Seasonal trends in lysogeny in an Appalachian oak-hickory forest soil.

Since 1989, investigations into viral ecology have revealed how bacteriophages can influence microbial dynamics within ecosystems at global scales. Most of the information we know about temperate phages, which can integrate themselves into the host genome and remain dormant via a process called lysogeny, has come from research in aquatic ecosystems. Soil environments remain under-studied, and more research is necessary to fully understand the range of impacts phage infections have on the soil bacteria they infect. The aims of this study were to compare the efficacy of different prophage-inducing agents and to elucidate potential temporal trends in lysogeny within a soil bacterial community. In addition to mitomycin C and acyl-homoserine lactones, our results indicated that halosulfuron methyl herbicides may also be potent inducing agents. In optimizing chemical induction assays, we determined that taking steps to reduce background virus particles and starve cells was critical in obtaining consistent results. A clear seasonal trend in inducible lysogeny was observed in an Appalachian oak-hickory forest soil. The average monthly air temperature was negatively correlated with inducible fraction and burst size, supporting the idea that lysogeny provides a mechanism for phage persistence when temperatures are low and host metabolism is slower. Furthermore, the inducible fraction was negatively correlated with both soil bacterial and soil viral abundance, supporting the idea that lysogeny provides a mechanism for temperate phage persistence when host density is lower. The present study is the first of its kind to reveal clear seasonal trends in inducible lysogeny in any soil.IMPORTANCELysogeny is a relationship in which certain viruses that infect bacteria (phages) may exist within their bacterial host cell as a segment of nucleic acid. In this state, the phage genome is protected from environmental damage and retains the potential to generate progeny particles in the future. It is thought that lysogeny provides a mechanism for long-term persistence for phages when host density is low or hosts are starved-two conditions likely to be found in soils. In the present study, we provide the first known evidence for a seasonal trend in lysogeny in a forest soil. Based on clear relationships observed between lysogeny, temperature, and soil microbial abundance, we find support for previous hypotheses regarding the factors governing lysogeny.

Understanding Viral Impacts in Soil Microbial Ecology Through the Persistence and Decay of Infectious Bacteriophages.

Marine bacteriophages have been well characterized in terms of decay rates, population dynamics in relation to their hosts, and their impacts on biogeochemical cycles in the global ocean. Knowledge in soil bacteriophage ecology lags considerably behind, with few studies documenting population dynamics with hosts and even fewer reporting phage decay rates. By using sterile soil or aquatic microcosms inoculated with single bacteriophage isolates, phage decay rates (loss of infectivity over time) were determined, independent of host interactions, for 5 model phage isolates. Decay rates varied by phage from 0.11-2.07% h-1 in soils to 0.07-0.28% h-1 in aquatic microcosms. For phages incubated in both soil and aquatic microcosms, the observed decay rate was consistently higher in soil microcosms than in aquatic microcosms by at least a factor of two. However, when decay rates for soil phage isolates in the present study were compared to those reported for marine and freshwater phage isolates from previous studies, the decay constants for soil phages were, on average, 4 times lower than those for aquatic phages. Slower rates of phage decay in soils indicate a lower turnover rate, which may have subsequent and potentially far-reaching impacts on virus-mediated mortality and bacterial activity. The wide range of decay rates observed in the present study and the lack of information on this critical aspect of virus-host dynamics in soil emphasizes the need for continued research in this field.

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 × 10(6) 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.

Sequence analysis of Discoknowium, an A5 mycobacteriophage.

Discoknowium is a temperate A5 bacteriophage that infects the bacterial host Mycobacterium smegmatis. Isolated from a rat fecal sample, Discoknowium's genome is 50,222 bp in length, contains 84 genes and 1 tRNA, and shares 82%-98% nucleotide identity with other A5 subcluster phages.

Acyl-homoserine lactones can induce virus production in lysogenic bacteria: an alternative paradigm for prophage induction.

Prophage typically are induced to a lytic cycle under stressful environmental conditions or when the host's survival is threatened. However, stress-independent, spontaneous induction also occurs in nature and may be cell density dependent, but the in vivo signal(s) that can trigger induction is unknown. In the present study, we report that acyl-homoserine lactones (AHL), the essential signaling molecules of quorum sensing in many gram-negative bacteria, can trigger phage production in soil and groundwater bacteria. This phenomenon also was operative in a lambda lysogen of Escherichia coli. In model coculture systems, we monitored the real-time AHL production from Pseudomonas aeruginosa PAO1 using an AHL bioluminescent sensor and demonstrated that lambda-prophage induction in E. coli was correlated with AHL production. As a working model in E. coli, we show that the induction responses of lambda with AHL remained unaffected when recA was deleted, suggesting that this mechanism does not involve an SOS response. In the same lambda lysogen we also demonstrated that sdiA, the AHL receptor, and rcsA, a positive transcriptional regulator of exopolysaccharide synthesis, are involved in the AHL-mediated induction process. These findings relate viral reproduction to chemical signals associated with high host cell abundance, suggesting an alternative paradigm for prophage induction.

