Microbial Observatory for Virioplankton Ecology

Microbial Observatory for Virioplankton Ecology (MOVE)

Data

An informed understanding of the impact of viral infection on microbial communities requires accurate estimation of the proportion of bacterial and phytoplankton biomass lost to viral infection. Of all the routes through which microbial biomass is transformed into dissolved organic matter perhaps the most efficient is viral infection, as all cellular components released during lysis, including viruses themselves, can be considered DOM. Thus, a critical parameter to determining the role of viral lysis in the marine carbon cycle is viral production. Most of the methods utilized for estimation of viral production rely on one or more poorly constrained conversion factors (e.g., burst size, latent period, generation time, or efficiency of tracer incorporation) in the calculation. Recently, two incubation-based methods for estimation of viral production were introduced which alleviate many of the problems inherent to other methods. Over the course of several cruises we have evaluated the efficacy of the fluorescently-labeled virus (FLV) and dilution approaches for estimates of viral production. The FLV approach is based on the well established tracer-dilution model in which changes in the ratio of total viruses to tracer viruses is used as an indicator of viral production and decay. The dilution approach, teleologically similar to approaches used for estimating phytoplankton growth rates, involves incubation of water samples in which the abundance of viruses has been reduced to at least 10% of ambient concentration.

While the dilution approach is technically and logistically less complicated than the FLV tracer method, estimates of viral production obtained are wildly inaccurate as evidenced by turnover time estimations. The source of this error may be the large loss of bacteria (ca. 80%) during vacuum filtration. Loss of bacteria necessitates use of a correction factor which greatly increases viral production estiamtes. We are currently investigating alternate means of viral dilution.

Although viruses are known to be abundant and dynamic in the Chesapeake Bay and other aquatic environments, the interaction between virus and its host is less understood due to the lack of knowledge on the phylogenetic diversity of virus and its host. Cyanomyophages are known to be dominant among cyanophage isolates that infect Synechococcus spp. To better understand the genetic diversity and population dynamics of cyanophages in the estuarine water, the viral capsid assembly protein gene g20 can be used as a marker molecule to monitor genetic variations of natural cyanomyophage communities in Chesapeake Bay. We’re investigating the distribution frequency of various genotypes of cyanophages in the Chesapeake Bay using PCR and TRFLP (Terminal Restriction Fragment Length Polymorphism) methods based on g20 sequences. T-RFLP has proven to be a rapid means of obtaining the genetic fingerprints of cyanomyophage communities in estuarine environments. Coordinately, Denaturing Gradient Gel Electrophoresis (DGGE) based on 16S rDNA gene sequence was applied to analyze the temporal and spatial variation of bacterial communities.

Flowchart of methods for investigation of cyanomyophage
community dynamics in the Bay.

       Phylogenetic affiliation of the Synechococcus isolates (IH24, IH26, IH40
        and IH44) from Chesapeake Bay with other known Synechococcus spp.
        strains based on RuBisCo gene sequences. Strains in red are phycoerythrin
        type while strains in green are phycocyanin type.

       Phylogenetic affiliation of the representative g20 clones (OTU1-OTU15)
       recovered from the Chesapeake Bay (blue type) with cyanomyophage isolates
       (red type) and representative clones from various natural environments
       previously studied .

Both TRFLP (g20 gene) and DGGE (16S rDNA) profiles indicate that cyanophage and bacterial communities are more dynamic temporally than spatially in the Chesapeake Bay.

While diatoms are the most conspicuous component of the phytoplankton assemblage in the main Bay dinoflagellates are key players in the annual biological cycle of the Chesapeake. Blooms of an array of dinoflagellate species occur throughout the Bay, but are especially pronounced along the western shore north of the Patuxent River. Even winter blooms of dinoflagellates including Heterocapsa spp. are not uncommon. Representative bloom-forming dinoflagellates in summer include Gyrodinium spp. and Scrippsiella spp. in the upper Bay and oligohaline portions of tributaries, Akashiwo sanguinea and Phaepolykrikos spp. in mesohaline waters, and Ceratium furca, Prorocentrum micans, and Cochliodinium heterolobatum in polyhaline regions and tributaries of the lower Bay.

Herbivory appears to be of minor importance in regulating the appearance or persistence of summer dinoflagellate blooms. Similarly, grazing has little impact on cyanobacterial blooms that form in oligohaline water of tributaries like the Potomac River. Ironically, no viral-host systems for viruses infecting dinoflagellates have been brought into culture. An important goal of MOVE is isolation of viruses infecting important dinoflagellate species occurring in Chesapeake Bay. The protistian culture collection maintained in the Coats laboratory contains representative species of these bloom forming microalgae including P. minimum, P. micans, Gymnodinium instriatum, Karlodinium micrum, Ceratium furca, and Akashiwo sanguinea).

Plaque assay showing the cyanophages which infect the Chesapeake Bay indigenous Synechococcus isolate IH44.

Cyanophages isolated from the Chesapeake Bay that infect indigenous Synechococcus isolates. The g20 amplicon was only obtained from the cyanomyophage IH44Ø9.

Conclusions

Process
The FLV approach is satisfying in that both decay and production estimates are obtained; however, these two estimates are often significantly different from one another. Preparation of tracer viruses which represent a viral community equivalent to those in situ is logistically difficult. This method requires longer incubations and significantly more microscope counts than the dilution approach.

The rate of virus reappearance is then used as a direct estimate of viral production. Once we have established which method is most suitable for estimation of viral production, this method will be used routinely for examination of changes in viral activity over the annual biological cycle of the Chesapeake.

Diversity and Composition
Unique and diverse g20 sequences were found in the Chesapeake Bay. Only one of 15 g20 genotypes was clustered with the known cyanomyophage isolates. Most g20 genotypes were not related to the g20 clonal sequences recovered from open ocean water samples, suggesting that Chesapeake Bay Synechococcus populations and the phage which infect them differ from those in the open ocean.

T-RFLP and DGGE profiles demonstrate that cyanomyophage populations and bacterial communities in the Bay changed more over seasonal rather than geographic scales. Cyanophages could play a significant role on regulating the structure of Synechococcus populations in Chesapeake Bay.

Isolation and characterization of cyanophages infecting Synechococcus strains in Chesapeake Bay should provide new insight on co-variation of virus-host populations.

We are currently developing a DGGE method based on the rbcL gene to specifically monitor the dynamics of Synechococcus populations in the Bay.

Environmental proteomics is working for the Chesapeake Bay microbial communities. Characterization of bacterial proteins recovered from seawater could shed light on their important microgeochemical function.