Diversity and Ecology of Viruses in Hyperarid Desert Soils

In recent years, remarkable progress has been made in the field of virus environmental ecology. In marine ecosystems, for example, viruses are now thought to play pivotal roles in the biogeochemical cycling of nutrients and to be mediators of microbial evolution through horizontal gene transfer. The diversity and ecology of viruses in soils are poorly understood, but evidence supports the view that the diversity and ecology of viruses in soils differ substantially from those in aquatic systems. Desert biomes cover ∼33% of global land masses, and yet the diversity and roles of viruses in these dominant ecosystems remain poorly understood. There is evidence that hot hyperarid desert soils are characterized by high levels of bacterial lysogens and low extracellular virus counts. In contrast, cold desert soils contain high extracellular virus titers. We suggest that the prevalence of microbial biofilms in hyperarid soils, combined with extreme thermal regimens, exerts strong selection pressures on both temperate and virulent viruses. Many desert soil virus sequences show low values of identity to virus genomes in public databases, suggesting the existence of distinct and as-yet-uncharacterized soil phylogenetic lineages (e.g., cyanophages). We strongly advocate for amplification-free metavirome analyses while encouraging the classical isolation of phages from dominant and culturable microbial isolates in order to populate sequence databases. This review provides an overview of recent advances in the study of viruses in hyperarid soils and of the factors that contribute to viral abundance and diversity in hot and cold deserts and offers technical recommendations for future studies.     

Resistance to Co-Occurring Phages Enables Marine Synechococcus Communities To Coexist with Cyanophages Abundant in Seawater

Recent reports documenting very high viral abundances in seawater have led to increased interest in the role of viruses in aquatic environments and a resurgence of the hypothesis that viruses are significant agents of bacterial mortality. Synechococcus spp., small unicellular cyanobacteria that are important primary producers at the base of the marine food web, were used to assess this hypothesis. We isolated a diverse group of Synechococcus phages that at times reach titers of between 10³ and 10⁴ cyanophages per ml in both inshore and offshore waters. However, despite their diversity and abundance, we present evidence in support of the hypothesis that lytic phages have a negligible effect in regulating the densities of marine Synechococcus populations. Our results indicate that these bacterial communities are dominated by cells resistant to their co-occurring phages and that these viruses are maintained by scavenging on the relatively rare sensitive cells in these communities.     

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 × 10⁹ to 4.17 × 10⁹ g⁻¹ dry weight) than in agricultural soils (8.7 × 10⁸ to 1.1 × 10⁹ 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.     

Selection and Characterization of Cyanophage Resistance in Marine Synechococcus Strains

Marine viruses are an important component of the microbial food web, influencing microbial diversity and contributing to bacterial mortality rates. Resistance to cooccurring cyanophages has been reported for natural communities of Synechococcus spp.; however, little is known about the nature of this resistance. This study examined the patterns of infectivity among cyanophage isolates and unicellular marine cyanobacteria (Synechococcus spp.). We selected for phage-resistant Synechococcus mutants, examined the mechanisms of phage resistance, and determined the extent of cross-resistance to other phages. Four strains of Synechococcus spp. (WH7803, WH8018, WH8012, and WH8101) and 32 previously isolated cyanomyophages were used to select for phage resistance. Phage-resistant Synechococcus mutants were recovered from 50 of the 101 susceptible phage-host pairs, and 23 of these strains were further characterized. Adsorption kinetic assays indicate that resistance is likely due to changes in host receptor sites that limit viral attachment. Our results also suggest that receptor mutations conferring this resistance are diverse. Nevertheless, selection for resistance to one phage frequently resulted in cross-resistance to other phages. On average, phage-resistant Synechococcus strains became resistant to eight other cyanophages; however, there was no significant correlation between the genetic similarity of the phages (based on g20 sequences) and cross-resistance. Likewise, host Synechococcus DNA-dependent RNA polymerase (rpoC1) genotypes could not be used to predict sensitivities to phages. The potential for the rapid evolution of multiple phage resistance may influence the population dynamics and diversity of both Synechococcus and cyanophages in marine waters.     

