Dynamics of autochthonous soil viral communities parallels dynamics of host communities under nutrient stimulation.

Viruses are highly abundant in soils with their numbers exceeding those of cooccurring bacterial cells by 10- to over 1000-fold. Water and organic matter content influence the magnitude of the viral-to-bacterial ratio in soils; thus, ecosystem type and land use shape interactions between viral and host microbial communities in soils. Less understood are the shorter term interactions between viral and host communities that ultimately maintain the large viral standing stock within soils. This study examined short-term dynamics of viral and bacterial communities in soils to determine whether the growth of soil bacterial communities results in the production of soil viruses, and if viral community responses occur within specific populations. In microcosms amended with different carbon sources, increases in viral abundance (VA) accompanied increases in bacterial abundance (BA) and bacterial respiration rate (BRR). The timing and intensity of increases in BA, VA and BRR were different across C sources suggesting differences in the predominant mode of viral replication within growth-stimulated bacterial populations. Moreover, compositional changes occurred in soil bacterial and viral communities indicating that new viral production arose from a subset of host populations. To our knowledge, these are the first observations of soil viral populations responding to short-term changes in soil bacterial communities.

Direct assessment of viral diversity in soils by random PCR amplification of polymorphic DNA.

Viruses are the most abundant and diverse biological entities within soils, yet their ecological impact is largely unknown. Defining how soil viral communities change with perturbation or across environments will contribute to understanding the larger ecological significance of soil viruses. A new approach to examining the composition of soil viral communities based on random PCR amplification of polymorphic DNA (RAPD-PCR) was developed. A key methodological improvement was the use of viral metagenomic sequence data for the design of RAPD-PCR primers. This metagenomically informed approach to primer design enabled the optimization of RAPD-PCR sensitivity for examining changes in soil viral communities. Initial application of RAPD-PCR viral fingerprinting to soil viral communities demonstrated that the composition of autochthonous soil viral assemblages noticeably changed over a distance of meters along a transect of Antarctic soils and across soils subjected to different land uses. For Antarctic soils, viral assemblages segregated upslope from the edge of dry valley lakes. In the case of temperate soils at the Kellogg Biological Station, viral communities clustered according to land use treatment. In both environments, soil viral communities changed along with environmental factors known to shape the composition of bacterial host communities. Overall, this work demonstrates that RAPD-PCR fingerprinting is an inexpensive, high-throughput means for addressing first-order questions of viral community dynamics within environmental samples and thus fills a methodological gap between narrow single-gene approaches and comprehensive shotgun metagenomic sequencing for the analysis of viral community diversity.

Evaluation of two approaches for assessing the genetic similarity of virioplankton populations as defined by genome size.

Viral production estimates show that virioplankton communities turn over rapidly in aquatic ecosystems. Thus, it is likely that the genetic identity of viral populations comprising the virioplankton also change over temporal and spatial scales, reflecting shifts in viral-host interactions. However, there are few approaches that can provide data on the genotypic identity of viral populations at low cost and with the sample throughput necessary to assess dynamic changes in the virioplankton. This study examined two of these approaches-T4-like major capsid protein (g23) gene polymorphism and randomly amplified polymorphic DNA-PCR (RAPD-PCR) fingerprinting-to ask how well each technique could track differences in virioplankton populations over time and geographic location. Seasonal changes in overall virioplankton composition were apparent from pulsed-field gel electrophoresis (PFGE) analysis. T4-like phages containing similar g23 proteins were found within both small- and large-genome populations, including populations from different geographic locations and times. The surprising occurrence of T4-like g23 within small genomic groups (23 to 64 kb) indicated that the genome size range of T4-like phages may be broader than previously believed. In contrast, RAPD-PCR fingerprinting detected high genotypic similarity within PFGE bands from the same location, time, and genome size class without the requirement for DNA sequencing. Unlike g23 polymorphism, RAPD-PCR fingerprints showed a greater temporal than geographic variation. Thus, while polymorphism in a viral signature gene, such as g23, can be a powerful tool for inferring evolutionary relationships, the degree to which this approach can capture fine-scale variability within virioplankton populations is less clear.

VIROME: a standard operating procedure for analysis of viral metagenome sequences.

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 predicted viral metagenomic ORF run through the VIROME pipeline is placed into one of seven ORF classes, thus, every sequence receives a meaningful annotation. Additionally, the pipeline includes quality control measures to remove contaminating and poor quality sequence and assesses the potential amount of cellular DNA contamination in a viral metagenome library by screening for rRNA genes. Access to the VIROME pipeline and analysis results are provided through a web-application interface that is dynamically linked to a relational back-end database. The VIROME web-application interface is designed to allow users flexibility in retrieving sequences (reads, ORFs, predicted peptides) and search results for focused secondary analyses.

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.

Phages across the biosphere: contrasts of viruses in soil and aquatic environments.

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.