Methods for the isolation of viruses from environmental samples.

Viruses are omnipresent and extraordinarily abundant in the microbial ecosystems of water, soil, and sediment. In nearly every reported case for aquatic and porous media environments (soils and sediments) viral abundance exceeds that of co-occurring host populations by 10-100-fold. If current estimates based on metagenome DNA sequence data are correct, then viruses represent the largest reservoir of unknown genetic diversity on Earth. Microscopy and molecular genetic tools have been critical in demonstrating that viruses are a dynamic component of microbial ecosystems capable of significantly influencing the productivity and population biology of their host communities. Moreover, these approaches have begun to describe and constrain the immense genetic diversity of viral communities. A critical first step in the application of many cultivation-independent approaches to virus ecology is obtaining a concentrate of viruses from an environmental sample. Culture-dependent methods also rely on viruses being present at a high enough abundance to detect. Here, methodological details for the isolation and concentration of viruses from water, soil, and aquatic sediment samples are covered in detail.

Prevalence of lysogeny among soil bacteria and presence of 16S rRNA and trzN genes in viral-community DNA.

Bacteriophages are very abundant in the biosphere, and viral infection is believed to affect the activity and genetic diversity of bacterial communities in aquatic environments. Lysogenic conversion, for example, can improve host fitness and lead to phage-mediated horizontal gene transfer. However, little is known about lysogeny and transduction in the soil environment. In this study we employed atrazine-impregnated Bio-Sep beads (a cell immobilization matrix) to sample active microbiota from soils with prior pesticide exposure history. Once recovered from soil, the bead communities were induced with mitomycin C (MC), and viral and bacterial abundances were determined to evaluate the incidence of inducible prophage in soil bacteria. The inducible fraction calculated within bead communities was high (ca. 85%) relative to other studies in aquatic and sedimentary environments. Moreover, the bacterial genes encoding 16S rRNA and trzN, a chlorohydrolase gene responsible for dehalogenation of atrazine, were detected by PCR in the viral DNA fraction purified from MC-induced bead communities. A diverse collection of actinobacterial 16S rRNA gene sequences occurred within the viral DNA fraction of induced, water-equilibrated beads. Similar results were observed in induced atrazine-equilibrated beads, where 77% of the cloned sequences were derived from actinobacterial lineages. Heterogeneous 16S rRNA gene sequences consisting of fragments from two different taxa were detected in the clone libraries. The results suggest that lysogeny is a prevalent reproductive strategy among soil bacteriophages and that the potential for horizontal gene transfer via transduction is significant in soil microbial communities.

Metagenomic characterization of Chesapeake Bay virioplankton.

Viruses are ubiquitous and abundant throughout the biosphere. In marine systems, virus-mediated processes can have significant impacts on microbial diversity and on global biogeocehmical cycling. However, viral genetic diversity remains poorly characterized. To address this shortcoming, a metagenomic library was constructed from Chesapeake Bay virioplankton. The resulting sequences constitute the largest collection of long-read double-stranded DNA (dsDNA) viral metagenome data reported to date. BLAST homology comparisons showed that Chesapeake Bay virioplankton contained a high proportion of unknown (homologous only to environmental sequences) and novel (no significant homolog) sequences. This analysis suggests that dsDNA viruses are likely one of the largest reservoirs of unknown genetic diversity in the biosphere. The taxonomic origin of BLAST homologs to viral library sequences agreed well with reported abundances of cooccurring bacterial subphyla within the estuary and indicated that cyanophages were abundant. However, the low proportion of Siphophage homologs contradicts a previous assertion that this family comprises most bacteriophage diversity. Identification and analyses of cyanobacterial homologs of the psbA gene illustrated the value of metagenomic studies of virioplankton. The phylogeny of inferred PsbA protein sequences suggested that Chesapeake Bay cyanophage strains are endemic in that environment. The ratio of psbA homologous sequences to total cyanophage sequences in the metagenome indicated that the psbA gene may be nearly universal in Chesapeake Bay cyanophage genomes. Furthermore, the low frequency of psbD homologs in the library supports the prediction that Chesapeake Bay cyanophage populations are dominated by Podoviridae.

Viruses in Soil Ecosystems: An Unknown Quantity Within an Unexplored Territory.