Diverse viromes in polar regions: A retrospective study of metagenomic data from Antarctic animal feces and Arctic frozen soil in 2012–2014

Antarctica and the Arctic are the coldest places, containing a high diversity of microorganisms, including viruses, which are important components of polar ecosystems. However, owing to the difficulties in obtaining access to animal and environmental samples, the current knowledge of viromes in polar regions is still limited. To better understand polar viromes, this study performed a retrospective analysis using metagenomic sequencing data of animal feces from Antarctica and frozen soil from the Arctic collected during 2012–2014. The results reveal diverse communities of DNA and RNA viruses from at least 23 families from Antarctic animal feces and 16 families from Arctic soils. Although the viral communities from Antarctica and the Arctic show a large diversity, they have genetic similarities with known viruses from different ecosystems and organisms with similar viral proteins. Phylogenetic analysis of Microviridae, Parvoviridae, and Larvidaviridae was further performed, and complete genomic sequences of two novel circular replication-associated protein (rep)-encoding single-stranded (CRESS) DNA viruses closely related to Circoviridae were identified. These results reveal the high diversity, complexity, and novelty of viral communities from polar regions, and suggested the genetic similarity and functional correlations of viromes between the Antarctica and Arctic. Variations in viral families in Arctic soils, Arctic freshwater, and Antarctic soils are discussed. These findings improve our understanding of polar viromes and suggest the importance of performing follow-up in-depth investigations of animal and environmental samples from Antarctica and the Arctic, which would reveal the substantial role of these viruses in the global viral community.     

Sequence Analysis of Marine Virus Communities Reveals that Groups of Related Algal Viruses Are Widely Distributed in Nature

Algal-virus-specific PCR primers were used to amplify DNA polymerase (pol) gene fragments from geographically isolated natural virus communities. Natural algal virus communities were obtained from coastal sites in the Pacific Ocean in British Columbia, Canada, and the Southern Ocean near the Antarctic peninsula. Genetic fingerprints of algal virus communities were generated using denaturing gradient gel electrophoresis (DGGE). Sequencing efforts recovered 33 sequences from the gradient gel. Of the 33 sequences examined, 25 encoded a conserved amino acid motif indicating that the sequences were pol gene fragments. Furthermore, the 25 pol sequences were related to pol gene fragments from known algal viruses. In addition, similar virus sequences (>98% sequence identity) were recovered from British Columbia and Antarctica. Results from this study demonstrate that DGGE with degenerate primers can be used to qualitatively fingerprint and assess genetic diversity in specific subsets of natural virus communities and that closely related viruses occur in distant geographic locations. DGGE is a powerful tool for genetically fingerprinting natural virus communities and may be used to examine how specific components of virus communities respond to experimental manipulations.     

Viral metagenomics analysis of planktonic viruses in East Lake, Wuhan, China

East Lake (Lake Donghu), located in Wuhan, China, is a typical city freshwater lake that has been experiencing eutrophic conditions and algal blooming during recent years. Marine and fresh water are considered to contain a large number of viruses. However, little is known about their genetic diversity because of the limited techniques for culturing viruses. In this study, we conducted a viral metagenomic analysis using a high-throughput sequencing technique with samples collected from East Lake in Spring, Summer, Autumn, and Winter. The libraries from four samples each generated 234,669, 71,837, 12,820, and 34,236 contigs (> 90 bp each), respectively. The genetic structure of the viral community revealed a high genetic diversity covering 23 viral families, with the majority of contigs homologous to DNA viruses, including members of Myoviridae, Podoviridae, Siphoviridae, Phycodnaviridae, and Microviridae, which infect bacteria or algae, and members of Circoviridae, which infect invertebrates and vertebrates. The highest viral genetic diversity occurred in samples collected in August, then December and June, and the least diversity in March. Most contigs have low-sequence identities with known viruses. PCR detection targeting the conserved sequences of genes (g20, psbA, psbD, and DNApol) of cyanophages further confirmed that there are novel cyanophages in the East Lake. Our viral metagenomic data provide the first preliminary understanding of the virome in one freshwater lake in China and would be helpful for novel virus discovery and the control of algal blooming in the future.     

