Viruses are the most abundant and diverse biological entities on the planet and constitute a sign... more Viruses are the most abundant and diverse biological entities on the planet and constitute a significant proportion of Earth’s genetic diversity. Most of this diversity is not represented by isolated viral-host systems and has only been observed through sequencing of viral metagenomes (viromes) from environmental samples. Viromes provide snapshots of viral genetic potential, and a wealth of information on viral community ecology. These data also provide opportunities for exploring the biochemistry of novel viral enzymes. The in vitro biochemical characteristics of novel viral DNA polymerases were explored, testing hypothesized differences in polymerase biochemistry according to protein sequence phylogeny. Forty-eight viral DNA Polymerase I (PolA) proteins from estuarine viromes, hot spring metagenomes, and reference viruses, encompassing a broad representation of currently known diversity, were synthesized, expressed, and purified. Novel functionality was shown in multiple PolAs. In...
Additional file 1: of Presence of pathogenic Escherichia coli is correlated with bacterial community diversity and composition on pre-harvest cattle hides
Shotgun metagenomics, which allows for broad sampling of viral diversity, has uncovered genes tha... more 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.
As reported in many aquatic environments, recent studies in terrestrial ecosystems implicate a ro... more As reported in many aquatic environments, recent studies in terrestrial ecosystems implicate a role for viruses in shaping the structure, function, and evolution of prokaryotic soil communities. However, given the heterogeneity of soil and the physical constraints (i.e., pore-scale hydrology and solid-phase adsorption of phage and host cells) on the mobility of viruses and bacteria, phage-host interactions likely differ from those in aquatic systems. In this study, temporal changes in the population dynamics of viruses and bacteria in soils under different land management practices were examined. The results showed that bacterial abundance was significantly and positively correlated to both virus and inducible prophage abundance. Bacterial and viral abundance were also correlated with soil organic carbon and nitrogen content as well as with C:N ratio. The seasonal variability in viral abundance increased with soil organic carbon content. The prokaryotic community structure was influenced more by land use than by seasonal variation though considerable variation was evident in the early plant successional and grassland sites. The free extracellular viral communities were also separated by land use, and the forest soil viral assemblage exhibiting the most seasonal variability was more distinct from the other sites. Viral assemblages from the agricultural soils exhibited the least seasonal variability. Similar patterns were observed for inducible prophage viral assemblages. Seasonal variability of viral assemblages was greater in mitomycin-C (mitC) induced prophages than in extracellular viruses irrespective of land use and management. Taken together, the data suggest that soil viral production and decay are likely balanced but there was clear evidence that the structure of viral assemblages is influenced by land use and by season.
It has been hypothesized that, by specifically lysing numerically dominant host strains, the viri... more It has been hypothesized that, by specifically lysing numerically dominant host strains, the virioplankton may play a role in maintaining clonal diversity of heterotrophic bacteria and phytoplankton populations. If viruses selectively lyse only those host species that are numerically dominant, then the number of a specific virus within the virioplankton would be expected to change dramatically over time and space, in coordination with changes in abundance of the host. In this study, the abundances of specific viruses in Chesapeake Bay water samples were monitored, using nucleic acid probes and hybridization analysis. Total virioplankton in a water sample was separated by pulsed-field gel electrophoresis and hybridized with nucleic acid probes specific to either single viral strains or a group of viruses with similar genome sizes. The abundances of specific viruses were inferred from the intensity of the hybridization signal. By using this technique, a virus comprising 1/1,000 of the...
Viral infection exerts selection pressure on marine microbes as viral-induced cell lysis causes 2... more 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 f...
Ribonucleotide reductases (RNRs) are ancient enzymes that catalyze the reduction of ribonucleotid... more 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 theProchlorococcusphage P-SSP7...
Chaperonins are protein-folding machinery found in all cellular life. Chaperonin genes have been ... more Chaperonins are protein-folding machinery found in all cellular life. Chaperonin genes have been documented within a few viruses, yet, surprisingly, analysis of metagenome sequence data indicated that chaperonin-carrying viruses are common and geographically widespread in marine ecosystems. Also unexpected was the discovery of viral chaperonin sequences related to thermosome proteins of archaea, indicating the presence of virioplankton populations infecting marine archaeal hosts. Virioplankton large subunit chaperonin sequences (GroELs) were divergent from bacterial sequences, indicating that viruses have carried this gene over long evolutionary time. Analysis of viral metagenome contigs indicated that: the order of large and small subunit genes was linked to the phylogeny of GroEL; both lytic and temperate phages may carry group I chaperonin genes; and viruses carrying a GroEL gene likely have large double-stranded DNA (dsDNA) genomes (470 kb). Given these connections, it is likely that chaperonins are critical to the biology and ecology of virioplankton populations that carry these genes. Moreover, these discoveries raise the intriguing possibility that viral chaperonins may more broadly alter the structure and function of viral and cellular proteins in infected host cells.