Viral abundance in soils can range from below detection limits in hot deserts to over 1 billion per gram in wetlands. Abundance appears to be strongly influenced by water availability and temperature, but a lack of informational standards creates difficulties for cross-study analysis. Soil viral diversity is severely underestimated and undersampled, although current measures of viral richness are higher for soils than for aquatic ecosystems. Both morphometric and metagenomic analyses have raised questions about the prevalence of nontailed, ssDNA viruses in soils. Soil is complex and critically important to terrestrial biodiversity and human civilization, but impacts of viral activities on soil ecosystem services are poorly understood. While information from aquatic systems and medical microbiology suggests the potential for viral influences on nutrient cycles, food web interactions, gene transfer, and other key processes in soils, very few empirical data are available. To understand the soil virome, much work remains.

Stormwater runoff drives viral community composition changes in inland freshwaters.

Storm events impact freshwater microbial communities by transporting terrestrial viruses and other microbes to freshwater systems, and by potentially resuspending microbes from bottom sediments. The magnitude of these impacts on freshwater ecosystems is unknown and largely unexplored. Field studies carried out at two discrete sites in coastal Virginia (USA) were used to characterize the viral load carried by runoff and to test the hypothesis that terrestrial viruses introduced through stormwater runoff change the composition of freshwater microbial communities. Field data gathered from an agricultural watershed indicated that primary runoff can contain viral densities approximating those of receiving waters. Furthermore, viruses attached to suspended colloids made up a large fraction of the total load, particularly in early stages of the storm. At a second field site (stormwater retention pond), RAPD-PCR profiling showed that the viral community of the pond changed dramatically over the course of two intense storms while relatively little change was observed over similar time scales in the absence of disturbance. Comparisons of planktonic and particle-associated viral communities revealed two completely distinct communities, suggesting that particle-associated viruses represent a potentially large and overlooked portion of aquatic viral abundance and diversity. Our findings show that stormwater runoff can quickly change the composition of freshwater microbial communities. Based on these findings, increased storms in the coastal mid-Atlantic region predicted by most climate change models will likely have important impacts on the structure and function of local freshwater microbial communities.

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.

Cluster K mycobacteriophages: insights into the evolutionary origins of mycobacteriophage TM4.

Five newly isolated mycobacteriophages--Angelica, CrimD, Adephagia, Anaya, and Pixie--have similar genomic architectures to mycobacteriophage TM4, a previously characterized phage that is widely used in mycobacterial genetics. The nucleotide sequence similarities warrant grouping these into Cluster K, with subdivision into three subclusters: K1, K2, and K3. Although the overall genome architectures of these phages are similar, TM4 appears to have lost at least two segments of its genome, a central region containing the integration apparatus, and a segment at the right end. This suggests that TM4 is a recent derivative of a temperate parent, resolving a long-standing conundrum about its biology, in that it was reportedly recovered from a lysogenic strain of Mycobacterium avium, but it is not capable of forming lysogens in any mycobacterial host. Like TM4, all of the Cluster K phages infect both fast- and slow-growing mycobacteria, and all of them--with the exception of TM4--form stable lysogens in both Mycobacterium smegmatis and Mycobacterium tuberculosis; immunity assays show that all five of these phages share the same immune specificity. TM4 infects these lysogens suggesting that it was either derived from a heteroimmune temperate parent or that it has acquired a virulent phenotype. We have also characterized a widely-used conditionally replicating derivative of TM4 and identified mutations conferring the temperature-sensitive phenotype. All of the Cluster K phages contain a series of well conserved 13 bp repeats associated with the translation initiation sites of a subset of the genes; approximately one half of these contain an additional sequence feature composed of imperfectly conserved 17 bp inverted repeats separated by a variable spacer. The K1 phages integrate into the host tmRNA and the Cluster K phages represent potential new tools for the genetics of M. tuberculosis and related species.

Cultivation-based assessment of lysogeny among soil bacteria.

Lysogeny has long been proposed as an important long-term maintenance strategy for autochthonous soil bacteriophages (phages). Whole genome sequence data indicate that prophage-derived sequences pervade prokaryotic genomes, but the connection between inferred prophage sequence and an active temperate phage is tenuous. Thus, definitive evidence of phage production from lysogenic prokaryotes will be critical in determining the presence and extent of temperate phage diversity existing as prophage within bacterial genomes and within environmental contexts such as soils. This study optimized methods for systematic and definitive determination of lysogeny within a collection of autochthonous soil bacteria. Twenty bacterial isolates from a range of Delaware soil environments (five from each soil) were treated with the inducing agents mitomycin C (MC) or UV light. Six isolates (30%) carried inducible temperate phages as evidenced by an increase in virus direct counts. The magnitude of induction response was highly dependent upon specific induction conditions, and corresponding burst sizes ranged from 1 to 176. Treatment with MC for 30 min yielded the largest induction responses for three of the six lysogens. Morphological analysis revealed that four of the lysogens produced lambda-like Siphoviridae particles, whereas two produced Myoviridae particles. Additionally, pulsed-field gel electrophoresis data indicated that two of the six lysogens were polylysogens, producing more than one distinct type of phage particle. These results suggest that lysogeny is relatively common among soil bacteria.