Desert farming benefits from microbial potential in arid soils and promotes diversity and plant health

Background

To convert deserts into arable, green landscapes is a global vision, and desert farming is a strong growing area of agriculture world-wide. However, its effect on diversity of soil microbial communities, which are responsible for important ecosystem services like plant health, is still not known.

Methodology/Principal Findings

We studied the impact of long-term agriculture on desert soil in one of the most prominent examples for organic desert farming in Sekem (Egypt). Using a polyphasic methodological approach to analyse microbial communities in soil as well as associated with cultivated plants, drastic effects caused by 30 years of agriculture were detected. Analysing bacterial fingerprints, we found statistically significant differences between agricultural and native desert soil of about 60%. A pyrosequencing-based analysis of the 16S rRNA gene regions showed higher diversity in agricultural than in desert soil (Shannon diversity indices: 11.21/7.90), and displayed structural differences. The proportion of Firmicutes in field soil was significantly higher (37%) than in the desert (11%). Bacillus and Paenibacillus play the key role: they represented 96% of the antagonists towards phytopathogens, and identical 16S rRNA sequences in the amplicon library and for isolates were detected. The proportion of antagonistic strains was doubled in field in comparison to desert soil (21.6%/12.4%); disease-suppressive bacteria were especially enriched in plant roots. On the opposite, several extremophilic bacterial groups, e.g., Acidimicrobium, Rubellimicrobium and Deinococcus-Thermus, disappeared from soil after agricultural use. The N-fixing Herbaspirillum group only occurred in desert soil. Soil bacterial communities were strongly driven by the a-biotic factors water supply and pH.

Conclusions/Significance

After long-term farming, a drastic shift in the bacterial communities in desert soil was observed. Bacterial communities in agricultural soil showed a higher diversity and a better ecosystem function for plant health but a loss of extremophilic bacteria. Interestingly, we detected that indigenous desert microorganisms promoted plant health in desert agro-ecosystems.     

Seasonal variations in PCR-DGGE fingerprinted viruses infecting phytoplankton in large and deep peri-alpine lakes

Double-stranded DNA viruses infecting eukaryotic algae (e.g., phycodnaviruses) and cyanobacteria (e.g., cyanophages) are now recognized as widespread and ubiquitous in aquatic environments. However, both the diversity and functional roles of these viruses in fresh waters are still poorly understood. We conducted a year-long study in 2011 of the community structure of planktonic virus groups in the upper lit layer of two important freshwater natural ecosystems in France, Lake Annecy (oligotrophic) and Lake Bourget (oligo-mesotrophic). Using PCR-DGGE to target a number of different structural and functional signature genes, i.e.,g20, g23, psbA, polB, and mcp, the phytoplankton viruses were shown to display temporal and spatial variability. There were marked seasonal changes in community structure for all viral groups in Lake Bourget, but only for T4-like myoviruses and psbA-containing cyanophages in Lake Annecy. The multivariate statistical analyses revealed that (1) various environmental factors can directly or indirectly explain the community structure observed for each phytoplankton viral group, and (2) temporal patterns of T4-like myovirus community structure were similar between the two lakes. In general, our results (1) suggest that the observed algal virus patterns were associated with significant shifts in phytoplankton biomass and/or structure, which in turn were shaped by the abiotic environment, and (2) support the Bank model proposed by Breitbart and Rohwer (Trends Microbiol 13:278–284, 2005). This study provides new evidence that freshwater lakes contain a significant diversity of algal viruses, and that the distribution of these viruses strongly mirrors that of their hosts.     

Dynamics and Distribution of Cyanophages and Their Effect on Marine Synechococcus spp.

