Viruses in the marine environment : abundance, distribution and contribution to the dissolved DNA pool

Viruses in the marine environment : abundance, distribution and contribution to the dissolved DNA pool

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Viruses in the marine environment : abundance, distribution and contribution to the dissolved DNA pool
Jiang, Chenyang
Place of Publication:
Tampa, Florida
University of South Florida
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ix, 103 leaves : ill. ; 29 cm.


Subjects / Keywords:
Viruses -- Ecology ( lcsh )
Viral pollution of water ( lcsh )
Lysogeny ( lcsh )
Dissertations, Academic -- Marine science -- Masters -- USF ( FTS )


General Note:
Thesis (M.S.)--University of South Florida, 1993. Includes bibliographical references (leaves 93-103).

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University of South Florida
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Universtity of South Florida
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
029496095 ( ALEPH )
29203752 ( OCLC )
F51-00103 ( USFLDC DOI )
f51.103 ( USFLDC Handle )

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VIRUSES IN THE MARINE ENVIRONMENT ABUNDANCE, DISTRIBUTION AND CONTRIBUTION TO THE DISSOLVED DNA POOL by CHENY ANG JIANG A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Marine Science University of South Florida May 1993 Major Professor : John H. Paul, Ph. D.


Graduate School University of South Florida Tampa, Florida CERTIFICATE OF APPROVAL Master's Thesis This is to certify that the Master' s Thesis of CHENY ANG JIANG with a major in Marine Science has been approved by the Examining Committee on May 4, 1993 as satisfactory for the thesis requirement for the Master of Science degree Examining Committee: hotessor: John H Paul, Ph. D. --,...---"1<:'""':7"-----=-=----. ili.emtSer: Jo9PB.Rose, Ph. D. Mefilber: Gabriel A. VargoJh. D


Chenyang Jiang 1993 All Rights Reserved


DEDICATION In fond memory, of my Grandfather, whose love and encouragement made me strong and determined when the going was rough and tough.


ACKNOWLEDGMENTS I wish to express my gratitude for my major professor Dr. John Paul w ithout whose concerned guidance, this thesis would not have come to f ruition. Special thanks to Dr. Joan Rose, Dr. Gabriel Vargo, Betty Loraamm, Jonathan Rast and crew members of the R/V Pelican, R/V Hatteras and R/V Bellows for all the help throughout this project. I will forever be appreciat ive of the helps from Scott Pichard, Marc Frischer Jennefier Thurmond, Chr i s Kellogg, Yanti an Lu, Lin Q i ao and many other friends. Their every smile has been a encouragement to me, helping me live through three years in the United States of America with happiness I extend my very sincere thanks.


TABLE OF CONTENTS LIST OF TABLES iii LIST OF FIGURES v ABSTRACT vii CHAPTER 1. INTRODUCTION 1 Viruses in Marine Environment 1 Methods for Detection of Marine Viruses 2 The Role of Viruses in Aquatic Ecosystems 6 Ecology of Marine Bacteriophage 1 0 The Goal of This Research 13 CHAPTER 2 MATERIALS AND METHODS 15 Phage and Bacterial Strains 16 Recovery of Phage and Calf Thymus DNA from Artificial Seawater 1 5 Field Sampling Sites 16 Effect of Prefiltration on Viral Recovery 25 Concentration of Samples 25 Direct Enumeration of Viruses 32 Measurement of Dissolved DNA 33 Separation of Soluble DNA and Viral DNA by Ultracentrifugation 34 Chemical Treatment of VFF-concentrated Dissolved DNA Samples and T2 Phage Lysates 34 DNA Measurement in Purified Viruses 35 Selection of Lysogenic Bacteria from Marine Isolates 37 Statistical Analysis 37 CHAPTER 3. RESULTS 38


Filtration Efficiency for Dissolved DNA and Viruses 38 Evaluation of Enumeration Techniques 41 Viral Abundance in Several Subtropical Marine Environments 44 Seasonal and Diel Viral Abundance in Tampa Bay 59 Separation of Viral DNA from Soluble DNA 64 Occurrence of Lysogeny Amongst Marine Bacterial Isolates 78 CHAPTER 4 DISCUSSIONS AND CONCLUSIONS 84 LIST OF REFERENCES 93 ii


LIST OF TABLES Table 1 Station Locations and Sampling Dates During the R/V Pelican Cruise of 1990. 20 Table 2 Station Locations and Sampl i ng Dates During the R/V Hatteras Cruise of 1990. 21 Table 3 Station Locations and Sampling Dates During the R/V Bellows Cruise of 1991 21 Table 4A. Station Locations and Sampling Dates During the R/V Pelican Cruise of 1992. 22 Table 4B. Station Locations and Sampling Depths along a Transect During the R/V Pelican Cru i se of 1992. 23 Table 5 Comparison of Filtration Efficiency of the Membrex VFF Benchmark, Membrex VFF Pacesetter and Amicon DC-1 0 Systems for Concentration of Dissolved DNA and Viruses From Sterile Artificial Seawater. 39 Table 6. Comparison of Membrex VFF Benchmark Membrex VFF Pacesetter and Am icon SWF DC-1 0 System for Concentration of Environmental Viral Populations 40 Table 7 Comparison of Methods to Enumerate Phage Particles. 42 Table 8 Effect of 0.2 pm Filtration on Recovery of Viruses Prior to VFF Concentration. 44 Table 9 Viral Abundance in Several Subtropical Marine Environments as Determined by Vortex Flow Filtration (VFF) and TEM. 45 Table 10. Partial Correlation Coefficients for the Seasonal Study in Tampa Bay 60 iii


Table 11. Partial Correlation Coefficients for the Diel Study in Tampa Bay 61 Table 12. Effect of Differential Centrifugation on Calf Thymus DNA and T2 phage in Artificial Seawater. 66 Table 13. Effect of Differential Centrifugation on VFFconcentrated Dissolved DNA and Viral Populations. 67 Table 14. Effect of Ethanol Precipitation on Dissolved DNA Measurement. 68 Table 15. Effect of Ethanol Precipitation on Calf Thymus DNA and T2 phage in Artificial Seawater that were Fractionated by Ultracentrifugation. 69 Table 16. Effect of Ethanol Precipitation on VFF concentrated Dissolved DNA Samples that were Fractionated by Ultracentrifugation. 71 Table 17. Comparison of Different Methods for T2 phage DNA Qua ntitatio n 74 Table 18. Effect of Chemical Treatment on VFF-concentrated Environmental Samples or T2 phage Lysates 75 Table 19. Effect of Ethanol Precipitation on Purified Phage Particles. 76 Table 20. Estimated Viral DNA Contribution to Dissolved DNA Content in Aquatic Environments. 77 Table 21. Selection of Lysogen From Bacterial Isolates by Inducement. 79 iv


LIST OF FIGURES Figure 1. Location of stations sampled in the Tampa Bay. 17 Figure 2. Location of stations sampled in the southeastern Gulf of Mexico and Caribbean. 18 Figure 3. Locations of stations sampled in Key Largo. 19 Figure 4. Schematic of Taylor vortices system during vortex flow filtration. 26 Figure 5. Membrex Benchmark system used for concentration of viruses and dissolved DNA from the marine environment. 27 Figure 6. Schematic diagram of a Pacesetter rotary biofiltration device. 29 Figure 7 Schematic diagram of an Amicon DC-1 0 system equipped with a spiral wound filter cartridge. 30 Figure 8. Electron micrographs of viral-like particles from marine environments. 48 Figure 9. The distribution of viral abundance along a transect from the estuarine environment (station 1) of Tampa Bay to the oligotrophic environment of the southeastern Gulf of Mexico (Station 9). 57 Figure 10. The vertical profile of the distribution of viral abundance at a station in the Gulf of Mexico (26', 84'). 58 Figure 11. Seasonal variation in viruses, bacteria and chlorophyll 2. concentrations. 62 v


Figure 12. Diel variation in viruses, bacteria, chlorophyll ,a and dissolved DNA concentrations. 63 Figure 13. Electron micrographs of DNA-containing membrane vesicle material. 72 Figure 14. Electron micrographs of temperate phages or bacteriocins induced from marine bacterial isolates. 80 vi


VIRUSES IN THE MARINE ENVIRONMENTABUNDANCE, DISTRIBUTION AND CONTRIBUTION TO THE DISSOLVED DNA POOL by CHENYANG JIANG An Abstract of a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Marine Science University of South Florida May 1993 Major Professor : John H Paul, Ph.D vii


The high abundance of viral populations in aquatic environments has come to light very recently (Bergh, 1989). Viruses, as obligate parasites, may play a significant role in controlling bacterial and phytoplankton populations in the world ocean. Also, since most of the viruses are less than 0.2 pm in size, they may contribute significantly to the dissolved organic carbon pool, and specifically, to the dissolved nucleic acid pools. In order to achieve a basic understanding of the role of viruses in the marine ecosystem, I employed a Membrex vortex flow filtration (VFF) system to concentrate viruses and dissolved DNA simultaneously from various aquati c environments. The resu lts of transmission electron microscopy direct enumeration of viruses in VFF-concentrated samples indicated that the surface viral concentrations ranged from 3.4 x 107 / ml in an estuarine environment i n the summer to the 1 .8 x 105 / ml in an oligotrophic oceanic environment. Subsurface euphotic zone measurements were similar or significant greater in viral concentrations as compare to the corresponding surface samples The lowest viral concentration of 1.3 x 104 /ml was found in the deep aphotic zone ( 1500m). Viral abundance in an estuarine environment showed a strong seasonal pattern, with highest abundance in the summer months and lowest in winter. The results from the diel study showed that the viral abundance in marine waters may change significantly on a time scale of hours. DNA in viral particles could not be simply separated from soluble DNA by differential ultracentrifugation because of the presence of macromolecular viii


DNA in the environmental samp l es Calculat ion of viral DNA content based on DNA content per phage and total phage counts suggested that viral DNA was usually contributed to a small fraction of the dissolved DNA, being as much as 20%, and in most cases, less than 5%. Temperate phage w ere found in an average of 43.1% of the mar i ne bacterial isolates investigated, indicat i ng that lysogeny is a common occurrence in the mar i ne environment. Abstract Approved: -"""Tt" _______ _,__ __________ Major Professor: John H. Paul, Ph. D Professor, Department of Marine Science Date Approved: __ 2_ 2 __ wt_a._ vc..._'""_ ix


1 CHAPTER 1. INTRODUCTION Historically, phytoplankton were the only component of microbial communities studied comprehensively by biological oceanographers. Bacteria, protozoa, viruses and fungi were only researched by "specialists. It was implicitly assumed that the oceanic productivity mechanisms and biogeochemical dynamics could be understood and modelled without taking into account the activities of these other components of the pelagic micro biota. This view was challenged by the quantitative studies of dissolved organic matter (DOM), bacterial biomass, and production, leading up to the discovery of the microbial loop (Azam et al., 1983). Recently it has been suggested that viruses may also play ecologically important roles in the microbial loop (Bergh et al., 1989; Proctor and Fuhrman, 1990; Suttle et al 1990), including a role in the mortality of bacteria and phytoplankton. Viruses in the Marine Environment The existence of viruses in marine environments has been known for nearly half a century (Zobell, 1946; Kriss and Rukina, 1947; Smith and Krueger, 1954; Spencer, 1955; 1960; Chaina, 1965; Johnson, 1968; Wiggins


2 and Liston, 1968; Hidaka, 1971; 1973; 1977; Stevenson and Albright, 1972; Kak imoto and Nagatomi, 1972; Zachary, 1974; Hidaka and lchida, 1976; Hidaka and Tokushige, 1978; Baross et al., 1978; Hidaka et al., 1979; Moebus, 198 0) Accurate methods to enumerate total viruses did not exist prior to 1988, and t:s timates based on plaque assays on defined hos t s implied that thei r occurrence was infrequent. Semiquantitative observations by transmission electron microscopy suggested that viruses were abundant in the mari ne environment (Johnson and S ieburth, 1978; Torrella and Morita, 1979; Proctor et al, 1988). Using u ltracentrifugation to concentrate viruses from natural waters, B e rgh et al. ( 1989) reported v i ral abundances in natural water a s hi g h as 2 5 x 108 / ml or 103 to 107 times more than had been reported bas e d on plaq u e assay. These observations have s ince been corroborated by other inves tigators us i ng other methods to concentrate viruses (Proctor and Fuhrman, 1990; Suttle et al., 1990). Like many other developments i n science, t he finding of abundant viral populations in the marine environment was based on the development of new methodologies. Methods for Detection of Marine Viruses The earliest method used to detect and enumerate mari ne viruses was plaque assay One assumption in this assay i s that each infective virus will form a clearing (plaque) on a lawn of host cells. This technique is relatively sens itive


