Here a virus, there a virus, everywhere the same virus?

There are an estimated 10(31) viruses on Earth, most of which are phages that infect bacteria. Metagenomic analyses have shown that environmental viral communities are incredibly diverse. There are an estimated 5000 viral genotypes in 200 liters of seawater and possibly a million different viral genotypes in one kilogram of marine sediment. By contrast, some culturing and molecular studies have found that viruses move between different biomes. Together, these findings suggest that viral diversity could be high on a local scale but relatively limited globally. Also, by moving between environments, viruses can facilitate horizontal gene transfer.     

Are we missing half of the viruses in the ocean?

Viruses are abundant in the ocean and a major driving force in plankton ecology and evolution. It has been assumed that most of the viruses in seawater contain DNA and infect bacteria, but RNA-containing viruses in the ocean, which almost exclusively infect eukaryotes, have never been quantified. We compared the total mass of RNA and DNA in the viral fraction harvested from seawater and using data on the mass of nucleic acid per RNA- or DNA-containing virion, estimated the abundances of each. Our data suggest that the abundance of RNA viruses rivaled or exceeded that of DNA viruses in samples of coastal seawater. The dominant RNA viruses in the samples were marine picorna-like viruses, which have small genomes and are at or below the detection limit of common fluorescence-based counting methods. If our results are typical, this means that counts of viruses and the rate measurements that depend on them, such as viral production, are significantly underestimated by current practices. As these RNA viruses infect eukaryotes, our data imply that protists contribute more to marine viral dynamics than one might expect based on their relatively low abundance. This conclusion is a departure from the prevailing view of viruses in the ocean, but is consistent with earlier theoretical predictions.     

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.     

Virus Decay and Its Causes in Coastal Waters

Recent evidence suggests that viruses play an influential role within the marine microbial food web. To understand this role, it is important to determine rates and mechanisms of virus removal and degradation. We used plaque assays to examine the decay of infectivity in lab-grown viruses seeded into natural seawater. The rates of loss of infectivity of native viruses from Santa Monica Bay and of nonnative viruses from the North Sea in the coastal seawater of Santa Monica Bay were determined. Viruses were seeded into fresh seawater that had been pretreated in various ways: filtration with a 0.2-(mu)m-pore-size filter to remove organisms, heat to denature enzymes, and dissolved organic matter enrichment to reconstitute enzyme activity. Seawater samples were then incubated in full sunlight, in the dark, or under glass to allow partitioning of causative agents of virus decay. Solar radiation always resulted in increased rates of loss of virus infectivity. Virus isolates which are native to Santa Monica Bay consistently degraded more slowly in full sunlight in untreated seawater (decay ranged from 4.1 to 7.2% h(sup-1)) than nonnative marine bacteriophages which were isolated from the North Sea (decay ranged from 6.6 to 11.1% h(sup-1)). All phages demonstrated susceptibility to degradation by heat-labile substances, as heat treatment reduced the decay rates to about 0.5 to 2.0% h(sup-1) in the dark. Filtration reduced decay rates by various amounts, averaging 20%. Heat-labile, high-molecular-weight dissolved material (>30 kDa, probably enzymes) appeared responsible for about 1/5 of the maximal decay. Solar radiation was responsible for about 1/3 to 2/3 of the maximal decay of nonnative viruses and about 1/4 to 1/3 of that of the native viruses, suggesting evolutionary adaptation to local light levels. Our results suggest that sunlight is an important contributing factor to virus decay but also point to the significance of particles and dissolved substances in seawater.     

Efficient dilution-to-extinction isolation of novel virus–host model systems for fastidious heterotrophic bacteria

Microbes and their associated viruses are key drivers of biogeochemical processes in marine and soil biomes. While viruses of phototrophic cyanobacteria are well-represented in model systems, challenges of isolating marine microbial heterotrophs and their viruses have hampered experimental approaches to quantify the importance of viruses in nutrient recycling. A resurgence in cultivation efforts has improved the availability of fastidious bacteria for hypothesis testing, but this has not been matched by similar efforts to cultivate their associated bacteriophages. Here, we describe a high-throughput method for isolating important virus–host systems for fastidious heterotrophic bacteria that couples advances in culturing of hosts with sequential enrichment and isolation of associated phages. Applied to six monthly samples from the Western English Channel, we first isolated one new member of the globally dominant bacterial SAR11 clade and three new members of the methylotrophic bacterial clade OM43. We used these as bait to isolate 117 new phages, including the first known siphophage-infecting SAR11, and the first isolated phage for OM43. Genomic analyses of 13 novel viruses revealed representatives of three new viral genera, and infection assays showed that the viruses infecting SAR11 have ecotype-specific host ranges. Similar to the abundant human-associated phage ɸCrAss001, infection dynamics within the majority of isolates suggested either prevalent lysogeny or chronic infection, despite a lack of associated genes, or host phenotypic bistability with lysis putatively maintained within a susceptible subpopulation. Broader representation of important virus–host systems in culture collections and genomic databases will improve both our understanding of virus–host interactions, and accuracy of computational approaches to evaluate ecological patterns from metagenomic data.Subject terms: Bacteriophages, Microbial ecology     