Cyanophages infecting marine Synechococcus cells were frequently very abundant and were found in every seawater sample along a transect in the western Gulf of Mexico and during a 28-month period in Aransas Pass, Tex. In Aransas Pass their abundance varied seasonally, with the lowest concentrations coincident with cooler water and lower salinity. Along the transect, viruses infecting Synechococcus strains DC2 and SYN48 ranged in concentration from a few hundred per milliliter at 97 m deep and 83 km offshore to ca. 4 × 10⁵ ml^-1 near the surface at stations within 18 km of the coast. The highest concentrations occurred at the surface, where salinity decreased from ca. 35.5 to 34 ppt and Synechococcus concentrations were greatest. Viruses infecting strains SNC1, SNC2, and 838BG were distributed in a similar manner but were much less abundant (<10 to >5 × 10³ ml^-1). When Synechococcus concentrations exceeded ca. 10³ ml^-1, cyanophage concentrations increased markedly (ca. 10² to > 10⁵ ml^-1), suggesting that a minimum host density was required for efficient viral propagation. Data on the decay rate of viral infectivity d (per day), as a function of solar irradiance I (millimoles of quanta per square meter per second), were used to develop a relationship (d = 0.2610I - 0.00718; r² = 0.69) for conservatively estimating the destruction of infectious viruses in the mixed layer of two offshore stations. Assuming that virus production balances losses and that the burst size is 250, ca. 5 to 7% of Synechococcus cells would be infected daily by viruses. Calculations based on contact rates between Synechococcus cells and infectious viruses produce similar results (5 to 14%). Moreover, balancing estimates of viral production with contact rates for the farthest offshore station required that most Synechococcus cells be susceptible to infection, that most contacts result in infection, and that the burst size be about 324 viruses per lytic event. In contrast, in nearshore waters, where ca. 80% of Synechococcus cells would be contacted daily by infectious cyanophages, only ca. 1% of the contacts would have to result in infection to balance the estimated virus removal rates. These results indicate that cyanophages are an abundant and dynamic component of marine planktonic communities and are probably responsible for lysing a small but significant portion of the Synechococcus population on a daily basis.     

Identification of a Diagnostic Marker To Detect Freshwater Cyanophages of Filamentous Cyanobacteria

Cyanophages are viruses that infect the cyanobacteria, globally important photosynthetic microorganisms. Cyanophages are considered significant components of microbial communities, playing major roles in influencing host community diversity and primary productivity, terminating cyanobacterial water blooms, and influencing biogeochemical cycles. Cyanophages are ubiquitous in both marine and freshwater systems; however, the majority of molecular research has been biased toward the study of marine cyanophages. In this study, a diagnostic probe was developed to detect freshwater cyanophages in natural waters. Oligonucleotide PCR-based primers were designed to specifically amplify the major capsid protein gene from previously characterized freshwater cyanomyoviruses that are infectious to the filamentous, nitrogen-fixing cyanobacterial genera Anabaena and Nostoc. The primers were also successful in yielding PCR products from mixed virus communities concentrated from water samples collected from freshwater lakes in the United Kingdom. The probes are thought to provide a useful tool for the investigation of cyanophage diversity in freshwater environments.     

Selection and characterization of cyanophage resistance in marine Synechococcus strains

Marine viruses are an important component of the microbial food web, influencing microbial diversity and contributing to bacterial mortality rates. Resistance to cooccurring cyanophages has been reported for natural communities of Synechococcus spp.; however, little is known about the nature of this resistance. This study examined the patterns of infectivity among cyanophage isolates and unicellular marine cyanobacteria (Synechococcus spp.). We selected for phage-resistant Synechococcus mutants, examined the mechanisms of phage resistance, and determined the extent of cross-resistance to other phages. Four strains of Synechococcus spp. (WH7803, WH8018, WH8012, and WH8101) and 32 previously isolated cyanomyophages were used to select for phage resistance. Phage-resistant Synechococcus mutants were recovered from 50 of the 101 susceptible phage-host pairs, and 23 of these strains were further characterized. Adsorption kinetic assays indicate that resistance is likely due to changes in host receptor sites that limit viral attachment. Our results also suggest that receptor mutations conferring this resistance are diverse. Nevertheless, selection for resistance to one phage frequently resulted in cross-resistance to other phages. On average, phage-resistant Synechococcus strains became resistant to eight other cyanophages; however, there was no significant correlation between the genetic similarity of the phages (based on g20 sequences) and cross-resistance. Likewise, host Synechococcus DNA-dependent RNA polymerase (rpoC1) genotypes could not be used to predict sensitivities to phages. The potential for the rapid evolution of multiple phage resistance may influence the population dynamics and diversity of both Synechococcus and cyanophages in marine waters.     