3 for detecting certain types of viruses in environmental samples Because bacteriophages are highly specific for their host strains (Baross et al., 1978; Spencer 1960; Moebus, 1981 ; 1983), viral detection by plaque assay relies on the use of a sensitive host. Env i ronmental factors such as pH, ionic strength, temperature, presence of organic cofactors and nutrients (Arderson, 1948; Adams 1959; Moebus, 1987) must be optimized for each phage -host system before the plaque assay can be conside r ed quantitative. Moreover, mutations in both hosts and phages could change host range specificities (Adams, 1959). Thus plaque assays cannot be expected to give information on the total number of viruses in a sample containing multiple viral types. The use of electron microscopy has enabled quantitation of the total number of viruses in environmental samples. The first report of the use of electron m icroscopy to enumerate total viruses from the marine environment was at the 1978 American Soc iety of Microbiology meeting (Johnson and Sieburth, 1978). This report indicated that the bacteriophages observed by transmission electron microscopy in offshore waters and bays were not a rare event. Torrella and Morita (1979) estimated the viral concentrations in Yaquina Bay to be over 104 per ml of seawater by electron microscopy direct enumeration of 0. 2 pm membrane filter concentrated samples It is necessary to concentrate viruses before enumeration by transmiss i on electron microscopy. There are two conventional methods used to concentrate


4 viruses from natural water samples: sample enrichment and adsorption-elution. The sample for enrichment is usually mixed with an appropriate nutrient broth and is inoculated with an exponentially growing culture of indicator bacteri a. This method has been demonstrated to be useful for viral isolation (Baross et al., 1978; Moebus, 1980; Ackermann and Nguyen, 1982) and viral morphology studies by electron microscopy (Ackermann and Nguyen, 1982; Frank and Moe bus, 1987). Because sample enrichment may change the composition of the phage populations, it is not suitable for the enumeration of total aquatic viruses. The primary adsorption and elution method for bacteriophages was described by Primrose and Day (1977). They concentrated viruses by adsorbing viral particles on a calcium phosphate suspension and then eluted them using a sodium phosphate buffer. The recovery of seeded coliphages ranged from ., 8 to 90% by this method. A variety of other adsorption elution methods have been used and evaluated for viral recovery (Rose et al., 1984; Sobsey and Hickey, 1985; Zerda et al., 1985). The adsorption elution procedure usually involved altering the sample pH and/or chemical composition, which may effect the biological composition of the sample. Naturally occurring organic matter in the sample was also found to interfere with virus adsorption and recovery from filters (Sobsey and Hickey, 1985). Ewert (1980a) adapted a sedimentation technique for assaying animal viruses (Sharp, 1960; 1965). Ewert ( 1980b) concentrated the total viruses


5 from sewage on dried agar by ultracentrifugation and then mounted the agar on electron microscopy specimen grids for direct observation. Bergh et al. (1989) modified this ultracentrifugation method by sedimenting viruses directly onto a formvar coated grid locating at the bottom of a centrifuge tube. Their results of direct viral enumeration indicated viral abundances in natural waters were 1 03 to 1 06 times more than had been reported based on plaque assay Recently, several investigators have reported concentration of viruses by hollow-fiber ultrafiltration or spiral cartridge ultrafiltration techni ques (Proctor and Fuhrman, 1990, Suttle et al., 1990; 1991 ). These systems employed low adsorbent ultrafilters (30 KD to 100 KD molecular weight cut off) to retain very small sized particles like viruses inside the filter without adsorbing on the filters. Thus, these ultrafilters can be used to process large volumes of water (201 00 liters) without clogging, and concentrating the samples to 10-30 mi. Direct enumeration of viruses in these concentrated samples indicated that viral concentrations ranged from 103 to 1 08/ml in the marine environments. A new nucleic acid staining method, using 4 '-6-diamidino 2 -phenylin dole (DAPI) to stain DNA contained in virus particles (Coleman et al., 1981 ), has been used to enumerate viruses from aquatic environments. In 1988, Sieburth et al. reported using epifluorescence microscopy to enumerate DAPI-stained virus-sized particles during a phytoplankton bloom in Narragansett Bay, Rhode Island Their results indicated that virus concentrations were 5 8 x 109 particles per liter of seawater in that estuary. This method has also been employed by


6 Suttle et al. (1990) to enumerate virus-like particles in concentrated 0.2 pm filtrates of seawater. Hara et al. (1991) reported viral concentrations of 1.0 to 1 6 x 1 06 per ml in Japanese coastal and offshore waters by epifluorescence microscopy of DAPI-stained samples. However, there are several problems associated with the DAPI-staining method preventing its wide use: ( 1). DAPI is specific for double-strand DNA (dsDNA), so only dsDNA viruses are DAPI positive. (2). Because of the small size of viruses, visualization is at the limit of resolution by light microscopy. (3) Because the fluorescence of viruses is so faint compared to brighter bacteria, it is often necessary to prefilter samples through a 0.2 pm filter, which may remove a significant amount of viruses (as shown in this study) The Role of Viruses in Aquatic Ecosystems The ecological role of viruses in aquatic ecosystems has been a point of interest for some time. Because viruses are obligate parasites, it has often been assumed that they control the population density and/or activity of their hosts in natural ecosystems Ahrens (1971) determined the seasonal fluctuation in the numbers of agrobacteria and their phage in the Kiel Bight. His studies indicated that an increase in the concentration of sensitive bacteria was a precondition for the growth of the bacteriophage population The detection of high viral concentrations in the marine environment


7 suggests that viruses may play a significant role in these microbial ecosystems (Proctor et al. 1988; Bergh, 1989). The proportion of marine microorganisms infected by viruses was investigated by examining sectioned cyanobacteria and heterotrophic bacteria for the presence of intracellular phage (Proctor and Fuhrman, 1990). These workers found that up to 7% of the cells contained recognizable phage particles. Assuming the final visible stage of phage assembly represents 10% of the latent period (Valentine and Chapman, 1966), it was estimated that up to between 30 and 70 % of microbial mortality could have been attributed to viral lysis The number of viruses in marine surface waters was found to change on a diurnal basis along with the number of bacteria and bacterial activity (Heldal and Bratbak, 1992). From these studies, viral decay rates of 0.05 h -1 to 1.1 h -1 were measured in a coastal marine environment. Therefore, it was suggested that phage may lyse 2 to 24% of the bacterial population per hour (Heldal and Bratbak, 1991 ). Although the first approach to incorporate viruses into the budget of microbial carbon(C) -transfer failed to balance the C-flow of viral lysis of bacteria (ie. viral lysis exceeded bacterial production by a factor of 6), the results support the idea that viral lysis may be a quantitatively significant process that needs to be incorporated into budgets of microbial C-transfer (Bratbak et al., 1992). It is becoming clear that protozoan grazing may not be the only major cause of bacterial mortality. Bacterial mortality is more likely combination by both protozoan grazing and viral lysis, yet predominance of one


8 process or the other may depend on the environmental conditions (ie. viral abundance vs protozoan abundance). Recent studies also der11onstrated that viruses can be a nutritional source for phagotrophic nanoflagellates and marine protists, and marine nanoflagellates might be a potential sink for viruses (Gonzalez et al., unpublished; Gonzalez and Suttle, unpublished) The utilization of viruses as carbon, nitrogen, and energy sources by bacteria was also reported (Findlay and Stotzky, 1992). While Borsheim et al. (1990) considered viruses from eukaryotic hosts to be of minor significance, Suttle et al. ( 1990) found a strong impact of the concentrated viral fraction from natural habitats on the mortality of laboratory cultures of unicellular eukaryotic phytoplankton such as diatoms, cryptophytes and prasinophytes. Also addition of concentrated environmental viral samples to seawater reduced primary production by as much as 78%. Other reports of the isolation and wide spread occurrence of algal viruses from aquatic environments (Meyer and Taylor, 1979; Sieburt h et al., 1988; Suttle et al., 1991; Muller, 1991; Cottrell and Suttle, 1991; Yamada et al., 1991) also suggest that eukaryotic viruses may be important in controlling primary production These recent findings led to the reconsideration of the current theories in aquatic microbial ecology which often assumed that bacterial production was balanced by grazing. Lysis of bacterial populations by viruses could possibly lead to the loss of organic carbon from transfer to higher trophic levels The


9 viral lysis of phytoplankton may provide another direct entrance for recently fixed carbon into the soluble pool for use by bacterioplankton. Another potential important role viruses may have in the oceans is as a component of the dissolved nucleic acids pool. Dissolved ( <0. 2 pm) DNA has been found in nearly all aquatic environments (DeFiaun et al., 1986; 1987; Paul et al., 1987; 1991a; Karl and Bailiff, 1989; BeeBee, 1991; Maruyana, et al., 1993). Reported concentrations ranged from 0.2 to 44 pg/liter, with the highest values being obtained for estuarine and river environments However, the methods used to measure dissolved DNA did not separate virions from soluble DNA. Determining the viral particles contribution to the dissolved DNA may be important for two reasons. First, dissolved exogenous DNA in the aquatic environment is susceptible to the extracellular nuclease degradation (Paul et al., 1987; 1989), serving as a nutrient source for the microbial utilization and as a source of nucleic acid precursors for macromolecular biosynthesis. Viral DNA, because of its encapsulation in viral capsids, would not be available to microorganisms as a direct nutrient source. Rather, viruses may contribute to the nutrient flow by lysis of the heterotrophic and autotrophic populations thereby producing dissolved DNA of microbial origins. Secondly, the mechanism of natural gene transfer may change with the composition of the dissolved DNA. Transformation and transduction are two of the three major genetic transfer mechanisms recognized in bacteria Many marine bacteria have been shown to be naturally transformable (Stewart and


10 Sinigalliano, 1990; Jeffrey et al., 1990; Frischer et al., 1990; Paul et al., 1991 b; 1992). Gene transfer via viral transduction was found amongst Pseudomonas strains in freshwater environments (Saye et al., 1987; 1990; Miller et al., 1990). Both processes could occur in the marine environment. An abundance of soluble DNA compared to virus particles favors gene transfer by transformation; the opposite situation favors gene transfer by transduction. Ecology of Marine Bacteriophage Phage can survive in the environment as free or attached virions or as prophage inside a lysogenic host (Moebus 1987). Free virions must maintain their activity until they come in contact with suitable host cells and initiate infection and replication. Although a couple of investigators have reported the stability of some marine phages in stock suspension for 6 months and up to 7 years (Hidaka, 1972; Moebus, 1987), the free viral populations in the marine environment were found to have rapid decay rates (Heldal and Bratbak, 1991; Noble et al., 1992; Suttle and Chen, unpublished) In other words, the lytic phase may not result in optimal phage survival in the environment. It has been hypothesized that bacteriophage couldn't affect the number or activity of bacteria in the environment (Wiggins et al., 1985), when their host population is lower than a threshold concentration (ca. 104 /ml). Further study of phage-host relationships under conditions simulating aquatic