Since 1982, specific serotypes of Shiga toxin-producing Escherichia coli (STEC) have been recogni... more Since 1982, specific serotypes of Shiga toxin-producing Escherichia coli (STEC) have been recognized as significant foodborne pathogens acquired from contaminated beef and, more recently, other food products. Cattle are the major reservoir hosts of these organisms, and while there have been advancements in food safety practices and industry standards, STEC still remains prevalent within beef cattle operations with cattle hides implicated as major sources of carcass contamination. To investigate whether the composition of hide-specific microbial communities are associated with STEC prevalence, 16S ribosomal RNA (rRNA) bacterial community profiles were obtained from hide and fecal samples collected from a large commercial feedlot over a 3-month period. These community data were examined amidst an extensive collection of prevalence data on a subgroup of STEC that cause illness in humans, referred to as enterohemorrhagic E. coli (EHEC). Fecal 16S rRNA gene OTUs (operational taxonomic un...
Marine viruses are critical drivers of ocean biogeochemistry and their abundances vary spatiotemp... more Marine viruses are critical drivers of ocean biogeochemistry and their abundances vary spatiotemporally in the global oceans, with upper estimates exceeding 10 8 per ml. Over many years, a consensus has emerged that virus abundances are typically 10-fold higher than prokaryote abundances. The use of a fixed-ratio suggests that the relationship between virus and prokaryote abundances is both predictable and linear. However, the true explanatory power of a linear relationship and its robustness across diverse ocean environments is unclear. Here, we compile 5671 prokaryote and virus abundance estimates from 25 distinct marine surveys to characterize the relationship between virus and prokaryote abundances. We find that the median virus-to-prokaryote ratio (VPR) is 10:1 and 16:1 in the near-and sub-surface oceans, respectively. Nonetheless, we observe substantial variation in the VPR and find either no or limited explanatory power using fixed-ratio models. Instead, virus abundances are better described as nonlinear, power-law functions of prokaryote abundances -particularly when considering relationships within distinct marine surveys. Estimated power-laws have scaling exponents that are typically less than 1, signifying that the VPR decreases with prokaryote density, rather than remaining fixed. The emergence of power-law scaling presents a challenge for mechanistic models seeking to understand the ecological causes and consequences of marine virusmicrobe interactions. Such power-law scaling also implies that efforts to average viral effects on microbial mortality and biogeochemical cycles using "representative" abundances or abundanceratios need to be refined if they are to be utilized to make quantitative predictions at regional or global ocean scales.
Marine viruses are critical drivers of ocean biogeochemistry and their abundances vary spatiotem-... more Marine viruses are critical drivers of ocean biogeochemistry and their abundances vary spatiotem- porally in the global oceans, with upper estimates exceeding 10 8 per ml. Over many years, a con- sensus has emerged that virus abundances are typically 10-fold higher than prokaryote abundances. The use of a fixed-ratio suggests that the relationship between virus and prokaryote abundances is both predictable and linear. However, the true explanatory power of a linear relationship and its robustness across diverse ocean environments is unclear. Here, we compile 5671 prokaryote and virus abundance estimates from 25 distinct marine surveys to characterize the relationship between virus and prokaryote abundances. We find that the median virus-to-prokaryote ratio (VPR) is 10:1 and 16:1 in the near- and sub-surface oceans, respectively. Nonetheless, we observe substantial variation in the VPR and find either no or limited explanatory power using fixed-ratio models. Instead, virus abundances...
Bacteriophage and Viral Ecology as Seen Through the Lens of Nucleic Acid Sequence Data
Microbial and Botanical Host Systems, 2011
... Homologues to gp 23 were nonexistent within a collection of 13,485 viral metagenome se-quence... more ... Homologues to gp 23 were nonexistent within a collection of 13,485 viral metagenome se-quences (8.45 Mb of DNA) in the Viral Infor-matics Resource for Metagenome Exploration database (VIROME) from viral assemblages under five different soil environments (Bhavsar et ...
