experimental sand columns under conditions of stormwater filtration relative to a clean-bed control. Our results demonstrate that these strains differentially affect E. coli retention, as was previously shown for a model colloid. To determine whether E. coli retention could be influenced by changes in relative abundance of strains within a microbial community, we selected two pairs of biofilm strains with the largest observed differences in E. coli retention and tested how changes in relative abundance of strain pairs in the biofilm affected E. coli retention. The results demonstrate that E. coli retention efficiency is influenced by the retention characteristics of the strains within biofilm microbial community, but individual strain characteristics influence retention in a manner that cannot be determined from changes in their relative abundance alone. This study demonstrates that changes in the relative abundance of specific members of a biofilm community can significantly alter filter performance, but these changes are not a simple function of strain-specific retention and the relative abundance. Our results suggest that the microbial community composition of biofilms should be considered when evaluating factors that influence filter performance. IMPORTANCE The retention efficiency of bacterial contaminants in biofilm-colonized biofilters is highly variable. Despite the increasing number of studies on the impact of biofilms in filters on bacterial retention, how individual bacterial strains within a biofilm community combine to influence bacterial retention is unknown. Here, we studied the retention of an E. coli K-12 strain, as a model bacterium, in columns colonized by four bacterial strains isolated from stormwater and P. aeruginosa, a model biofilm-forming strain. Simplified two-strain biofilm communities composed of combinations of the strains were used to determine how relative abundance of biofilm strains affects filter performance. Our results provide insight into how biofilm microbial composition influences bacterial retention in filters and whether it is possible to predict bacterial retention efficiency in biofilm-colonized filters from the relative abundance of individual members and the retention characteristics of cultured isolates.

Interaction dynamics and virus-host range for estuarine actinophages captured by epicPCR.

Viruses impact microbial diversity, gene flow and function through virus-host interactions. Although metagenomics surveys are rapidly cataloguing viral diversity, methods are needed to capture specific virus-host interactions in situ. Here, we leveraged metagenomics and repurposed emulsion paired isolation-concatenation PCR (epicPCR) to investigate viral diversity and virus-host interactions in situ over time in an estuarine environment. The method fuses a phage marker, the ribonucleotide reductase gene, with the host 16S rRNA gene of infected bacterial cells within emulsion droplets providing single-cell resolution for dozens of samples. EpicPCR captured in situ virus-host interactions for viral clades with no closely related database representatives. Abundant freshwater Actinobacteria lineages, in particular Rhodoluna sp., were the most common hosts for these poorly characterized viruses, with interactions correlated with environmental factors. Multiple methods used to identify virus-host interactions, including epicPCR, identified different and largely non-overlapping interactions within the vast virus-host interaction space. Tracking virus-host interaction dynamics also revealed that multi-host viruses had significantly longer periods with observed virus-host interactions, whereas single-host viruses were observed interacting with hosts at lower minimum abundances, suggesting more efficient interactions. Capturing in situ interactions with epicPCR revealed environmental and ecological factors shaping virus-host interactions, highlighting epicPCR as a valuable technique in viral ecology.

Oyster Calcifying Fluid Harbors Persistent and Dynamic Autochthonous Bacterial Populations That May Aid in Shell Formation.

The eastern oyster (Crassostrea virginica) is a keystone species in estuarine environments but faces threats to shell formation associated with warming temperatures and acidification. Extrapallial fluid (EF), which is responsible for shell formation, harbors diverse and abundant microbial communities. Commensal microbial communities are vital to host health and fitness, yet long-term studies investigating temporal responses of the EF microbiome and its function in oyster fitness are lacking. In this study, bacterial communities of oyster EF and the water column were characterized monthly from October 2010 to September 2011. We investigated the selection, composition, and dynamics of resident and transient community members, evaluated the impact of temperature on EF microbial communities, and examined the functional role of the EF microbiome. Oyster EF communities were significantly different from the water column and were enriched for several taxa, including the Deltaproteobacteria, Epsilonproteobacteria, and Gammaproteobacteria. Overall, 94 resident members were identified in oyster EF. These members were persistent and abundant, comprising on average 33% of EF communities. Resident EF communities formed high-temperature and low-temperature groups and were more abundant overall at colder temperatures. Oyster EF resident communities were predicted to be enriched for dissimilatory nitrate reduction, nitrogen fixation, nitrification, and sulfite reductase genes. Sulfate and nitrate reduction may have a synergistic effect on calcium carbonate precipitation and indirectly aid in shell formation. Therefore, the potential role of the oyster EF microbiome in shell formation warrants further investigation as oysters and other shellfish face the future impacts of ocean warming and acidification.