11 environments indicated that phage replications were more likely affected by the host cells' physiological conditions rather than cell density (Kokjohn et al., 1991). No limitation or threshold value of physiologically competent host-cell density was observed for phage replication. However, phage replication was limited in starved host cells. Kokjohn et al. (1991) also observed that bacteriophage exposed to starved cells appears to establish a pseudolysogenic relationship with its host. Because cells existing in natural aquatic ecosystems are subject to nutrient deprivation (Rozak and Colwell, 1987), Kok john et al. suggested that the virulence of the viruses may be an artifact of the laboratory, where they are routinely exposed to physiologically competent hosts. "Lysogenization" is a process where the injected phage DNA enters into a semistable state within the bacterium, rather than initiating the lytic process (Lwoff, 1953). The phage genome is either integrated into the bacterial genome or is maintained in the cytoplasm in the form of a plasmid or integrated into other temperate phage genome. The integrated viral DNA, then known as a prophage, will replicate in the course of cell division and will be contained in the progeny of the originally infected cell (Barkstale and Arden, 1974; David et al. 1980; Freifelder, 1983). Bacteria carrying prophage, known as lysogens, are immune to lytic infection by free-phage of that species. However, at rates that depend on environmental conditions, lysogens can be induced to go through the lytic cycle, releasing free phage particles called temperate phage (Ackermann and DuBow, 1987). Lysogenic bacteria were also found to


12 reproduce faster than their nonlysogenic parents (Edline et al. 1977; 1975). The lysogenic state is beneficial to both the phage and its host: it ensures perpetuation of phage and confers at least one new property to the bacteri um, antiphage immunity, which has an evident survival value (Ackermann and DuBow, 1987). Lysogens occur in many branches of the eubacteria (e. g. Ordogh and Szende, 1961; Zimmerer et al., 1966; Gabrilovich et al., 1968; Hongo et al., 1968; Kasathlya, 1970; Rautenstein et al., 1970; Pulverer, 1974; Manthey and Pulverer, 1975; Karawanov, 1976; Huggins and Sand i ne, 1977; Grange and Bird, 1978; Bernhelmer, 1979; Steensma and Robertson, 1983; Marti n et al. 1984; Shlmodori et al., 1984), including cyanobacteria (Stafferman et al. 1969; Gromov, 1983). The natural occurrence of lysogeny in a freshwater environment was evaluated recently. Between 1-7% of the Pseudomonas aeruginosa isolates were tested positive by the criterion of release of plaque forming units (PFU) infectious on laboratory strains of P. aerugi n osa ( R eplicon and Miller, 1990). Colony hybridizati on using various DNA prob e s s peci f i c for P. aeruginosa phages revealed that up to 70% of freshwater Pseudomonas isolates contained phage-specific DNA (Ogunseitan et al., 1990; 1992). Very little is known concerning lysogeny in marine bacteria. I have found only one report of a temperate marine phage that was isolated from marine mud. It was found to lysogenize between 10 and 16% of its host cells (Hidaka and Shirahama, 1974). Although about one half of the bacteri a were estimated


13 to be lysogens (Ackermann and DuBow, 1987), we do not know i f lysogeny i s as common with nonmarine species. Also, the influence of ecologically important factors, such as radiation, nutrient, salinity, pH, temperature, on the establishment of lysogeny or on the relative frequency of prophage entering the lytic cycle is not known. The Goal of This Research I became interested in marine viruses because of the question of the viral contribution to the dissolved DNA pool. The abundance of viruses in the marine environment suggests that v i ral DNA may be quantitatively significant in terms of other forms of nucleic acids in seawater. To investigate what proportion of the dissolved DNA was attributabl e to nucleic ac i ds encapsulated in virus particles I adapted vortex flow filtration technology to the simultaneous concentration of viruses and free (soluble) DNA from the dissolved ( <0. 2 pm) fraction of seawater samples The goals of this research are: (1). To develop methods for concentration and enumeration of total viruses from aquatic environments. (2). To determine the concentration and spatial distribution of viruses in various subtropical marine environments (ie. estuarine, coastal oceanic, oligotrophic oceanic)


14 (3). To determine the temporal distribution of viral abundance in the marine environment b y Investigating seasonal and die I viral abundance variation. (4). To develop a method for separation and quantitation of viral DNA and "free" DNA, thereby determining the viral contribution to the dissolved DNA pool. (5). To evaluate the frequency of occurrence of lysogeny in marine bacterial isolates.


15 CHAPTER 2. MATERIALS AND METHODS Pha ge and Bacterial Strains T2 phage was propagated in Escherichia coli B. (ATCC 11303B2) by top agar overlay. Plates were incubated overnight at 37C. The phagecontaining celllysates were eluted from the plates with sterile 0.5 M TrisHCI (pH 8.0), sterile filtered, and stored at 4C until use. Indigenous marine bacterial isolates designated #16 (tentatively identified to be Vibrio parahaemolyticus by Microbial ID. Inc, Newark, DE) and #13 (unidentified) were isolated from a Membrex concentrated (see below) St. Petersburg Pier water sample. 16 phage infecting bacterium #16 and 13C phage infecting bacterium #13 were also isolated from a concentrated St. Petersburg Pier water sampled two weeks after the isolation of their hosts by top agar overlay. The propagation of 16 phage and 13C phage in their respective bacterial hosts were performed as described above except that the incubation temperature was at 26 C instead of 37C.


16 Recovery of Phage and Calf Thymus DNA from Artificial Seawater T2 phage, q> 16 phage (final concentrat i on 1 08pfu/ml each) or calf thymus DNA (1 0 to 30 pg/L) was added to autoclaved, sterile filtered artificial seawater (ASWJP). Phage titers were determined in the arti ficial seawater before and after concentration by ultrafiltration (see below). DNA content in concentrated samples was determined by the fluorometric Hoechst 33258 method (Paul and Myers, 1982). Field Sampling Sites Freshwater samples were collected from the Medard Reservoir, Valrico, Fla. Estuarine surface water samples were taken from Tampa Bay at Bayboro Harbor, North Shore Park, St. Petersburg Pier, Hooker's Point, and from the Gulf of Mexico at the Fort Desoto Fish i ng Pier St. Petersburg, Fla. (Figure 1 ). Offshore surface water samples from the southeastern Gulf of Mexico Dry Tortugas and Bahamas were collected during four cruises (Figure 2) between 1990 and 1992 Cruise dates and sampling locations are described in Tables 1 through 4A. Samples along a transect from Tampa Bay across the West Florida shelf to southeastern Gulf of Mexico were taken for a survey of viral abundance during a cruise aboard the R/V Pelican in 1992. Two to three depths were sampled at each station alone this transect (Table 4B). A depth profile


17 Figure 1 Location of stations sampled in Tampa Bay.


G u L F OF ,, ATLANTIC \ .. ;:. ......... f r f t LJrrL IV.ll.u, 1ioll1'Jt l ... :: t Q r ..... /: 0 t ""' '"' l .; :. -. . . o '\ -"VVvf:l.. M E X I C o : : ;: t 0 .. 0 o q f to../ .. f Il.. :::.:. / f e.-,...... ;:., /' b ,:> ...... ... . --"_........ I' 0 It /'./' s s '{ 1l ,../' t ... ....... ..,. CAvs..u. ............ --.. L\NI( i .... ........ .._ ... / /' ----0 C> . ..... 7 : ..,1--.., lJ -1tv I( 0 s \ Figure 2. Location of sampling stations in southeastern Gulf of Mexico and Caribbean. represents the sampling stations during a cruise on board R/V Pelican from June 22 to June 30, 1990; 0 represents the sampling stations during a cruise on board R/V Hatteras from August 30 to September 7, 1990; .& represents the sampl i ng stations during a cruise on board R/V Bellows from August 6 to August 7 1991; represent the sampling stations during a cruise on board R / V Pelican f rom June 14 to June 28, 1992. (X)


19 _,.')-' 8025 8020 Longitude W Figure 3 Locations of stations sampled in Key Largo.


Table 1 Station Locations and Sampling Dates During the R/V Pelican Cruise of 1990. Station Description Date Location Environment P901 Tampa Bay June 22 27'00, 82'80 estuarine P90-2 Gulf of Mexico,St2 Jun e 23 26'00, 85'00 oligotrophi c oceanic P90-3 Gulf of Mexico,St3 June 24 24'70, 85'91 oligotrophic oceanic P905 Loggerhead Key, Dry Tortugas June 26 ca 24', 82' coastal oceanic P90-7 Florida Bay June 30 24'00, 81 '25 coastal oceanic N 0


Table 2. Station Locations and Sampling Dates During the R/V Hatteras Cruise of 1990. Station Description Date Location Environment H90-1 Sea Buoy, Miami August 30 25'00, 80'00 coastal oceanic H90-1 b Outfall Miami August 30 25', 80' sewage outfall H90-2 N W.Prov.Channel, Bahamas August 31 26', 78' oligo oceanic H90-3 1500m depth, Atlantic Ocean Sept 1 2 5 48,, 7 6 51 Deep sea H90-4 Mama Rhoda Rocks, Bahamas Sept.1 25', 78'60 coastal oceanic H90-5 Mangrove swamp, Joulter's Cay Bahamas Sept 2 25'07, 78'33 coastal oceanic Table 3 Station Locations and Sampling Dates During the R/V Bellows cruise of 1991. Station Description Date Location Environment B91-3 Gulf of Mexico,St 3. August 6 27'01, 83 19'98 oligotrophic oceanic B91-7 Gulf of Mexico,St 7. August 7 26'93, 84'10 oligotrophic oceanic N


Table 4A. Station Locations and Sampling Dates During the R/V Pelican Cruise of 1992. Station Description Date Location P92-11 Loggerhead Key, Dry Tortugas June 20 24' ,82' P92-13A Garden Key, Dry Tortugas June 21 24',82' P92-13B Mote, Garden Key, Dry Tortugas June 21 24' ,82' P92-14A Key West Harbor June 22 24, 81 P92-14B Sand Key Key West June 22 24,81 P92-15 Seagrass bed, Bimini Harbor June 23 25',79' P92-16 Mangrove swamp, Bimini Harbor June 23 25',79' P92-17 Concrete ship, near Turtle Rocks Bahamas June 24 25',79' P92-19 South Cat Cay, Bahamas June 25 25',79' P92-20 Gulf Stream June 27 25' 179 16' Environment coastal oceanic coastal oceanic eutrophic coastal oceanic coastal oceanic coastal oceanic coastal oceanic coastal oceanic coastal oceanic oligotro. oceanic N N


23 Table 48. Station Locations and Sampling Depths Along a Transect During the R/V Pelican Cruise of 1992 Station Date Locations Depth Environment P92-1 a June 15 88 Channel Last Marker 0.5 estuarine P92-1 b June 15 88 Channel Last Marker 3.4 P92-2a June 15 27' 1 82 1.14 estuarine P92-2b June 15 2 7 O 3 5 I 1 8 2 O 43 I 4.43 P92-3a June 15 27 15'1 83 17' 1-2 coastal oceanic P92-3b June 15 27'183' 30 P92-4a June 16 26 56' 1 83 1-2 oligo. oceanic P92-4b June 16 26 56' 1 83 60 P92-5a June 16 26'1 84' 1-2 oligo. oceanic P92-5b June 16 26'1 84' 100 P92 6a June 17 2 6 O 21 I 1 84 O 30' 1-2 oligo. oceanic P92-6b June 17 26 21 I 1 84' 100 P92-6c June 17 2 6 O 21 I 1 84 O 30' 200 P92-7a June 17 26', 84' 1-2 oligo. oceanic P92 -7b June 17 26'1 84' 250 P92-7c June 17 26'1 84' 1500 P92-8a June 18 25'1 85' 1-2 oligo oceanic P92-8b June 18 25', 85' 85 P92-8c June 18 25'1 85' 2500 P92-9a June 18 25'1 84' 1-2 oligo. oceanic P92-9b June 18 25'1 84' 118


24 was also taken at station #1 0 (26', 84') during this cruise The depths sampled i ncluded 3m, 25m, 50m, 68m, 1OOm, 500m, 1 OOOm, and 2500m. Surface water samples were also collected from the Key Largo area in January 1992. The locations of stations sampled appear in Figure 3. Station 1 and 2 were in a man-made canal. Station 6 was located outside the reef break at the edge of Florida shelf break. Samples were also taken from the seagrass environment of Hawk Channel (St.3), the coral-reef environment of French Reef (St. 5) and Mosquito Bank (St.4), the mangrove environment of Blackwater Sound (St. 1 0), and Tarpon Sound (St. 9), an enclosed embayment on the west side of Key Largo. Water samples were taken with Niskin bottles or acid washed carboys on cruises and at Key Largo offshore stations, and with a dip bucket for inshore sampling. Samples for a study of seasonal viral abundance were taken from the St. Petersburg city Pier, Tampa Bay every two weeks for 13 months, starting in December 1990 and ending in December 1991. In addition to viral direct counts (see below), bacterial direct counts, chlorophyll .a. concentration, temperature and salinity were also determined for each sample. One-hundred-eighty liters of seawater was also collected for a diel study from the St. Petersburg city Pier on August 3, 1992 by surface pumping. The water was placed in a 200 liter polyethylene tank. The top of the tank was covered by neutral density screening to reduce light intensity. A refrigerated recirculator (Coolflow, Neslab Instrument Inc., Portmouth, NH) was used to