Ecological investigations rely on data describing the biomass, diversity, and composition of livi... more Ecological investigations rely on data describing the biomass, diversity, and composition of living things. In the case of microbial communities, these data are primarily gathered using microscopy and molecular genetic approaches. The diminutive size of viruses means that obtaining genetic material sufficient for molecular approaches for examining the diversity and composition of aquatic viral assemblages can be challenging. Moreover, in procedures for the isolation and cultivation of novel viruses from natural waters, high-density viral inocula provide the best chance for success. To address the need for samples containing a high-density of viruses, investigators have used tangential-flow filtration (TFF) to concentrate viruses from large-volume (>20 L) water samples. This report outlines procedures for the preparation of viral concentrates from large volume water samples using TFF and discusses the effect of concentration procedures on viral recovery and downstream molecular genetic analyses.
Viral lysis of microbial hosts releases organic matter that can then be assimilated by nontargete... more Viral lysis of microbial hosts releases organic matter that can then be assimilated by nontargeted microorganisms. Quantitative estimates of virus-mediated recycling of carbon in marine waters, first established in the late 1990s, were originally extrapolated from marine host and virus densities, host carbon content and inferred viral lysis rates. Yet, these estimates did not explicitly incorporate the cascade of complex feedbacks associated with virus-mediated lysis. To evaluate the role of viruses in shaping community structure and ecosystem functioning, we extend dynamic multitrophic ecosystem models to include a virus component, specifically parameterized for processes taking place in the ocean euphotic zone. Crucially, we are able to solve this model analytically, facilitating evaluation of model behavior under many alternative parameterizations. Analyses reveal that the addition of a virus component promotes the emergence of complex communities. In addition, biomass partitioni...
One consistent finding among studies using shotgun metagenomics to analyze whole viral communitie... more One consistent finding among studies using shotgun metagenomics to analyze whole viral communities is that most viral sequences show no significant homology to known sequences. Thus, bioinformatic analyses based on sequence collections such as GenBank nr, which are largely comprised of sequences from known organisms, tend to ignore a majority of sequences within most shotgun viral metagenome libraries. Here we describe a bioinformatic pipeline, the Viral Informatics Resource for Metagenome Exploration (VIROME), that emphasizes the classification of viral metagenome sequences (predicted open-reading frames) based on homology search results against both known and environmental sequences. Functional and taxonomic information is derived from five annotated sequence databases which are linked to the UniRef 100 database. Environmental classifications are obtained from hits against a custom database, MetaGenomes On-Line, which contains 49 million predicted environmental peptides. Each pred...
Despite the predominance of aquatic environments on the planet Earth, microbial abundance and div... more Despite the predominance of aquatic environments on the planet Earth, microbial abundance and diversity within soil environments exceed that of the aquatic realm. Most of what we know of viral ecology within natural systems has come through investigations of aquatic environments. However, the 'aquatic-bias' in viral ecology is beginning to change as the cultivation-independent approaches, which revealed the extraordinary abundance and diversity of viruses within aquatic systems, are now being applied to soils. This review briefly summarizes recent investigations of viral abundance and diversity in soil environments.
Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicon... more Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa-a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3-V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with .1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure.
SUMMARYThe discovery that viruses may be the most abundant organisms in natural waters, surpassin... more SUMMARYThe discovery that viruses may be the most abundant organisms in natural waters, surpassing the number of bacteria by an order of magnitude, has inspired a resurgence of interest in viruses in the aquatic environment. Surprisingly little was known of the interaction of viruses and their hosts in nature. In the decade since the reports of extraordinarily large virus populations were published, enumeration of viruses in aquatic environments has demonstrated that the virioplankton are dynamic components of the plankton, changing dramatically in number with geographical location and season. The evidence to date suggests that virioplankton communities are composed principally of bacteriophages and, to a lesser extent, eukaryotic algal viruses. The influence of viral infection and lysis on bacterial and phytoplankton host communities was measurable after new methods were developed and prior knowledge of bacteriophage biology was incorporated into concepts of parasite and host commu...
Isotopic analysis of cellular biomass has greatly improved our understanding of carbon cycling in... more 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 aluminumcoated glass wool, in the lab with 0.1 and 1.0 µm microspheres and E. 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 , 117 L 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 5955 liters, 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.
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Papers by K. Wommack