Prevalence of Viral Photosynthetic and Capsid Protein Genes from Cyanophages in Two Large and Deep Perialpine Lakes

Cyanophages are important components of aquatic ecosystems, but their genetic diversity has been little investigated in freshwaters. A yearlong survey was conducted in surface waters of the two largest natural perialpine lakes in France (Lake Annecy and Lake Bourget) to investigate part of this cyanophage diversity through the analysis of both structural (e.g., g20) and functional (e.g., psbA) genes. We found that these cyanophage signature genes were prevalent throughout the year but that the community compositions of g20 cyanomyoviruses were significantly different between the two lakes. In contrast, psbA-containing cyanophages seemed to be more similar between the two ecosystems. We also found that a large proportion of g20 sequences grouped with cyanomyophage isolates. psbA sequences, belonging to phages of Synechococcus spp., were characterized by distinct triplet motifs (with a novel viral triplet motif, EFE). Thus, our results show that cyanophages (i) are a diverse viral community in alpine lakes and (ii) are clearly distinct from some other freshwater and marine environments, suggesting the influence of unique biogeographic factors.     

Marine viruses,a genetic reservoir revealed by targeted viromics

Metagenomics has opened new windows on investigating viral diversity and functions. Viromic studies typically require large sample volumes and filtration through 0.2 μm pore-size filters, consequently excluding or under-sampling tailed and very large viruses. We have optimized a targeted viromic approach that employs fluorescence-activated sorting and whole genome amplification to produce dsDNA-enriched libraries from discrete viral populations from a 1-ml water sample. Using this approach on an environmental sample from the Patagonian Shelf, we produced three distinct libraries. One of the virus libraries was dominated (79.65% of sequences with known viral homology) by giant viruses from the Mimiviridae and Phycodnaviridae families, while the two other viromes were dominated by smaller phycodnaviruses, cyanophages and other bacteriophages. The estimated genotypic richness and diversity in our sorted viromes, with 52–163 estimated genotypes, was much lower than in previous virome reports. Fragment recruitment of metagenome reads to selected reference viral genomes yields high genome coverage, suggesting little amplification and sequencing bias against some genomic regions. These results underscore the value of our approach as an effective way to target and investigate specific virus groups. In particular, it will help reveal the diversity and abundance of giant viruses in marine ecosystems.     

Phylogenetic Diversity of Marine Cyanophage Isolates and Natural Virus Communities as Revealed by Sequences of Viral Capsid Assembly Protein Gene g20

In order to characterize the genetic diversity and phylogenetic affiliations of marine cyanophage isolates and natural cyanophage assemblages, oligonucleotide primers CPS1 and CPS8 were designed to specifically amplify ca. 592-bp fragments of the gene for viral capsid assembly protein g20. Phylogenetic analysis of isolated cyanophages revealed that the marine cyanophages were highly diverse yet more closely related to each other than to enteric coliphage T4. Genetically related marine cyanophage isolates were widely distributed without significant geographic segregation (i.e., no correlation between genetic variation and geographic distance). Cloning and sequencing analysis of six natural virus concentrates from estuarine and oligotrophic offshore environments revealed nine phylogenetic groups in a total of 114 different g20 homologs, with up to six clusters and 29 genotypes encountered in a single sample. The composition and structure of natural cyanophage communities in the estuary and open-ocean samples were different from each other, with unique phylogenetic clusters found for each environment. Changes in clonal diversity were also observed from the surface waters to the deep chlorophyll maximum layer in the open ocean. Only three clusters contained known cyanophage isolates, while the identities of the other six clusters remain unknown. Whether or not these unidentified groups are composed of bacteriophages that infect different Synechococcus groups or other closely related cyanobacteria remains to be determined. The high genetic diversity of marine cyanophage assemblages revealed by the g20 sequences suggests that marine viruses can potentially play important roles in regulating microbial genetic diversity.     