25 recirculate anti-freeze coolant within a 15-meter-long, 0.8 em diameter tygon tubing inside the tank to regulate the water temperature. Samples were taken every 4 hours for 36 hours for viruses, bacteria, chlorophyll a, dissolved DNA, temperature and salinity measurements. Effect of Prefiltration on Viral Recovery Natural water samples or sterile artificial seawater containing T2 phage were filtered through Whatman GF/D and 0.2 pm Nuclepore filters at a vacuum of < 150 mm Hg prior to VFF concentration (see below). A replicate sample was also concentrated by VFF without prefiltration. At the Loggerhead Key station during the Pel i can cruise of 1990, a third sample was also passed through a 142 mm Millipore 0.22 pm GV Durapore filter using positive filtration pressure. The samples were immediately concentrated by VFF (see below). Concentration of Samples A Benchmark Rotary Biofiltration unit (Membrex Inc., Garfield, NJ) was used for vortex flow filtration (VFF) concentration of samples. Vortex flow filtration is a filtration technology based on Taylor vortices (Taylor, 1923). The vortices are established by rotation of a cylindrical filter inside a second cylinder (Figure 4). The sample is fed under pressure between these two cylindrical


26 TAYLOR VORTICES Apphtd Prusu,. Figure 4. Schematic of Taylor vortices during vortex flow filtration. Taylor vortices are created by rotating a cylinder at a critical velocity within a narrow annu lar gap. These vortices are pairs of counter-rotating 'rings' whose diameter is equivalent to the annular gap. This system dramatically reduces the problems of membrane fouling and concentration polarization.


Aetentate T u b i ng Feed Solution Vessel T r ansdu c er Co nnecto r Peristaltic Pump Tub i ng Stopcock and Check V alve Permea t e T u b ing Permeate Collection Vessel 27 Figure 5. Membrex Benchmark system used for concentration of viruses and dissolved DNA from marine environments The system is set up i n a rec i rculation mode. The water sample is pumped into the Taylor vortices filtration cylinder by a low shear peristaltic pump. The filtrate goes to waste through the permeate tubing, and the viruses and dissolved DNA reta in ed by the filter (termed retentate) returned to the feed solution vessel until less than 50 ml retentate are left. This system has been successfully employed to concentrate viruses from 1 100 liters of sample.


28 surfaces, forcing fluid across the filter surface and into the inner cylinder for collection or out to waste. The vortices constantly keep the filter surface clean which prevents clogging. For this study, the system was set up in the recirculation configuration (Figure 5) using either a 200 cm2 or 400 cm2 filter. The 400 cm2 filter allowed a greater sample filtration rate. Filtration was performed at 7 to 8 PSI for the 100 KD and 10 to 12 PSI for the 30 KD filters with a filter rotation speed of 2000 RPM (200 cm2 filter) or 1500 RPM (400 cm2 filter). The sample was pumped in from the feed vessel in the recirculation mode until less than 50 ml of sample was left in the reservoir (Figure 5). At this time the feed port was closed, an air pump was fitted to the retentate line, and the remaining sample in the reservoir was collected using a sterile 60 cc syringe. The sample was fed into the filter cylinder as the retentate volume was reduced under positive air pressure to 15-25 ml (200 cm2 filter) or 35-50 ml (400 cm2 filter). The concentrated retentate was either used directly in dissolved DNA measurements (see below) or fixed with EM grade glutaraldehyde (2%) and further concentrated for TEM by ultracentrifugation for 90 min at 201,000 x G For concentration of larger sample volumes, a Pacesetter unit (Membrex Inc ) with a 100 KD filter was used. This system is based on the same design as the Benchmark but equipped with a 2300 cm2 filter (Figure 6). The operating procedure of this system is the same as the Benchmark except that, when the


Pressure Vent Valve Permeate Tube Permeate Contr ol Box Motor El DO OCJ 29 Pressure Gauge Pressure Regulator F ilter Module Rotor Membrane Cartr idge Drain Tube Feed Tube Peristaltic Pump Seawater Figure 6 Schematic diagram of a Pacesetter rotary biofiltration device. The filter module houses a single membrane cartridge with an area of 2 5 sq. ft (2300 cm2). The sample is drawn in by a peristaltic pump, and recirculated concentrated sample returns to the feed vessel through the retentate line. The filtrate is passed through the permeate line to waste. The system was used to concentrate 20 to over 300 liters of sample from the marine environment.


30 Back ,.,. ..,,. Valve Figure 7. Schematic diagram of an Am icon DC-1 0 system equipped with a spiral wound filter cartridge. This system is set up in a recirculation mode. The water sample is pumped into the low absorbent spiral wound filter cartridge under positive pressure, and the retentate flows back to the reservoir. The filtrate goes out to waste. This system was used to concentrate 20 to 120 liters of water samples to about 750 ml with a flow rate at 2.2 L/min.


31 feed vessel was empty (Figure 6), the retentate (now reduced to a liter volume) was collected for plaque titer, or further concentrated by the Benchmark to 35 to 50 ml for TEM viral direct counts. An Amicon spiral wound filtration DC-10 unit (Amicon Inc. Beverly, MA) was also used for concentration of large volume water samples (20-120 liters) on the Pelican cruise of 1992 in the Bahamas and Gulf of Mexico. This system employed a YM low absorbent membrane ultrafilter which enabled filtration of large water volumes without clogging The system was set up in the recirculation configuration (Figure 7) using a 30 KD molecular weight cutoff spiral wound cartridge filter. Filtration was performed at 25 psi for the inlet pressure and 22.5 psi for the outlet pressure (Figure 7) The permeate flow rate was at about 2.2 L/min. The water sample in the reservoir was pumped into the filter cartridge in the recirculation mode until about 750 ml of retentate was left in the reservoir. The concentrated samples (collected from the drain valve) were either used directly for plaque assay or further concentrated by a Membrex Benchmark system for TEM The concentration efficiencies of both the Pacesetter and the Amicon DC-1 0 systems were compared to the Benchmark system for the concentration of the same sample. The recoveries of the other filtration systems for the concentration of environmental viruses were expressed as a percentage of the recovery obtained with the Benchmark with a 100 KD filter system.


32 Direct Enumeration of Viruses For transmission electron microscopy enumeration of viruses, concentrated samples were diluted (3x to 30x) with Dl to decrease salt content and one pi was spotted onto a butyl-methacrylate-collodion coated copper grid and allowed to dry. The area of the drop was subsequently measured with a 1 OX optical micrometer. The samples were stained with a drop of 2% uranyl sulfate (Polysciences, Warrington, PA) for approximately 30 seconds, and viewed with a Hitachi H500 TEM. Counting was performed at 48,000 X or 36,000 X magnification and photography was performed at 48,000 to 150,000 X magnification. The magnification of the TEM was checked using catalase crystals for the high magnifications and calibration grids for the lower magnifications. For epifluorescence microscopy enumeration of viruses, samples were stained with 105 M DAPI (4',6'-diamidino-2-phenylindole; Aldrich Chern. Co.) for at least 20 min at room temperature in the dark. Ten pi of the stained suspension was spotted onto a clean glass slide and a 22 mm2 cover glass was (Corning No. 1 1/2) placed on top of the drop. The slides were viewed with an Olympus BH-2 epifluorescence microscope under UV (360 nm) excitation using a UV Fluorite 1 OOx objective. Viral particles were found to adhere approximately equally to both the cover glass or the slide, therefore the value obtained was multiplied by two to account for the surface not counted in any


33 one field. Measurement of Dissolved DNA For the disso l ved DNA measureme nt, water samples were filtered through GF/D and 0.2 pm Nuclepore filters at a vacuum of < 150 mm Hg Both water samples and filtrates were kept on ice during the filtration process to minimize enzymatic hydrolysis of DNA (Paul et al. 1987). F ive replicate filtrates (0.1 to 1.0 Leach) were ethanol precipitated and the precipitate solubi lized as previously described (DeFiaun et al., 1986). Two of these replicate samples were spiked with calf thymus DNA to determine the efficiency of recovery. DNA content was determined in concentrated extracts by the Hoechst 33258 method (Paul and Myers, 1982) and subsamples were DNase treated to detect nonspecific fluorescence. For detection of dissolved DNA by VFF filtration, 0 2 pmfiltered seawater was concentrated by VFF as described above. At some stations, when small sample volumes (0 5 to 1 L) were used, two of the five replicate samples were spiked with calf thymus DNA prior to VFF filtration to correct for losses in recovery. All the samples were kept cold during the process. DNA concentrations were measured in the retentate by fluorometric DNA determination and replicate retentate samp l es were DNase treated and corrected for nonspecific fluorescence For some samples, a portion of the


34 retentate (usually 5.0 ml) was precipitated by two volumes of ethanol at -20C for at least 12 hours. The precipitate was harvested by centrifugation, and dissolved in 1 xSSC (0.15M NaCI, 0.015 M sodium citrate, pH 7 0) for determination of DNA. Separation of Soluble DNA and Viral DNA by Ultracentrifugation Aliquots of artificial seawater spiked with T2 phage ( 1 010pfu/ml) and/or calf thymus DNA (12 pg/ml -98 pg/ml) were centrifuged at 201 K x G, 1 03K x G, 37K x G, and 25.8K x G for 90 minutes The DNA concentrations were determined in the supernatant and pellet fractions, and in some samples DNA concentrations were measured again after ethanol precipitation. Membrex concentrated 0.2 pm filtered seawater samples were centrifuged at 201 Kx G, 16. 5K x G, 11.2K x G. Viral direct counts were measured for all the pellet fractions. DNA contents were measured in both the supernatant and pellet fractions and, in some samples, remeasured after ethanol precipitation. Chemical Treatment of VFF-concentrated Dissolved DNA Samples and T2 Phage Lysates Heparin (final concentration of 5 units/ml, Sigma chemical Co.) was added to the environmental retentates to release histone-bound DNA material.


