Viruses are all around us. The first indication of the amazing abundance of viruses within marine and freshwater environments came through direct observations of water samples using powerful

electron microscopes. The productive waters of estuaries, like the Chesapeake Bay, typically

contain around 1 to 10 million virus particles per drop (milliliter). This means that the Chesapeake,

virus particles. Large numbers such as these

are typically used in astronomy when

describing the number of stars in the Milky

of earth put together, microbial ecologists

estimate that our biosphere contains around

used to describe the amount of stars in the

universe, can also be used to describe the

abundance of viruses on planet Earth.

 

 

We now refer to free-floating (planktonic) viruses

within natural waters as virioplankton. This is

similar to calling planktonic bacteria-bacterio-

plankton or planktonic unicellular algae-phyto-

plankton. The discovery, over 15 years ago, of

the vast abundance of virioplankton lead to two central questions on the influence of viruses on co-existing populations of bacterio- and phytoplankton:

 

1) How does viral infection influence the net productivity (growth) of bacterio- and phytoplankton?

2) How do viruses alter the genetic composition of bacterio- and phytoplankton communities?

 

 

 

 

 

    

    The discovery and later acknowledgement that viruses are the most abundant class of microorganisms in aquatic environments is perhaps the best example of our ignorance as to the true nature of microbial communities. In the years since this epiphany, we have learned that in some environments viral infection accounts for a significant proportion of daily bacterial mortality; making viral lysis the most efficient means of transforming biomass into dissolved organic matter.  Moreover, as viral infection is generally very host-specific, this process may be important in shaping the composition and diversity of co-existing plankton communities. To date the majority of virio-

plankton studies have concentrated on methods development; simpleenumeration and observational studies; and, to a more limited extent, examination of virioplankton diversity and com-

munity structure. In only a few cases, mainly focusing on enumeration, have virioplankton populations been examined in the context of an annual cycle. An important goal of this microbial observatory is to investigate the role of viruses in the annual biological cycle of a temperate estuary, the Chesapeake Bay. In this effort we will apply a suite of recently developed analyses to characterize the productivity, diversity and composition of Chesapeake Bay virioplankton over annual cycles. Coincident with these analyses will be efforts to bring novel phycoviruses and cyanophages into culture.

 

     Initial investigations have focused on comparative studies of three methods for estimation of viral production (TdR incorporation, fluorescently-labeled virus (FLV) tracer, and dilution); isolation of cyanophage infecting strains of the unicellular cyanobacteria, Synechoccocus; and characterization of cyanophage and bacterioplankton populations using culture-independent approaches. Estimates of virioplankton production varied widely between the methods ranging from 0.1 to 7 x 106 viruses ml-1 h-1 for the same water sample. From these initial trials it appears the dilution approach will suffice for routine estimation of virioplankton production, however, each method has significant shortcomings. Several phages infecting green strains of Synechoccocus from Chesapeake Bay were isolated and characterized according to microscopy, plaque morphology and molecular phylogeny of the g20 gene. Terminal restriction fragment length polymorphism (TRFLP) analysis of g20 amplicons from cyanophage in Baltimore Harbor indicated dramatic seasonal variation in populations of these viruses. Similarly, populations of bacterioplankton showed strong seasonality in community structure as assessed by 16S rRNA denaturing gradient gel electrophoresis (DGGE). Finally, we are developing a novel approach to phenotypic characterization of bacterioplankton using proteomic tools. From a combination of 2-D gel electrophoresis and MALDI TOF MS several proteins were identified from bacterioplankton concentrates collected from Chesapeake Bay water samples. It is our hope that this approach will eventually lead to new insights on the connection between the structure of microbial communities (viruses, bacteria and phytoplankton) and the biogeochemical function they perform.