Rebekah R. Helton

Lysogenic virus-host interactions predominate at deep-sea diffuse-flow hydrothermal vents.

The consequences of viral infection within microbial communities are dependent on the nature of the viral life cycle. Among the possible outcomes is the substantial influence of temperate viruses on the phenotypes of lysogenic prokaryotes through various forms of genetic exchange. To date, no marine microbial ecosystem has consistently shown a predisposition for containing significant numbers of inducible temperate viruses. Here, we show that deep-sea diffuse-flow hydrothermal vent waters display a consistently high incidence of lysogenic hosts and harbor substantial populations of temperate viruses. Genetic fingerprinting and initial metagenomic analyses indicate that temperate viruses in vent waters appear to be a less diverse subset of the larger virioplankton community and that these viral populations contain an extraordinarily high frequency of novel genes. Thus, it appears likely that temperate viruses are key players in the ecology of prokaryotes within the extreme geothermal ecosystems of the deep sea.

Assessment of Factors Influencing Direct Enumeration of Viruses within Estuarine Sediments

ABSTRACT Accurate enumeration of viruses within environmental samples is critical for investigations of the ecological role of viruses and viral infection within microbial communities. This report evaluates differences in viral and bacterial direct counts between estuarine sediment samples which were either immediately processed onboard ship or frozen at −20°C and later processed. Viral and bacterial abundances were recorded at three stations spanning the length of the Chesapeake Bay in April and June 2003 within three sediment fractions: pore water (PW), whole sediment (WS), and sediment after pore water removal (AP). No significant difference in viral abundance was apparent between extracts from fresh or frozen sediments. In contrast, bacterial abundance was significantly lower in the samples subjected to freezing. Both bacterial and viral abundance showed significant differences between sediment fractions (PW, WS, or AP) regardless of the fresh or frozen status. Although pore water viral abundance has been used in the past as a measurement of viral abundance in sediments, this fraction accounted for only ca. 5% of the total sediment viral abundance across all samples. The effect of refrigerated storage of sediment viral extracts was also examined and showed that, within the first 2 h, viral abundance decreased ca. 30% in formalin-fixed extracts and 66% in unfixed extracts. Finally, the reliability of direct viral enumeration via epifluorescence microscopy was tested by using DNase treatment of WS extractions. These tests indicated that a large fraction (>86%) of the small SYBR gold fluorescing particles are likely viruses.

Repeating patterns of virioplankton production within an estuarine ecosystem

The Chesapeake Bay, a seasonally variable temperate estuary, provides a natural laboratory for examining the fluctuations and impacts of viral lysis on aquatic microorganisms. Viral abundance (VA) and viral production (VP) were monitored in the Chesapeake Bay over 4 1/2 annual cycles, producing a unique, long-term, interannual study of virioplankton production. High and dynamic VP rates, averaging 7.9 × 106 viruses per mL per h, indicate that viral lysis impacts a significant fraction of microorganisms in the Chesapeake. Viral-mediated bacterial mortality, VA, VP, and organic carbon release all displayed similar interannual and seasonal trends with higher values in 2003 and 2006 than in 2004 and 2005 and peaks in early spring and summer. Surprisingly, higher rates of viral lysis occurred in winter, resulting in a magnified effect of viral lysis on bacterioplankton during times of reduced productivity. Viral lysis directly impacted the organic carbon pool, contributing on average 76 μg of C per L per d, an amount capable of sustaining ∼55% of Chesapeake Bay bacterial production. The observed repeating interannual patterns of VP and lysis are likely interlinked with seasonal cycles of host abundance and diversity, which are in turn driven by annual cycles in environmental conditions, emphasizing the complex interplay of seasonality and microbial ecology in the Chesapeake Bay.