35 The mixtures were incubated at room temperature for 1 hour before DNA concentrations were measured A cell-free T2 phage lysate {in 0.5M Tris pH 8.0) was divided into four aliquots to test for the effect of chemical treatment on dissolved DNA measurements. One aliquot was amended with 5mM EDTA, 0.5% SDS and 50 pg/ml of Proteinase K {Sigma chemical Co ); the other aliquot was adjusted to pH 8 5 using 0.1 N NaOH and 50 pg/ml of Lipase {Sigma chemical Co ) was added Both samples were incubated at 37C for 1 hour with gentle shaking The third aliquot of T2 phage lysate was extracted with an equal volume of chloroform. The last aliquot remained untreated DNA contents were determined in all four aliquots of the phage lysate by the Hoechst 33258 method {Paul and Myers, 1982) before and after ethanol precipitation. DNA Measurement in Purified Viruses T2 phage and 4> 16 phage were purified from cell lysates following standard A phage purification protocols {Sambrook et al., 1989). Viral lysates were clarified by centrifugation at 8,000 rpm (SS-34 rotor, Sorvall Inc.) for 5 min Supernatants were pooled and treated with DNase I {50 pg/ml) and RNase {5 pg/ml) at room temperature for 1 hr, followed by salt (NaCI, 1M) precipitation and PEG (polyethylene glycol8000, 1 0%) precipitation. The phage pellets were suspended in SM buffer (0.1 M NaCI, 8mM MgS047H20, 50mM


36 Tris-CI, 0.01% gelatin), and extracted with equal volume of chloroform. To the aqueous phase 0.5 g/ml of cesium chloride was added then carefully layered onto a cesium chloride step gradient (bottom to top, 1 7g/ml, 1.50g/ml, 1.45g/ml). The viral band was collected by puncturing the side of the centrifuge tube after centrifugation at 22,000 rpm for 2 hours (Beckerman, SW41 rotor) After dialyzing against 1000 the volumes of dialysis buffer (1 0 mM NaCI, 50mM Tris-CI pH 8.0, 1 OmM MgCI2), purified phages were collected in microfuge tubes and stored at 4 C for future uses. Purified phages were diluted with Dl and sonicated at 55 HZ with a sonic cleaner (Ultrasonic, Fisher Scientific) for 2-5 minutes before counting by TEM. The DNA content was determined before and after ethanol precipitation. A second aliquot of the purified phages was incubated with 50 pg/ml DNase I at room temperature for 2 hours, followed by heat treatment (65 C for 2 hours) using a thermocycler (TwinBiock, Ericomp, Inc.). The DNA content was then determined prior to and after ethanol precipitation of the heated samples. Viral DNA was extracted from the purified viral particles by EDTA (20 mM), proteinase K (50 pg/ml ) and SDS (0.5%) treatment, and purified by phenolchloroform extraction and ethanol precipitation. Purified phage DNA was quantified by UV absorbance at OD260 and OD280 using a Cary 17D spectrophotometer (Varian Instrument Group, Sugarland, TX) and by the Hoechst method (Paul and Myers, 1982). T2 phage DNA was also determined by the ethidium bromide fluorometric method (Sambrook et al., 1989) and


37 diphenylamine colorimetric method (Burton 1955). Selection of Lysogenic Bacteria from Marine Isolates. Fifty one marine isolates from different environments were tested for the presence of inducible prophage. Mitomycin was added to mid-log phase bacterial cultures (00550 0 6) to a final concentration of 0 5 pg/ ml. The culture was further incubated and cell density monitored every 2 hours for the first 6 hours, and finally after overnight incubation. The cultures in which the density decreased, or remained the same, upon Mitomycin C addition were centrifuged at 8,000 rpm (SS-34 rotor, Sorvall Inc.) for 5 minutes and the supernatants collected for detection of temperate phage particles by TEM. Statistical Analysis An analyses of variance (ANOVA) and a multiple regression program by Human Systems Dynamics Inc., Northridge, CA was used for the statistical data analysis. Multiple comparisons were performed by Tukey test (Zar 1984).


38 CHAPTER 3 RESULTS Filtration Efficiency for Dissolved DNA and Viruses The results of the filtration efficiency experiments for the Membrex VFF Benchma : : Membrex Pacesetter and Amicon SWF DC-1 0 systems used for concentrating dissolved DNA and viruses from artificial seawater are shown in Table 5. The Benchmark system recoveries for T2 phage and Cl> 16 phage ranged from 68% to 91.6%, with no difference observe d between the 100 KD and 30 KD filters. The Pacesetter recovered only 17.5% of T2 phage, and the recovery of Cl> 16 phage by the Amicon DC 1 0 system was 30%. Because viral enumeration in these studies was performed by plaque titer, the Benchmark concentra ti o n procedure apparently had l ittle effect on the biological activity (infectivity ) of the virus. The DNA r e coveries with the 1 OOKD and 50KD filters (12% and 20% respectively) in the Benchmark system were significantly lower than those obtained with 30KD f ilter (avg. 80% ) In all ensuing studies, the 30 KD filter w a s used for concentrating dissolved DNA. If viral enumeration was required only, then the 100 KD filter was used, which filtered at a flow rate approximately three times that of the 30 KD filter (7 l i ters / h compared to 2.5 liters / h for the 30KD filter).


39 Table 5 Comparison of Filtration Efficiency of the Membrex VFF Benchmark, Membrex VFF Pacesetter and Amicon SWF DC-1 0 Systems for Concentration of Dissolved DNA and Viruses from Sterile Artificial Seawater. Sample F i lter Ret. Initial Titer 1Final Titer Recovery and Cutoff or DNA cone or DNA cone. efficiency Date (KD) (107pfu / ml (1 07pfu/ml (%) or pg/L) or pg/L) T2 Phage2 Benchmark 3/7/90 100 10 8.1 81 7 /24/90 100 11.7. 6 8.4. 6 72 7 /12/90 30 20.8.3 14. 1 .6 68 7 /24/90 30 11.7.6 8.2.4 70 Pacesette r 100 15.5.9 2.7.8 17.5 ct> 1 6 phage2 Benchmark 100 9 .47 0.5 8.67.71 91.6 Am icon 30 8 5 8 2.55 .4 30 Calf Thymus DNA3 Benchmark 3 /13/90 100 30 3.54 12 6/7/90 50 20 3.91 20 3 /19/90 30 30 23.7 1 79 6/7/90 30 20 15.9 80 1 Final phage titers or DNA concentrations were corrected for concentration factors. 2Phage recovery was from 2 or 3 liters autoclaved, sterile filtered artificia l seawater (ASWJP) for Benchmark, 100 liters for Pacesetter, 20 liters for SWF DC1 0 and phage titer determined by plaque assay. 3C alf thymus DNA was added to two or three liters autoclaved, sterile filtered ASWJP and the DNA content determined fluorometrically using Hoechst 33258 (Paul and Myers, 1982).


Table 6 Comparison of Membrex VFF Benchmark, Membrex VFF Pacesetter and Am icon SWF DC-1 0 Systems for Concentration of Environmental Viral Populations Sample Site St. Pete Pier, Tampa Bay Hookers point, Tampa Bay N.W. Prov.Channel Bahamas St.Pete Pier, Tampa Bay Loggerhead Key, Dry T ortugas Bim i ni Harbor, Seagrass bed System Benchmark Benchmark Benchmark Pacesetter Benchmark Pacesetter Benchmark DC-10 Benchmark DC-10 Benchmark DC-10 Retention Flow Rate cutoff (KD) (L!Hr) 100 6-8 30 4-5 100 6-8 100 60-70 100 6-8 100 60-70 100 6-8 30 130-140 100 6-8 30 130-140 100 6-8 30 130-140 Initial Vol (l) 5 5 20 90.5 25 71. 8 20 20 40 40 20 20 1V ira l Cone 105/ ml 12.0. 4 9.5. 5 13.3.55 4.3.26 0 .42.06 0 .084.02 20.8.26 7 .7.16 0.85.12 0.48.13 0 .67.12 0.45 .14 Recovery % 100 79. 2 100 32. 3 100 20 100 37.0 100 56. 5 100 67.2 1Viral concentrations were determined by TEM direct counts in the concentrated retentates Assuming the recovery of membrex Benchmark with a 100 KD filter was 100%, the recoveries of the other systems were calculated accordingly.


41 To compare the efficiency of the three systems for concentration of aquatic viral populations, water samples were concentrated by either the Pacesetter or the Ami con DC-1 0 system and results compared to those obtained for the Benchmark using the same sample (Table 6). Total virus numbers were determined by viral direct counts using the TEM. No significant difference was found between the 30 KD and 1 00 KD filters using the Benchmark alone. The Pacesetter yielded an average 26.2% of the recovery of the Benchmark system. The recoveries of the Am icon DC 1 0 system ranged from 36.9% to 67% (average 53.6%) of the Benchmark recoveries. Since the Pacesetter and Amicon DC1 0 systems have approximately 1 Ox and approximately 20x faster flow rates, respectively, compared with the Benchmark system, they were used to concentrate viruses from large volumes of oligotrophic or deep sea samples on the cruises The final viral counts obtained with these systems were corrected for the average efficiency of the system (26.2% for the Pacesetter and 53.6% for the Amicon DC-10). Evaluation of Enumeration Techni ques The results of comparison of the three methods used to enumerate phage particles are shown in Table 7. When T2 phage was employed, the results of DAPI direct counts and TEM direct counts were compared with those obtained by plaque titers. The results by all three methods fell in the same order of


Table 7. Comparison of Methods to Enumerate Phage Particles Enumeration by: Experiment or Sample Plaque Assay TEM (106 viruses/ml SO) A. T2 phage in cell-free lysates 1 Expt 1 Expt 2 Expt 3 41 000 11 000 29,000 3,600 35,000 B. T2 Phage in artificial seawater3 Expt 4 71.7.0 Expt 5 82 24.2 Expt 6 84.2 36. 0 C. Natural Populations from the Gulf of Mexico4 Mouth of Tampa Bay, St 1 Gulf of Mexico, St 2 Gulf of Mexico, St 3 NO NO NO NO NO NO 85.9.9(120) 67.0(81.7) 62. 9 55.6(74. 7) 1.8.34 0 .28.136 0.26.09 OAPIOC 50,400 21 00( 123)2 18,000 2100 (62. 1) 22,000 2200 (68. 9) 29.2 6.6 (40. 7) 114.0 (139) 117.0.3(139) 0.18.095 0.0154 0.007 0 .017 .004 1Counting and plaque assays performed directly in phage-host system medium without concentration. 2Values in parenthesis are % plaque assay values 3For experiments 4,5, and 6, T2 phage was added to autoclaved, filter sterilized artificial seawater and the phage then concentrated by VFF. "Samples were prefiltered through 0.2 pm filters prior to VFF, which accounts for the low viral titers. N


43 magnitude for T2 phage samples. The absolute value of the percent difference from the plaque titer was calculated as: o% = I (DC/PA.1 00)-1 00 I where DC is the DAPI or TEM direct counts and PA is the plaque assay counts. The absolute value was 39.2 11.6% for DAPI counts and 21.2 3 .65% for TEM counts, respectively. The results of TEM direct counts were generally closer to plaque titers than DAPI counts. For the enumeration of viruses in environmental samples, the results of DAPI direct counts were more than one order magnitude lower than those obtained by TEM counts. T hi s may be caused by weak fluorescence of the stained natural viruses and smaller size of some natural viruses, as compared to T2 phage Thus, TEM was used for viral enumeration in all the environmental samples. For all the dissolved DNA measurements, water samples were filtered through 0.2 pm Nuclepore filters to remove bacterioplankton and phytoplankton. The effect of 0.2 pm filtration on viral abundance was investigated using T2 phage in artificial seawater and natural viral populations in environmental water samples (Table 8). The effect of filtration on T2 phage abundance was variable, removing as much as 50 to 99% (mean 78.9%) of the pfu as compared to unfiltered samples. The effect on natural phage populations was also variable, having either no effect or removing 90% of viral populations. The least effect was in offshore waters, and the greatest effect was in nearshore waters. A similar effect on phage abundance was noticed for


44 Table 8. Effect of 0.2 pm Filtration on Recovery of Viruses Prior to VFF Concentration. Sample Unfiltered 0.2 pm filtered 106 viruses/ml1 A. T2 Phage in Artificial Seawater Expt 1 Expt 2 Expt 3 963 81 16 141 B. Natural Phage Populations Mouth of Tampa Bay Gulf of Mexico St2 Gulf of Mexico St3 Dry Tortugas 17.4.5 0 .24.06 0.44.5 1.5.1 1 All virus abundances determined by TEM 117 26 0.23.1 71.7."' 1 .78.34 0.28.14 0 .26.09 0 .14.03 0.19.122 %of unfiltered 12.1 0.28 50.9 10.2 117 59.1 9.3 12.7 2Value determined for prefiltration with a 14Z mm Dura pore filter. filtration through 142 mm Dura pore filters (removal 87.3% of the phage population), a procedure w hich has been used by other investigators prior to ultrafiltration (Proctor and Fuhrman, 1990; Suttle et al., 1990). For all data, the average phage abundance decreased 66% after filtration. Viral Abundance in Several Subtropical Marine Environments The water samples taken from estuarine, coastal oceanic, oligotrophic oceanic environments were concentrated by VFF and the TEM viral direct count