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.

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.

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.

Counts and sequences, observations that continue to change our understanding of viruses in nature.

The discovery of abundant viruses in the oceans and on land has ushered in a quarter century of groundbreaking advancements in our understanding of viruses within ecosystems. Two types of observations from environmental samples--direct counts of viral particles and viral metagenomic sequences--have been critical to these discoveries. Accurate direct counts have established ecosystem-scale trends in the impacts of viral infection on microbial host populations and have shown that viral communities within aquatic and soil environments respond to both short term and seasonal environmental change. Direct counts have been critical for estimating viral production rate, a measurement essential to quantifying the implications of viral infection for the biogeochemical cycling of nutrients within ecosystems. While direct counts have defined the magnitude of viral processes; shotgun sequences of environmental viral DNA--virome sequences--have enabled researchers to estimate the diversity and composition of natural viral communities. Virome-enabled studies have found the virioplankton to contain thousands of viral genotypes in communities where the most dominant viral population accounts for a small fraction of total abundance followed by a long tail of diverse populations. Detailed examination of long virome sequences has led to new understanding of genotype-to-phenotype connections within marine viruses and revealed that viruses carry metabolic genes that are important to maintaining cellular energy during viral replication. Increased access to long virome sequences will undoubtedly reveal more genetic secrets of viruses and enable us to build a genomics rulebook for predicting key biological and ecological features of unknown viruses.

Shotgun metagenomics indicates novel family A DNA polymerases predominate within marine virioplankton.

Virioplankton have a significant role in marine ecosystems, yet we know little of the predominant biological characteristics of aquatic viruses that influence the flow of nutrients and energy through microbial communities. Family A DNA polymerases, critical to DNA replication and repair in prokaryotes, are found in many tailed bacteriophages. The essential role of DNA polymerase in viral replication makes it a useful target for connecting viral diversity with an important biological feature of viruses. Capturing the full diversity of this polymorphic gene by targeted approaches has been difficult; thus, full-length DNA polymerase genes were assembled out of virioplankton shotgun metagenomic sequence libraries (viromes). Within the viromes novel DNA polymerases were common and found in both double-stranded (ds) DNA and single-stranded (ss) DNA libraries. Finding DNA polymerase genes in ssDNA viral libraries was unexpected, as no such genes have been previously reported from ssDNA phage. Surprisingly, the most common virioplankton DNA polymerases were related to a siphovirus infecting an α-proteobacterial symbiont of a marine sponge and not the podoviral T7-like polymerases seen in many other studies. Amino acids predictive of catalytic efficiency and fidelity linked perfectly to the environmental clades, indicating that most DNA polymerase-carrying virioplankton utilize a lower efficiency, higher fidelity enzyme. Comparisons with previously reported, PCR-amplified DNA polymerase sequences indicated that the most common virioplankton metagenomic DNA polymerases formed a new group that included siphoviruses. These data indicate that slower-replicating, lytic or lysogenic phage populations rather than fast-replicating, highly lytic phages may predominate within the virioplankton.

A carbon free filter for collection of large volume samples of cellular biomass from oligotrophic waters.

Isotopic analysis of cellular biomass has greatly improved our understanding of carbon cycling in the environment. Compound specific radiocarbon analysis (CSRA) of cellular biomass is being increasingly applied in a number of fields. However, it is often difficult to collect sufficient cellular biomass for analysis from oligotrophic waters because easy-to-use filtering methods that are free of carbon contaminants do not exist. The goal of this work was to develop a new column based filter to autonomously collect high volume samples of biomass from oligotrophic waters for CSRA using material that can be baked at 450°C to remove potential organic contaminants. A series of filter materials were tested, including uncoated sand, ferrihydrite-coated sand, goethite-coated sand, aluminum-coated sand, uncoated glass wool, ferrihydrite-coated glass wool, and aluminum-coated glass wool, in the lab with 0.1 and 1.0 µm microspheres and Escherichia coli. Results indicated that aluminum-coated glass wool was the most efficient filter and that the retention capacity of the filter far exceeded the biomass requirements for CSRA. Results from laboratory tests indicate that for oligotrophic waters with 1×10(5) cells ml(-1), 117l of water would need to be filtered to collect 100 µg of PLFA for bulk PLFA analysis and 2000 l for analysis of individual PLFAs. For field sampling, filtration tests on South African mine water indicated that after filtering 5955l, 450 µg of total PLFAs were present, ample biomass for radiocarbon analysis. In summary, we have developed a filter that is easy to use and deploy for collection of biomass for CSRA including total and individual PLFAs.