Contrasting genomic patterns and infection strategies of two co‐existing Bacteroidetes podovirus genera

Bacterial viruses (phages) are abundant, ecologically important biological entities. However, our understanding of their impact is limited by model systems that are primarily not well represented in nature, e.g. Enterophages and their hosts. Here, we investigate genomic characteristics and infection strategies among six aquatic Bacteroidetes phages that represent two genera of exceptionally large (～70–75 kb genome) podoviruses, which were isolated from the same seawater sample using Cellulophaga baltica as host. Quantitative host range studies reveal that these genera have contrasting narrow (specialist) and broad (generalist) host ranges, with one‐step growth curves revealing reduced burst sizes for the generalist phages. Genomic comparisons suggest candidate genes in each genus that might explain this host range variation, as well as provide hypotheses about receptors in the hosts. One generalist phage, φ38:1, was more deeply characterized, as its infection strategy switched from lytic on its original host to either inefficient lytic or lysogenic on an alternative host. If lysogenic, this phage was maintained extrachromosomally in the alternative host and could not be induced by mitomycin C. This work provides fundamental knowledge regarding phage‐host ranges and their genomic drivers while also exploring the ‘host environment’ as a driver for switching phage replication mode.     

Determination of bacterial number and biomass in the marine environment.

Three techniques for the measurement of bacterial numbers and biomass in the marine environment are described. Two are direct methods for counting bacteria. The first employs an epifluorescence microscope to view bacteria that have been concentrated on membrane filters and stained with acridine orange. The second uses a transmission electron microscope for observing replicas of bacteria that are concentrated on membrane filters. The other technique uses Limulus amebocyte lysate, an aqueous extract from the amebocytes of the horseshoe crab, Limulus polyphemus, to quantitate lipopolysaccharide (LPS) in seawater samples. The biomass of gram-negative (LPS containing) bacteria was shown to be related to the LPS content of the samples. A factor of 6.35 was determined for converting LPS to bacterial carbon.     

Determination of bacterial number and biomass in the marine environment.

Three techniques for the measurement of bacterial numbers and biomass in the marine environment are described. Two are direct methods for counting bacteria. The first employs an epifluorescence microscope to view bacteria that have been concentrated on membrane filters and stained with acridine orange. The second uses a transmission electron microscope for observing replicas of bacteria that are concentrated on membrane filters. The other technique uses Limulus amebocyte lysate, an aqueous extract from the amebocytes of the horseshoe crab, Limulus polyphemus, to quantitate lipopolysaccharide (LPS) in seawater samples. The biomass of gram-negative (LPS containing) bacteria was shown to be related to the LPS content of the samples. A factor of 6.35 was determined for converting LPS to bacterial carbon.     

In situ survival of Vibrio cholerae and Escherichia coli in tropical coral reefs.

Vibrio cholerae and Escherichia coli were inoculated into membrane diffusion chambers and placed around two small coral reef islands in Puerto Rico and monitored for 5 days. Several chambers were also buried in the sands of one of the reefs. Both E. coli and V. cholerae densities declined by 2 orders of magnitude, as measured by direct particle counts with a Coulter Counter (Coulter Electronics, Inc., Hialeah, Fla.). However, the density of neither bacteria changed dramatically when the same samples were analyzed by epifluorescent direct counts. Differences in the two direct count methods were accounted for by changes in cell morphology that occurred in both bacteria after exposure to seawater. Morphological changes occurred more rapidly in E. coli compared with those in V. cholerae. Bacteria in chambers exposed to sediment did not show significant changes in morphology and had only a slight decline in density. Physiological activity declined by more than 40% for both bacteria within 24 h. The decline in activity was less severe in the sediments. Tropical coral reef sands and turtle grass beds were shown to be less stressful environments for V. cholerae and E. coli than would have been predicted from temperature and microcosm studies. V. cholerae can survive the in situ conditions of a tropical coral reef and could become a source of bacterial contamination for fish and shellfish in this environment. The simultaneous monitoring of E. coli levels established that this bacteria can not be used as an indicator of V. cholerae or other fecal-borne pathogens in coral reef environments because of the greater stress these environments put on E. coli. Both bacteria could be of greater public health importance in tropical marine areas than previously imagined.     