45 Table 9 Viral Abundance in Several Subtropical Marine Env i ronments as Determined by Vortex Flow Filtration (VFF) and TEM Sampling Location Initial Vol. Vol. After Viral Abund of Sample VFF (ml) (106/ ml) in (liters) Initial val Estuarine environments : Tampa Bay Bayboro Harbor 3 25. 0 34.0 14.0 Mouth of Tampa Bay 3 21.3 17.4 2.5 Hookers Point 20 57. 0 13.3 5.5 St Pete Pier (summer) 5 52.3 32.4 6 8 St Pete Pier (winter) 5 51.0 2 2 1 .03 Pelican 92 St. 2a 5 55. 0 4.65 1 1 Coastal oceanic environments : 1Sewage outfall, Miami 306 50.0 4.66 0.15 Mangrove lagoon, Blackwater Sound, Key Largo 10 46. 5 11.9 0 6 Seagrass bed, Hawk channel, Key Largo 10 48 3.2 1.2 Fre n c h reef, Key Largo 20 36 1.7 0.32 M os q uito Bank, Key Largo 20 40 1.9 0.3 Key \'lest Harbor 10 52 4.27 1 4 Florida Bay 20 18. 9 2.0 0.1 Sand Key, Key West 40 55 0.73 0 .07 Moat of Garden Key, Dry Tortugas 10 48.5 16.7.25 Garden Key, Dry Tortugas 40 48 2.41 0 .55 Loggerhead Key, Dry Tortugas 15 8 4 1.5 1 0 2 0 64 0.85 0.12


46 Table 9. (continued) Concrete ship, Bahamas 20 51 1.77 0.55 Mangrove Swamp, Joulter's Cay, Bahamas 10 50 2.3 1 1 Mangrove Swamp, Bimini Harbor 20 70 11.2 0.19 Seagrass Bed, Bimini Harbor 20 61 0.67 0.10 South Cat Cay, Bahamas 20 63.5 0.18 0.03 Oligotrophic offshore environments: NW Providence Channel, Bahamas 25 36.4 0.42 0 .06 Gulf of Mexico, St. 2 20 17.4 0 .24 0.06 Gulf of Mexico, St. 3 30 '16.0 0.44 0.15 Gulf of Mexico, St. 5a 20 38.0 0.85 0 .13 Gulf of Mexico, St. 7a 40 49. 0 0.49 0.06 Gulf of Mexico, St. 9a 40 50. 0 0 .53 0.04 Gulf Stream 40 61.5 0.18 0 .03 Deep sea environments: 1 Atlantic Ocean, 1500m 192.8 53.6 0.11 0.07 Gulf of Mexico St 7c, 1 500m 100 40. 5 0.013 O.CXE Gulf of Mexico St Be, 2500m 100 44 0.006 0.011 Gulf of Mexico St 1 Of, 500m 100 40 0.05 0.001 2Gulf of Mexico St 1 Og, 1 OOOm 100 44. 5 0.014 0.01 1Sample concentrated by use of a Pacesetter (large scale) VFF 2Sample concentrated by use of an Amicon DC1 0 device. device.


47 data appear in Table 9 Viral concentrations in estuarine samples ranged from 2.2 x 106 to 3.4 x 107 viruses /ml (mean 1.5 x 107/ml). The lowest value from the Petersburg Pier sample, Tampa Bay, was taken in December. The coastal oceanic samples (all measured during summer except samples from Key Largo area whi ch were taken in January) had viral concentrations ranging from 1 8 x 105 to 1.67 x 107 viruses/ml (mean 3.96 x 106/ml). The 1.67 x 107 value was found in an enclosed seawater moat around Garden Key, Dry Tortugas, and was nearly seven times higher than the viral concentration outside the moat ( 2.41 x 1 06/ml). The sample collected off the beach of an uninhabited island (South Cat Cay, Bahamas) on a stormy day had the lowest viral concentration (1.8 x 1 05/ml) of all the coastal environments sampled. The viral direct count was 1 .19 x 107 /ml in the mangrove fringed Blackwater Sound, Key Largo, which was higher than all the nearby oceanic environments off Key Largo. A mangrove fringed lagoon in Joulter's Cay, Bahamas, had nearly five fold more viral particles (2.3x1 06/ml) compared to the adjacent oceanic environment (4.2x105/ml). The viral concentration found in a mangrove swamp at Bimini Harbor was 17 fold that of over a seagrass bed at Bimini Harbor. The oligotrophic offshore oceanic samples (also sampled in summer) had less than 8.5x1 05 viruses/mi. The viral like particles in deep sea environments (below 500m) were between 1.3x104 and 1 .1x105 /nil (mean 4.46 x 104/ml). Figure 8{a-h) shows electron photo-micrographs of viral-like particles in the marine environment. The head size of the viral-like particles in the


Figure 8 (a-h). Electron micrographs of viral like particles in marine environments. (a). Tailed and non-tailed phages from the sample collected from Bayboro Harbor, Tampa Bay. (x178,000) Figure 8b. Electron micrograph of a bacterium and attached bacteriophage-like particles. This sample was taken from coastal oceanic environment of Floric 3 Bay (x166, 800)


49 Sa


Figure 8c. Electron micrograph of a giant phage-like particle (ca. 170nm in head size, 576 nm in tail length). This sample was taken from a surface station (26 00', 84') in the oligotrophic environment of southeastern Gulf of Mexico. (x84,706) Figure 8d. Electron micrograph of a bacterium and ta i led vi r al particles, which were found in a subsurface sample (50 m below surface) at a Gulf of Mexico station (26', 84'). (x106,800)


51 8d 0 .1U'n


Figure 8e. Electron micrograph of viral like particles and a bacterium. The sample was taken off miami near a sewage outfall. {x84,000) Figure Sf. Electron micrograph of cp 13 A tailed phage particles, which were isolated from St. petersburg Pier water sample using ind i genous bacterial host #13. {x175,000)




Figure 8g. Electron micrograph of <1> 13 C non-tailed phage particles, which were also isolated from the St. petersburg Pier surface water using indigenous bacterial host #13. (x176,470) Figure 8h. Electron micrograph of a group of viral-like particles found in a sample collected 1500 meter below the surface in the Atlantic Ocean east of Northeast Providence Channel.(x180,000)


55 8g


56 .micrographs ranged from 35.9 nm to 176 nm (average 80.65 41.1 nm), and the tail length ranged from 84.1 nm to 576 nm. Because no systematic attempt was made to size phage i n all samples counted, these photographs may not be representative of the natural phage population. Large, tailed phage were more likely to be photographed than small tai lless forms. No filamentous viruses were observed, in part because of the difficulty in identifying them in the complex mixtures of cell types and particles observed in microbial populations. In addition to the survey of viral abundance in a variety of subtropical marine environments, the distribution of viral abundance was investigated along a transect from Tampa Bay to the offshore Gulf of Mexico (Figure 2, St. 1 to St 9) during the Pel ican cruise of 1992. The results indicated that viral concentrations decreased from inshore to offshore (Figure 9) The surface water (upper 2m) viral concentration in the estuarine station 1 sample (2.07 x 107 / ml) was higher (p<0.001) than that of in the station 2 sample (4.65 x 106 / ml) and, both samples had significantly greater (p < 0 .001) viral concentrations compared to the offshore samp l es (St.3 to St.9, ranging from 3 8 to 8 .45 x 105 / ml) No significant difference (p > 0.5) in viral concentration was detected for all the offshore environments sampled (St.3 to St.9). Six out of 9 stations in the transect showed no significant difference (p > 0 05) in viral concentrations between the intermediate depth (3.4m to 250m below surface) and the surface waters (St.1 ,2,4,5,8, 9). Only at station 3 was the viral concentration in the intermediate water ( 1.6 x 106 /ml) more abundant (p < 0 .01) compared to the


E -<0 w 0 T"" (/) ..... r:: ::J 0 0 ..... 0 Q) .... 0 as .... 5 I 10 I I .. I ; I [1'''' '1 . ::: ;: .. 1 I . 0 1 0 01 V :!H Z : !!L:::&: ! % 1 2 3 4 5 6 7 8 9 Station Number I water D Inter. water .Deep water Figure 9. The distribution of viral abundance along a transect from the estuarine environment (station 1) of Tampa Bay to the oligotrophic environment of the southeastern Gulf of Mexico (Station 9) Surface water is from the upper 2 meter of the water column; intermediate water is from 3.4 m to 250 m below the surface; deep water is from 200 m to 2500 m below the surface (when samples were taken from the third depth) (11 --.J


Depth in meters 0 20 40 60 80 100 1000 2 4 6 8 Viral Direct Counts (1 OES/ml) -+-Viral Direct Count 10 Figure 10. The vertical profile of the distribution of viral abundance at a station in the Gulf of Mexico (26 ', 84'). The viral direct counts decreased from the surface to the depth with a subsurface maxima viral concentration at 50 m depth. (J1 0)


59 surface water. Chlorophyll Q. concentrations were also significantly higher in this intermediate water sample (data not shown). The intermediate waters taken at St. 6 and St. 7 had significantly lower (p < 0 .001) viral concentrations (2.44 to 6.88 x 104 /ml) compared to the surface samples (4.87 to 5.4 x 105 /ml). All the deep water samples {200-2500m below surface) had significantly lower (p<0.001) viral concentrations (1.3 to 3 7 x 104 /ml) compared to the corresponding surface samples A vertical profile {Figure 1 0) sampled in the Gulf of Mexico at station 10 during the Pelican cruise of 1992 showed that the viral population decreas e d from surface (6 x 1 05/ml) to depth { 1.4 x 1 04/ ml) with a subsurface maximum (p < 0.001) v i ral concentrati on {8.6 x 1 05/ ml) at the 50 meter depth. This depth profile resembles the profile for the dissolved DNA and particulate DNA. A primary productivity maximum was also found in the 50 meter subsurface samples (data not shown). Seasonal and Diel Viral Abundance in Tampa Bay The results of a seasonal viral abundance study indicated that the viral population had a strong se a sonal pattern (Figure 11) with the highest concentration in the summer (wet season) and lowest in the winter (dry season). Viral abundance increased by one order of magnitude (from 0 2 to 2.3 x 107 / ml) beginni ng in May. This increase was preceded by a doubling in Table


1 0. Partial Correlation Coefficients for the Seasonal Study in Tampa Bay VDC VDC 1.000 BDC Chi .2 Temp. Salinity 0.561 0.725 0.649 -0.803 BDC 1.000 0.513 0.793 -0.518 Chlg_ 1.000 0.588 -0.750 Temp 1.000 -0.534 Salinity 1 .000 60 bacterial abundance from mid-April to the end of April. The increase in bacterialabundance was preceded by an increase in water temperature from 19C to 25C at the end of March. A secondary increase in viral abundance in early September may have resulted from either a bacterial bloom in August or a phytoplankton bloom (as indicated by increased chlorophyllst). The second viral peak (3.0 x 107 /ml) also coincided with the lowest salinity, resulting from late summer rains. A multiple correlation analysis for the seasonal study data (Table 1 0) showed that viral direct counts were inversely correlated with salinity (r =-0.803) and positively correlated with chlorophyll .2 concentration (r=0.725). Bacterial abundance was best correlated with temperature (r = 0. 793). Salinity


61 also inversely correlated with the chlorophyll .a. concentration (r = -0. 750). The results of stepwise solution of multiple correlation analysis indicated the following relationship: VDC = -.223(Salinity} + .676(Temp} + 58.09 (where VDC is in 1 06 /ml, salinity is in parts per thousand, and temperature is in centigrade; n=25, r=0.8507, F=31.428}. This result implies that the variation in viral direct count could be explained by the variation in salinity and temperature. Table 11. Partial Correlation Coefficients for the Diel Study in Tampa Bay VDC BDC Chlg_ DONA T. s. VDC 1.000 BDC 0 .226 1.000 Chi ,a 0.280 0.697 1.000 DONA 0.186 0.165 0.309 1.000 T -0 .052 0.368 0.359 0.225 1.000 s -0.235 -0.429 -0.526 0.525 0.067 1.000 The results of a diel viral abundanr.P. st11rlv in S:in .... 1., ....


D Ja F M A My Ju Jy A S 0 N D Month 62 Figure 11 Seasonal variat i on i n viruses, bacteria and chlorophyll .a concentration. Samples were taken from St. Petersburg Pier by semi-monthly sampling for 13 month, starting at December 1990 and ending at December 1991. VDC is viral direct counts; BDC is bacterial direct counts; Chla is chlorophyll a; T is temperature; S is salinity.