Seasonal Dynamics and Metagenomic Characterization of Estuarine Viriobenthos Assemblages by Randomly Amplified Polymorphic DNA PCR

ABSTRACT Direct enumeration and genetic analyses indicate that aquatic sediments harbor abundant and diverse viral communities. Thus far, synecological analysis of estuarine sediment viral diversity over an annual cycle has not been reported. This oversight is due in large part to a lack of molecular genetic approaches for assessing viral diversity within a large collection of environmental samples. Here, randomly amplified polymorphic DNA PCR (RAPD-PCR) was used to examine viral genotypic diversity within Chesapeake Bay sediments. Using a single 10-mer oligonucleotide primer for all samples, RAPD-PCR analysis of sediment viral assemblages yielded unique banding patterns across spatial and temporal scales, with the occurrence of specific bands varying among the sample set. Cluster analysis of RAPD-PCR amplicon banding patterns indicated that sediment viral assemblages changed with season and to a lesser extent with geographic location. Sequence analysis of RAPD-PCR amplicons revealed that 76% of sediment viral sequences were not homologous to any sequence in the GenBank nonredundant protein database. Of the GenBank sequence homologs, the majority belonged to viruses within the Podoviridae (24%) and Myoviridae (22%) viral families, which agrees with the previously observed frequencies of these morphological families in Chesapeake Bay sediments. Furthermore, the majority of the sediment viral sequences homologous to GenBank nonredundant protein sequences were phages or prophages (57%). Hence, RAPD-PCR proved to be a reliable and useful approach for characterization of viral assemblages and the genetic diversity of viruses within aquatic sediments.

Estimates of viral abundance in soils are strongly influenced by extraction and enumeration methods

Viruses are highly abundant in temperate soils, ranging from 107 to 109 g−1, and outnumbering soil bacteria from 5- to over 1,000-fold. In order to determine the potential impacts of viruses on soil microbial communities, it is important to establish reliable methods for comparing changes in viral abundances within and across soil samples. The goals of this study were to optimize extraction-enumeration methods to accurately determine viral abundances in a range of soil types, to evaluate the feasibility of simultaneously enumerating bacterial cells and virus particles using a single extraction procedure, and to assess the utility of flow cytometry (FCM) for enumerating virus particles in soil extracts. Comparisons of extraction approaches indicated that sonication or blender extraction of soils with potassium citrate buffer yielded the highest viral abundances for most soil types. Combined viral and bacterial extractions underestimate abundances compared to separately-optimized extractions for each. Flow cytometric counts were anywhere between 350- and 1,400-fold higher than epifluorescence microscopy (EFM)-based counts for the same soil. Trends in viral abundance across soil types were different from those via EFM, and different relationships between viral abundance and soil properties were observed depending on the enumeration method. Thus, FCM is not currently recommended for enumeration of viruses in soil extracts. Based on EFM results, soil moisture and organic matter content were the most important factors determining viral abundance in soils.

Bias in bacteriophage morphological classification by transmission electron microscopy due to breakage or loss of tail structures.

Virtually every study that has used transmission electron microscopy (TEM) to estimate viral diversity has acknowledged that loss of phage tails during sample preparation may have biased the results. However, the magnitude of this potential bias has yet to be constrained. To characterize biases in virus morphological diversity due to tail loss, six phage strains representing the order Caudovirales were inoculated into sterile sediments and soils. Phage particles were then extracted using standard methods. Morphologies of extracted phage particles were compared to those of unmanipulated control samples to determine the extent of tail breakage incurred by extraction procedures. Podoviruses exhibited the smallest frequency of tail loss during extraction (1.2–14%), myoviruses were moderately susceptible to tail breakage (15–40%), and siphoviruses were highly susceptible (32–76%). Thus, TEM assessments of viral diversity in soils or sediments by distribution of tail morphologies may be biased toward podoviruses and virions lacking tails, while simultaneously underestimating the abundance of siphoviruses. However, since the majority of viral capsids observed under TEM were intact, estimates of viral diversity based on the distribution of capsid diameters may provide a more reliable basis for morphological comparisons within and across ecosystems. Microsc. Res. Tech., 2011.