In situ survival of Vibrio cholerae and Escherichia coli in tropical coral reefs

Vibrio cholerae and Escherichia coli were inoculated into membrane diffusion chambers and placed around two small coral reef islands in Puerto Rico and monitored for 5 days. Several chambers were also buried in the sands of one of the reefs. Both E. coli and V. cholerae densities declined by 2 orders of magnitude, as measured by direct particle counts with a Coulter Counter (Coulter Electronics, Inc., Hialeah, Fla.). However, the density of neither bacteria changed dramatically when the same samples were analyzed by epifluorescent direct counts. Differences in the two direct count methods were accounted for by changes in cell morphology that occurred in both bacteria after exposure to seawater. Morphological changes occurred more rapidly in E. coli compared with those in V. cholerae. Bacteria in chambers exposed to sediment did not show significant changes in morphology and had only a slight decline in density. Physiological activity declined by more than 40% for both bacteria within 24 h. The decline in activity was less severe in the sediments. Tropical coral reef sands and turtle grass beds were shown to be less stressful environments for V. cholerae and E. coli than would have been predicted from temperature and microcosm studies. V. cholerae can survive the in situ conditions of a tropical coral reef and could become a source of bacterial contamination for fish and shellfish in this environment. The simultaneous monitoring of E. coli levels established that this bacteria can not be used as an indicator of V. cholerae or other fecal-borne pathogens in coral reef environments because of the greater stress these environments put on E. coli. Both bacteria could be of greater public health importance in tropical marine areas than previously imagined.     

Biological quality of seawater evaluated in situ with embryo-larval test of Crassostrea gigas and Mytilus galloprovincialis]

Embryos and larvae of bivalves are frequently used in marine ecotoxicology for the purpose of assessing seawater quality, because they are very sensitive to pollutants and provide rapid responses. Laboratory studies, however, cannot accurately simulate natural conditions. We conducted bivalve embryo-larval studies in situ at the marina of Arcachon (south-west French Atlantic coast), in order to assess 'biological quality' of the water. One experiment conducted in winter 1999 (temperatures of 10 degrees C) with embryos of the Mediterranean mussel, Mytilus galloprovincialis, has shown that such tests are practicable in winter at low temperatures. This study did not show any deterioration in 'biological quality' of the water. Four series of experiments were subsequently performed during summer 2000 (ambient water temperatures of 19 to 22.4 degrees C) with embryos of the Japanese oyster, Crassostrea gigas. The results show that the 'sea water biological quality' deteriorates from the port entrance towards its inner part. To our knowledge, this is the first investigation of the marine environment in which bivalve embryos have been used in situ. They are very suitable for this type of study, because bivalve embryos and larvae are more sensitive to pollutants than the adults, and also because they belong to euryhaline species and the embryos tolerate summer temperatures (both species) as well as winter temperatures (mussels), allowing biomonitoring to be conducted all over the year.     

Significance of Bacteriophages for Controlling Bacterioplankton Growth in a Mesotrophic Lake

Bacterium-specific viruses have attracted much interest in aquatic microbial ecology because they have been shown to be about 10 times more abundant than planktonic bacteria. So far most of the studies of interactions of planktonic bacteria and viruses have been done in marine environments, and very little is known about these interactions in lakes. Therefore, we studied phage proliferation in Lake Constance, a large mesotrophic lake in Germany. We enumerated bacteria and quantified the fraction of bacteria with mature intracellular phage particles and the number of free viruses by transmission electron microscopy. Between the end of March and early August 1992, peaks of bacterial abundance were followed in 1 to 2 weeks by peaks in the fraction of bacteria containing visible phage particles (0 to 1.7%) and in the number of free viruses (1 x 10(sup7) to 4 x 10(sup7) ml(sup-1)). We estimated that 1 to 17% +/- 12% of all bacteria were phage infected, implying that phage-induced mortality was <34% +/- 24% of total mortality. A direct comparison between phage-induced mortality, the net decrease of bacterial numbers, and bacterial growth rates indicated that phage-induced mortality accounted for <11% of total bacterial mortality during the phytoplankton spring bloom and 18 to 21% following the bloom. Estimated burst sizes ranged from 21 to 121 phages. Phage production rates of 0.5 x 10(sup6) to 2.5 x 10(sup6) ml(sup-1) day(sup-1) accounted for 70 to 380% of the observed net increase rates of free phages, implying high rates of simultaneous phage decay. The cyclic dynamics between bacteria and phages and the varying size structure of the intracellular mature phage particles suggested that phage infection was important in structuring the bacterial host assemblage during the study period.     