4 voc 3 E 6 E 0 ... BOC 63 Figure 1 2 Die l variat i o n in viruses bacteria and dissolved DNA concentrati ons Estuar i ne water from St. Petersbu r g Pier were collected in a 200 liter polyethylene tank for the diel study. Samples were taken every 4 hours for biological parameters determinat i on DONA i s dissolved DNA; the rest are the same as in f i gure 11.


64 diel rhythm of viral abundance was observed A significant increase (p < 0.01) in viral abundance (3. 7 x 1 07/ml) occurred at midnight, 32 hours into the dielstudy. This increase in viral population was preceded by an increase in bacterial (p < 0.01) and chlorophyiiQ. (p < 0.025) concentrations. The increases in bacteria l and chlorophyll 2. concentrations may have been caused by the elevation of water temperature in the afternoon. Dissolved DNA values did follow a diel rhythm, w ith maximum values found at 28 hour (8pm) into the diel study. The fact that viral direct counts did not correlate with this peak suggests that dissolved DNA was not due to DNA in v i ral particles. The multiple correlation analysis of the data (Table 11) indicated that there was no significant correlation (n = 10, r = 0.4723, F = 0.230, p > 0 5) within any of the parameters in this study. Separation of Viral DNA from Soluble DNA In an attempt to separate DNA enclosed in viral particles from soluble DNA, I employed differential centrifugation. Table 12 shows the effect of differential centrifugation on calf thymus DNA and T2 phage in artificial seawater. Artificial seawater spiked with calf thymus DNA was centrifuged at 201 K x G, 1 03K x G, 37K x G and 25.8K x G The centrifugation speed of 201 K x G resulted in the least amount of calf thymus DNA in the supernatant (6.1 %), while at a speed of 25.8KxG, over 65% of the spiked DNA remained


65 in the supernatant. The centrifugation speeds of 201 K x G and 25.8K x G were also used for sedimentation of T2 phage. An average of over 90% of the phages were found i n the pellet fractions for both speeds Thus, a centrifugation speed of 25.8K x G or lower should enable separation of T2 phage from the majority of soluble calf thymus DNA left in the supernatant. This protocol might also be able to separate dissolved DNA from viral particles in samples from the marine env i ronment. The results of different i al centrifugation of VFF-concentrated dissolved DNA from aquatic environments (Table 13) ind i cated that the difference between the centrifugation speed of 201 K x G and 11.2K x G was not great in terms of viral direct counts. However, changing of the centrifugation speed from 201 K x G to 11.2K x G resulted in a decrease of the dissolved DNA portion in the pellet from 94.8% to 69.2% for the estuarine sample from Fort Desoto. This result suggests that a lot of dissolved DNA in the pellet is not viral DNA in the estuarine environmental samples Ethanol precipitation of the VFFconcentrated environmental dissolved DNA samples or cell-free phage lysates resulted in an increase in the DNA measurement by Hoechst 33258 stain (Table 14). This increase in the DNA measurement ranged from a 1.63 to 7.83 fold, with the highest increase found in the viral abundant estuarine samples of Fort Desoto. Because this increase in the DNA measurement was also found in cell-free phage lysates after ethanol precipitation, it suggested that the dissolved DNA from the environments


66 Table 12. Effect of Differential Centrifugation on Calf Thymus DNA and T2 Phage in Artificial Seawater Treatment Pellet Calf Thymus DNA (0.5 -300 KB) 201 KxG 103 KxG 37 KxG 25.8 KxG T2 phage1 201 KxG, run 1 201 KxG, run 2. 25.8 KxG 60. 6 62.4 26.9 23. 5 82.1 >99 >99 %In: Supernatant 6.1 24.9 45.2 65. 0 1.3 0 .77 <0.1 Unaccount. 33.7 12.7 27.9 11.5 15.6 0 0 1 phage abundance determined by plaque titer the supernatant.


67 Table 13. Effect of Differential Centr i fugation on VFF-concentrated Dissolved DNA and V i ral Populations. Sample 1VFF DNA % 2Viral Counts (pg/ml) (1 06/ml) Fort Desoto 11.2 KxG Super. 0.52 30. 7 NO 11 .2 KxG Pellet 1.17 69.2 19. 4 8.7 16.5 KxG Super. 0.21 10. 3 NO 16.5 KxG Pellet 1.83 89.7 16. 9 6 6 201 KxG Super. 0.13 5.23 NO 201 KxG Pellet 2.37 94.8 23. 8 3.9 Offshore Gulf of Mexico, St. 3 11.2 KxG Super. 0 .38 57.6 NO 11. 2 KxG Pellet 0 .28 42.4 1.05 0 .25 201 KxG Super. 0.33 50.8 NO 201 KxG Pellet 0.32 49. 2 1.61 0.44 1 DNA concentration was determined in VFF-concentrated retentate directly. 2Viral counts were determi ned by TEM in the pellet fractions.


Table 14. Effect of Ethanol Precipitation on Dissolved DNA Measurement Sample DNA Cone. (pg/L) Aft/Before VDC Before EtOH Aft EtOH (106/ml) Medard Reservoir 4.2 0.4 12.1 1.2 2.9 4.9. 5 BBH of Tampa Bay 3.1 0.17 5.47 1.76 4.2.7 7.9 1.0 13.2 2.1 1.67 NO 12.2 1.1 21.5 3.0 1.76 NO Mouth of Tampa Bay 5.04 1.48 11.05 0 .99 2.19 1.8.34 Ft. DeSoto 2.06 11.7 5.68 9.77. 4 1.25 9.64 7.72 23.8.0 0.94 7.36 7.83 13.8.21 Sea Buoy, Miami 2.6 1.26 7.46 1.14 2.87 0.74.2 Chub Cay, Bahamas 0.9 0.67 1.82 0.93 2.02 0.4 Gulf of Mexico, St. 3a 1.96 4.41 2.25 1.61 .4 Gulf of Mexico, St. 7 0.83 1.96 2.33 0.49. 2 T2 Phage lysate 5.59 0.06 12.5 0.58 2 .24 7170 5.21 0.12 12.17 .28 2.34 8000 3.15 0.02 9.54 0.19 3.03 35300 Cl 13 C phage lysate 0.41 0.67 1.63 NO en Ql)


69 Table 15. Effect of Ethanol Precipitation on Calf Thymus DNA and T2 phage in Artificial Seawater that were Fractionated by Ultracentrifugation. Sample Calf Thymus DNA 201 KxG Pellet 201 KxG P. Et. ppt T2 phage (4.3x1 09 } 201 KxG pellet 201 KxG P. Et. ppt Total DONA (pg) 24 18.9 37.4 139.0 % DNase Digest. DNase Digest. (pg) 97.5 97.3 33 87.4 23.4 18.4 12.4 121.6 Viral DNA release by ethanol precipitation= 121.6-12.4 = 109.2 pg DNA. Calculated viral DNA content= 4.3x109 phage x 1.6x10"18 g DNA/phage = 0.68 pg DNA behaved like viral DNA. The measurements of calf thymus DNA did not increase after ethanol precipitation and were nearly 100% DNase digestible (Table 15), indicating that the increase in DNA concentration was not caused by artifacts during ethanol precipitation. A similar phenomenon was also found in a supernatant fraction of the environmental dissolved DNA sample (Table 16) suggesting only soluble DNA remained in the supernatant fraction. The increase in the DNA


70 measurement and DNase sensitivity after ethanol precipitation were found both in the T2 phage lysate (Table 15) and the environmental dissolved DNA pellet fractions (Table 16). These results suggested that the pellet fractions of the environmental dissolved DNA behaved like viral DNA preparations. Ethanol precipitation might cause the release of viral DNA from viral protein capsids making the DNA more accessible to Hoechst stain and DNase If the increased DNA measurement was only from the released viral DNA, it suggested that 109.2 JJg out of 121.6 JJg (89.8%) total DNA was from viral DNA enclosed in T2 particles (Table 15), 0 .525 JJg out of 0.57 JJg (92.1 %) total DNA in offshore Gulf of Mexico station 7 and, 0 .22 JJg out of 0.54 JJg (40. 7%) total DNA in offshore Gulf of Mexico station 3 (Table 16) are from encapsuled viral DNA. However, the calculations of viral DNA content based on the DNA per phage and total phage counts did not agree with those estimations. Using 1.6x1 o-16 g DNA per T2 phage (Freifelder, 1987), the T2 viral DNA should contribute 0.68 JJg (0.56%) of the total dissolved DNA, or about 160 times less than the result from DNA fluorescence measurements. The average DNA content of 9x1 o-17 g DNA per phage (Freifelder, 1987) was used to calculate the viral DNA content in the pellets of environmental samples. The calculated viral DNA content was 1 2 times less than the estimation of viral DNA from DNA fluorescence measurement for the offshore Gulf of Mexico station 7, and 1.5 times less for the offshore Gulf of Mexico station 3, respectively. One possibility of over-estimation of the DNA content by Hoechst


71 Table 1 6. Effect of Ethanol Precipitation on VFF-concentrated Dissolved DNA Samples that were Fractionated by Ultracentr i fugation Sample Total DNA % DNase Digestible (pg/L) Offshore Gulf of Mexico, ST.7 11.2 KxG Super 0.27 63 11.2 KxG S Eppt 0.17 82.3 11.2 KxG Pellet 0.27 48 11.2 KxG P,Eppt 0 .45 93 201 KxG Pellet 0.21 21 201 KxG P,Eppt 0 .625 91.2 DNase Digestible {pg/L) 0.17 0.14 0.13 0.42 0.045 0.57 VDC 0 .97.54 0.49 .19 Viral DNA release by ethanol precipitation = 0.57 0.045 =0.525 pg Calculated viral DNA content= 0.49x109 x 9x10-17 g DNA/phage= 0.044pg. Offshore Gulf of Mexico, St.3 11 2 KxG Pellet 11.2 KxG P,Eppt 201 KxG Pellet 201 KxG P,Eppt 0.6 0.89 0 .49 0.58 46.6 95.5 65.3 93.1 0 .28 0 .85 0.32 0.54 1 .05 1 .61 Viral DNA release by ethanol precipitation = 0 .54-0 .32 = 0.22 pg Calculated viral DNA content = 1 .61x109 x 9x10"17 g DNA/phage = 0.145 pg DNA


Figure 13 (a,b) Electron micrographs of DNA containing membrane vesicle material. (a). From a sample of St Petersburg Pier {x87 ,000) Figure 13b. Electron micrograph of DNA contained membrane vesicle-like material in T2 phage lysate (x229,500)




74 fluorometry may be caused by the special affinity of Hoechst for phage DNA. However, the comparison of the results {Table 17) for the purified T2 phage DNA {0D260 I OD280 ""1.8) measured by four methods {A260 UV absorbance, Hoechst stain, ethidium bromide stain and diphenylamine colorimetry) indicated that there was no significant difference {P<0. 2) between the Hoechst stainingmethod and the other DNA determination methods. Thus, overestimation of the viral DNA content by Hoechst fluorometry seems unlikely Table 17. Comparison of Different Methods for T2 Phage DNA Quantitation. Sample Method DNA content OD2eo/OD280 {pg/ml) T2 DNA Exp.1 A 260 422. 0 1.775 Hoechst 521.8 125. 4 Et-Br 534.5 85.6 Diphenylamine 347. 6 22.5 T2 DNA Exp. 2 A260 223.13 6.89 1.76 Hoechst 282.2 58. 4 The other possibility of over-estimation of the viral DNA content may be because there are other forms of "bound DNA in seawater that behave like virus particles. The DNA may be "bound" to histones or histone-like protein polyphenolic or humic-like proteins. Additionally, DNA-containing membrane vesicles are commonly produced by gram negative bacteria (Dorward and Garon, 1990, Mayrand and Greninier, 1989), and such membrane-like vesicle