Metagenomic and Small-Subunit rRNA Analyses Reveal the Genetic Diversity of Bacteria, Archaea, Fungi, and Viruses in Soil

Recent studies have highlighted the surprising richness of soil bacterial communities; however, bacteria are not the only microorganisms found in soil. To our knowledge, no study has compared the diversities of the four major microbial taxa, i.e., bacteria, archaea, fungi, and viruses, from an individual soil sample. We used metagenomic and small-subunit RNA-based sequence analysis techniques to compare the estimated richness and evenness of these groups in prairie, desert, and rainforest soils. By grouping sequences at the 97% sequence similarity level (an operational taxonomic unit [OTU]), we found that the archaeal and fungal communities were consistently less even than the bacterial communities. Although total richness levels are difficult to estimate with a high degree of certainty, the estimated number of unique archaeal or fungal OTUs appears to rival or exceed the number of unique bacterial OTUs in each of the collected soils. In this first study to comprehensively survey viral communities using a metagenomic approach, we found that soil viruses are taxonomically diverse and distinct from the communities of viruses found in other environments that have been surveyed using a similar approach. Within each of the four microbial groups, we observed minimal taxonomic overlap between sites, suggesting that soil archaea, bacteria, fungi, and viruses are globally as well as locally diverse.     

Metagenomic and small-subunit rRNA analyses reveal the genetic diversity of bacteria, archaea, fungi, and viruses in soil

Recent studies have highlighted the surprising richness of soil bacterial communities; however, bacteria are not the only microorganisms found in soil. To our knowledge, no study has compared the diversities of the four major microbial taxa, i.e., bacteria, archaea, fungi, and viruses, from an individual soil sample. We used metagenomic and small-subunit RNA-based sequence analysis techniques to compare the estimated richness and evenness of these groups in prairie, desert, and rainforest soils. By grouping sequences at the 97% sequence similarity level (an operational taxonomic unit [OTU]), we found that the archaeal and fungal communities were consistently less even than the bacterial communities. Although total richness levels are difficult to estimate with a high degree of certainty, the estimated number of unique archaeal or fungal OTUs appears to rival or exceed the number of unique bacterial OTUs in each of the collected soils. In this first study to comprehensively survey viral communities using a metagenomic approach, we found that soil viruses are taxonomically diverse and distinct from the communities of viruses found in other environments that have been surveyed using a similar approach. Within each of the four microbial groups, we observed minimal taxonomic overlap between sites, suggesting that soil archaea, bacteria, fungi, and viruses are globally as well as locally diverse.     

Phylogenetic Diversity of Sequences of Cyanophage Photosynthetic Gene psbA in Marine and Freshwaters

Many cyanophage isolates which infect the marine cyanobacteria Synechococcus spp. and Prochlorococcus spp. contain a gene homologous to psbA, which codes for the D1 protein involved in photosynthesis. In the present study, cyanophage psbA gene fragments were readily amplified from freshwater and marine samples, confirming their widespread occurrence in aquatic communities. Phylogenetic analyses demonstrated that sequences from freshwaters have an evolutionary history that is distinct from that of their marine counterparts. Similarly, sequences from cyanophages infecting Prochlorococcus and Synechococcus spp. were readily discriminated, as were sequences from podoviruses and myoviruses. Viral psbA sequences from the same geographic origins clustered within different clades. For example, cyanophage psbA sequences from the Arctic Ocean fell within the Synechococcus as well as Prochlorococcus phage groups. Moreover, as psbA sequences are not confined to a single family of phages, they provide an additional genetic marker that can be used to explore the diversity and evolutionary history of cyanophages in aquatic environments.