Effect of distance from the polluting focus on relative concentrations of Bacteroides fragilis phages and coliphages in mussels.

Concentrations of fecal bacteria, somatic and F-specific coliphages, and phages infecting Bacteroides fragilis in naturally occurring black mussels (Mytilus edulis) were determined. Mussels were collected over a 7-month period at four sampling sites with different levels of fecal pollution. Concentrations of both fecal bacteria and bacteriophages in mussel meat paralleled the concentration of fecal bacteria in the overlying waters. Mussels bioaccumulated efficiently, although with different efficiencies, all of the microorganisms studied. Ratios comparing the levels of microorganisms in mussels were determined. These ratios changed in mussels collected at the different sites. They suggest that bacteriophages infecting B. fragilis and somatic coliphages have the lowest decay rates among the microorganisms studied, with the exception of Clostridium perfringens. On the contrary, concentrations of F-specific coliphages showed a greater rate of decay than the other bacteriophages at sites more distant from the focus of contamination. Additionally, levels of enteroviruses were studied in a number of samples, and in these samples, the B. fragilis bacteriophages clearly outnumbered the enteroviruses. The results of this study indicate that, under the environmental conditions studied, the fate of phages infecting B. fragilis released into the marine environment resembles that of human viruses more than any other microorganism examined.     

Efficient method to isolate and purify viruses of bacteria from marine environments

Aim:  To isolate viruses of specific heterotrophic bacterial strains from marine environments using a host addition/virus amplification protocol (HAVAP) for use in phage/host systems.
Methods and Results:  Bacteria-free seawater samples containing natural viruses assemblages were inoculated with a single laboratory grown bacterial host of interest in a nutrient-enriched [peptone, Fe(III) and yeast extract] seawater suspension. These conditions enhanced the replication of only those virus(s) capable of infecting the host bacterium. After incubation, free viruses were recovered at concentrations ranging 10⁵–10¹⁰ infectious virus particles per ml of seawater. Using this approach, 15 viruses were isolated and represented 12 unique phage/host systems. Two of the hosts tested were infected by more than one virus.
Conclusions:  Isolation of high concentrations of specific viruses is possible even if their initial concentrations in native waters are low. This approach allows the recovery of phage/host systems that may not be numerically dominant.
Significance and Impact of the Study:  This host enrichment protocol for virus detection and isolation is well-suited for aquatic viral ecology studies that require phage/host systems.     

Comparison between two methods of assaying relative microbial activity in marine environments.

Two methods for determining relative microbial activity in the marine environment were compared. In one method, a single concentration of a labeled substrate was used to calculate rates of substrate utilization; in the other, multiple concentrations of the same substrate (heterotrophic activity method) were used to calculate maximum potential substrate utilization rates. These studies were made on 232 seawater and 79 sediment samples taken from a variety of marine environments. The highest correlations between these two methods were seen in the sediment samples tested. The lowest correlation coerfficient seen in the sediment samples was 0.90, and the highest was 0.98. In seawater samples (six studies), the lowest correlation coefficient was 0.77 and the highest was 0.95. The correlation between these two methods was also substrate concentration dependent. Higher correlation coefficients were observed when higher substrate concentrations were used. Under certain conditions, these two methods appear to be comparable for estimating relative levels of microbial activity in the marine environment.     

Comparison between two methods of assaying relative microbial activity in marine environments.

Two methods for determining relative microbial activity in the marine environment were compared. In one method, a single concentration of a labeled substrate was used to calculate rates of substrate utilization; in the other, multiple concentrations of the same substrate (heterotrophic activity method) were used to calculate maximum potential substrate utilization rates. These studies were made on 232 seawater and 79 sediment samples taken from a variety of marine environments. The highest correlations between these two methods were seen in the sediment samples tested. The lowest correlation coerfficient seen in the sediment samples was 0.90, and the highest was 0.98. In seawater samples (six studies), the lowest correlation coefficient was 0.77 and the highest was 0.95. The correlation between these two methods was also substrate concentration dependent. Higher correlation coefficients were observed when higher substrate concentrations were used. Under certain conditions, these two methods appear to be comparable for estimating relative levels of microbial activity in the marine environment.