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.

Incidence of lysogeny within temperate and extreme soil environments.

A companion study indicated that approximately 30% of cultivable soil bacteria may contain inducible prophage; however, the degree to which this cultivation-based estimate applies to autochthonous communities of soil bacteria is unknown. To estimate the prevalence of lysogeny within soil bacterial communities, induction assays were carried out by extracting bacteria from soil and subsequently exposing extracts to mitomycin C (MC; 0.5 microg ml(-1)), or by exposing bacteria to MC (1.0 microg ml(-1)) through direct addition to soil slurries. Induction was assessed as an increase in viral direct counts relative to those obtained in controls, as detected by epifluorescence microscopy. Extracting bacteria from soils followed by 18 h MC exposure generated significantly higher prophage induction than all other treatments (P < 0.05). For three Antarctic soil samples, estimates of inducible fraction (IF) were statistically indistinguishable across two independent assays (P = 0.82), indicating that this approach is highly reproducible. Although IF was lower in Antarctic soils (4-20%) and higher in temperate Delaware soils (22-68%), no clear correlations were found between lysogeny and soil physical properties. For Delaware soils, IF estimates were similar between whole soil assays (44%) and cultivation-based approaches (30%). While these data suggest that lysogeny is common among soil bacteria, the specific factors which promote temperate interactions remain unclear.

Abundance and diversity of viruses in six Delaware soils.

The importance of viruses in marine microbial ecology has been established over the past decade. Specifically, viruses influence bacterial abundance and community composition through lysis and alter bacterial genetic diversity through transduction and lysogenic conversion. By contrast, the abundance and distribution of viruses in soils are almost completely unknown. This study describes the abundance and diversity of autochthonous viruses in six Delaware soils: two agricultural soils, two coastal plain forest soils, and two piedmont forest soils. Viral abundance was measured using epifluorescence microscopy, while viral diversity was assessed from morphological data obtained through transmission electron microscopy. Extracted soil virus communities were dominated by bacteriophages that demonstrated a wide range of capsid diameters (20 nm to 160 nm) and morphologies, including filamentous forms and phages with elongated capsids. The reciprocal Simpson's index suggests that forest soils harbor more diverse assemblages of viruses, particularly in terms of morphological distribution. Repeated extractions of virus-like particles (VLPs) from soils indicated that the initial round of extraction removes approximately 70% of extractable viruses. Higher VLP abundances were observed in forest soils (1.31 x 10(9) to 4.17 x 10(9) g(-1) dry weight) than in agricultural soils (8.7 x 10(8) to 1.1 x 10(9) g(-1) dry weight). Soil VLP abundance was significantly correlated to moisture content (r = 0.988) but not to soil texture. Land use (agricultural or forested) was significantly correlated to both bacterial (r = 0.885) and viral (r = 0.812) abundances, as were soil organic matter and water content. Thus, land use is a significant factor influencing viral abundance and diversity in soils.

Sampling natural viral communities from soil for culture-independent analyses.

An essential first step in investigations of viruses in soil is the evaluation of viral recovery methods suitable for subsequent culture-independent analyses. In this study, four elution buffers (10% beef extract, 250 mM glycine buffer, 10 mM sodium pyrophosphate, and 1% potassium citrate) and three enumeration techniques (plaque assay, epifluorescence microscopy [EFM], and transmission electron microscopy [TEM]) were compared to determine the best method of extracting autochthonous bacteriophages from two Delaware agricultural soils. Beef extract and glycine buffer were the most effective in eluting viable phages inoculated into soils (up to 29% recovery); however, extraction efficiency varied significantly with phage strain. Potassium citrate eluted the highest numbers of virus-like particles from both soils based on enumerations by EFM (mean, 5.3 x 10(8) g of dry soil(-1)), but specific soil-eluant combinations posed significant problems to enumeration by EFM. Observations of virus-like particles under TEM gave confidence that the particles were, in fact, phages, but TEM enumerations yielded measurements of phage abundance (mean, 1.5 x 10(8) g of dry soil(-1)) that were about five times lower. Clearly, the measurement of phage abundance in soils varies with both the extraction and enumeration methodology; thus, it is important to assess multiple extraction and enumeration approaches prior to undertaking ecological studies of phages in a particular soil.