75 Table 18. Effect of Chemical Treatment on VFF-concentrated Environmental Samples or T2 Phage Lysates. Sample Treatment Total DNA o/o DNase DNase DNase Digestible digestible Digest. pg/ml pg/ml Aft.Ept. Ft. Desoto Ret. Untreat. 0.31 26.6 0.08 Heparin 0.27 37.4 0.10 T2 lysate Untreat. 9.29 19.8 1.84 3.49 Proteinase K 10.27 0 0 3.48 Lipase 8.19 18.4 1.51 4.25 Chloroform 7.53 18.6 1.40 2.89 structures were observed under TEM in both phage lysates and environmental concentrated samples (Figure 13). Dissolved DNA may be composed of soluble DNA, viral particles and DNA protected by membranes or proteins. In an effort to test this hypothesis, experiments were conducted using Heparin, Lipase, Proteinase K and chloroform to treat VFF-concentrated environmental samplesor T2 cell-free lysates (Table 18) in order to release "bound" DNA other than viral DNA. No increases of DNase digestibility were detected by these treatments. Proteinase K treated samples resulted in 0% DNase sensitivity which may be due to the degradation of DNase by the residual Proteinase K in the sample. The increase of the digestible DNA measurement was observed in the untreated T2 phage sample as well as chemical treated samples after ethanol precipitation. The results of these experiments suggested that either the "bound" DNA was insensitive to these treatments or they were not performed


76 Table 19. Effect of Ethanol Precipitation on Purified Phage Particles Sample DNA con DNA incr. 1VDC 2VDDNA Bet. Et .ppt Aft. Et .ppt {pg/ml) (pg/ml) (lml) {pg/ml) T2 phage 47.1 94.2 47. 1 10.05x1010 16.08 Cl>16 phage 32.7 134.25 101.6 3.5x1 012 195. 3 1viraLconcentration determined by TEM direct counts 2viral DNA calculated by multiply viral concentration with DNA content per phage; 1 6 X 1 016 g/T2 phage (Freifelder, 1987) and 5 .58 X 1 0 "17 g/16 phage (Kellogg unpubl i shed) at the right conditions Another possibility explaining the over-estimation of viral DNA in cell-free phage lysates may have been the presence of host DNA contamination of phage preparations In order to eliminate the effect of nonviral DNA on DNA measurements, ces i um chloride purified T2 phage and Cl> 16 phage preparat i onswere used to quantitate viral DNA contents (Table 19). Increases in DNA measurement after ethanol precipitation were observed in both purified phage samples. For the purified Cl> 16 phage sample, the increased DNA value was similar to the calculated vi ral DNA content using the value of Cl> 16 genome as determ i ned by Kellogg (unpublished data) For the purified T2 phage sample the increased DNA value was about 2.8 fold higher as compare to the calculated viral content using the published value of T2 phage genome size


77 Table 20. Estimated Viral DNA Contribution to Dissolved DNA Content in Aquatic Environments Sample 1Diss. DNA 2Viral DC 3Est. Viral % Diss. (pg/L) (106/ml) DNA Cont. DNA (pg/L) Ft. DeSoto, 5/28 11.7 9.77 1.36 0.88 9 0 Ft. DeSoto, 7/25 9 .63 23. 8 3.9 2.14 22.2 Ft. DeSoto, 10/2 7.63 13 8 0.21 1.24 16.8 St. Pete Pier 4.4 6 .30 2.01 0.57 13. 0 Medard Reservoir 12.1 4.9 2.5 0.44 3.6 Bayboro Harbor 5.34 4 2 0 7 0.38 7.1 Mouth of Tampa Bay 11.03 1.8 0.34 0.16 1.5 Gulf of Mexico, St. 3 9.4 1.61 0.44 0.14 1.5 Gulf of Mexico, St. 7 4.9 0.97 0.59 0.087 1.78 Sea Buoy Miami 11.5 0.74 0.15 0.067 0.58 1Dissolved DNA determined by ethanol precipitation method (DeFiaun et al., 1986) 2Virus counts are for 0.2 pm filtered samples 3Determined by multiplying viral direct counts by an average phage DNA content (9 X 1 0"17 g/phage, Freifelder, 1987) (Freifelder, 1987). However, this increased value was much less (60 times) than that was found in unpurified samples. I suspected that there was still a little contamination of host DNA in the purified preparation. The overall results


78 of thi s study indicated that the over-estimation of viral DNA after ethanol precipitation was from the viral-like extracellular host DNA. The viral DNA contribution to the dissolved DNA content of aquatic environments was estimated based on viral direct counts (Table 20). The average DNA content per phage of 9 x 1 o-1 7 g DNA (Freifelder 1987) was used for the envi ronmental viral DNA calculations. Viral DNA contributed 0.58 to 22.2% of the total dissolved DNA. The greatest viral DNA contribution to d i ssolved DNA was found in estuar ine environments i n the summer. of Lysogeny amongst Marine Bacterial Isolates Bacteria isolated from estuarine, coastal oceanic environments and benthic invertebrates were screened for lysogeny by Mitomycin C. induction of prophage (Table 21). Eighteen point two percent to 100% of bacteria tested were found to contain i n ducible prophage or bacteriocins (Figure 14 a-d). Of all 51 iso l ates, 43.1% were lysogens. These results suggest that temperate phage and lysogenic hosts are common occurrences in the marine environment.


79 Table 21 Selection o f Lysogen From Bacterial Isolates by Mitomycin C Inducement. Site No. of isolates No. of lysogens %lysogenic St. Pete Pier Tampa Bay 10 4 40 North Shore Beach St. Pete, T a mpa Bay 11 2 18. 2 Key Largo Canal St.1 6 2 33. 3 Key Largo, Canal St.2 4 1 25 Hawk Channel Key L a rgo 5 3 60 Mosquito Bank, Key Largo 2 1 50 Algae Reef Key L a rgo 4 2 50 T a rpon Sound, Key L a rgo 3 3 100 Blackwater S o und K e y Largo 4 3 75 Spong e, Key Largo St. 6 2 1 50 Total 51 22 43. 1


Figure 14 (a-d) Electron micrographs of temperate phages or bacteriocin induced from marine bacterial isolates (a). From a marine lysogeny host #5 isolated from St. Petersburg P i er Figure 14b. Electron micrograph of bacteriocin induced from a marine isolate #5 by mitomyci n C inducement. (x225,000)


8 1


F i gure 14c. E lectron m i crog r aph of a temperate phage-like particle induced from a bac terial isolate from the reef environment of Mosquito Bank off Key Largo ( x286,000) Figure 14d. Electron micrograph of a temperate phage-like particle induced from a marine isolate #19 off St. Petersburg, Pier (x305,000)


83 14d


84 CHAPTER 4 DISCUSSION AND CONCLUSIONS The Benchmark VFF device was shown to be an efficient and rapid method to concentrate phage particles and dissolved DNA from seawater. The efficiency of harvesting T2 phage, 16 phage and calf thymus DNA was between 70 and 80%. Although Benchmark VFF with any of the pore-sized ultrafilters ( 100, 50 and 30 KD) could be used to efficiently harvest viruses, only the 30 KD filter collected DNA efficiently. The Pacesetter VFF system with a 100 KD filter recovered less than 30% of environmental viral populations, when compared to a Benchmark VFF device. Because it has over 10 times faster filtration rate than the Benchmark system, it is therefore practical to harvest several hundreds of liters of seawater. The Amicon SWF DC-1 0 is an advanced version of hollow fiber filtration system and has been used to concentrate the viral population for quantitative studies (Proctor and Fuhrman 1990; Suttle et at., 1990, 1991). This study indicated that the SWF DC-10 system recovered 30% of 16 viruses during a study of seeded viruses in seawater. The recovery of environmental v i ral populations varied between 30% and 67% as compared to the Benchmark VFF system. The decrease in viral abundance, caused by 0 2 pm filtration, is not surprising because collection of viruses on 0.2 pm filters has been used for


85 some time by aquatic virologists (Gerba and Goyal, 1982). Viruses are known to adsorb to parti culate matter ( > 0.2 pm), and thereby would be retained on 0.2 pm filters A greater percentage of i ruses passed the filters in samples from oligotrophic waters, perhaps because of the smaller amount of particul ate matter available for viral adsorption. These results are important because most of the viruses (average 60%, and often 90%) are retained on the 0 2 pm filters in the process of filtering water for dissolved DNA measurements, and therefore would not be measured as dissolved DNA. A second result of these findings is that if samples are 0.2 pm prefiltered to remove bacteria and other microorganisms for v i ral enumeration, underestimation of the total viral population may occur. The higher viral concentration in estuarine environments was not unexpected because bacterial abundance has always thought to be the precondition for viral abundance (Ahrens, 1971 ). A significant linear relationship had been reported between phage and bacterial abundance (Paul et al, 1991 c). However, this log/log relationship may only reflect differences between offshore and nearshore environments. The decrease of all the biological parameters as a function of distance from the shore resulted in the significant correlation The multiple regression analysis of the seasonal viral abundance data indicated that variations in viral concentrations could be explained by variate in salinity and temperature in an estuarine environment The strong inverse correlation (r =0 .803) between viral abundance and salinity was found both in the estuarine

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86 samples and samples taken from Key Largo area (data not shown). One possible explanation might be that rivers transported viruses into the estuaries. A second explanation is that the input of nutrients from rivers resulted in a general increase in micarobial production, therefore caused the phage abundance in the estuaries. A further study of freshwater input of viruses will answer this question. The greater viral abundance in the coastal environment was found in mangrove-fringed bodies of water. Amongst those samples, the only salinity measured was in Blackwater Sound, Key Largo. This salinity was much lower compared to the other samples off the Key Largo area (data not shown). It suggests that there may be freshwater inputs in that environment. The other possibility might be that extra nutrient input from detrital (ie. mangrove leaves) food web in the environment supported active heterotrophic populations. Viral concentrations may not only be affected by their biological environment but also by physical conditions of the environment. For example, the sample collected off the beach of South Cat Cay, Bahamas, on a stormy day had the lowest viral concentration of all the coastal environments sampled. This may have been caused by the strong currents mixing the coastal water with oligotrophic offshore water or deep water. On the other hand, the highest viral concentration for a coastal environment was found in an enclosed seawater moat around Fort Jefferson (Garden Key, Dry Tortugas) which had little water movement. The nutrients in runoff from the island may have

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87 supported growth of phytoplankton, bacterial and viral populations Although bacteriophages have been isolated from marine sediment below 800m depth (Wiebe and Liston, 1968), no estimation of viral concentration in the deep sea environments has been presented yet. The TEM observation in this study indicated that virus-like particles in deep sea environments were between 104 to 105 /mi. Other investigators (Proctor and Fuhrman, 1991) also demonstrated the presence of phage in samples from sediment traps moored at a depth of 400m. They suggested that from 2 to 37% of the bacteria in traps had been killed by phage infection. Previous reports of viral abundance are based on random sampling of vari o u s environments. Although these reports are of great value, a clear picture of viral abundance as a function of environment or trophic status has not yet come forth. The data presented in this study fills this need. The results of transect sampling for viral abundance study indicated that surface viral concentrations decreased from estuarine to offshore environments with no great difference among the offshore stations in Gulf of Mexico. Subsurface euphotic zone ( < 100-150 m) measurements had similar or significantly greater viral concentrations than corresponding surface waters. The lowest viral counts were found in the aphotic zone The depth profile of viral distribution indicated that viral population decreased from the surface to depth with a subsurface maximum viral concentration present at 50 meters depth. The decrease of viral abundance from nearshore to offshore can be

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88 explained as relationship of any of the biological parameters as a function of distance from the shore. The distribution of viral abundance with depth may have been caused by biological, chemical, and physical parameters. In general, there is a gradual decline in bacterial numbers down the water column (e. g. Simidu et al. 1983), albeit with a slight increase at the thermocl i ne (e.g. Ezura et al., 1974). The subsurface maxima of phytoplankton parameters and the subsequent decrease below the euphotic zone has also been well documented (Dortch, 1987; Li and wood, 1988). Therefore, the distribution of viruses will follow that of their host populat i ons In terms of physical parameters, solar radiat i on may be an important factor affecting viral vertical distribution under normal subtropical marine environmental conditions. Noble et al. (1992) and Suttle and Chen (1992) have demonstrated in the laboratory that marine viruses are sensitive to solar radiation (A< 320 nm). Infectivities of seeded viral populations declined rapidly in the seawater under subtrop i cal sea surface light intensities (1872 pmol quanta m2 s1 ) Thus, it is suggested that the surface water viral populations may have a lower infectivity and faster decay rate caused by solar radiation than the deeper populations. The subsurface maximum viral concentration may have resulted from the optimal environmental conditions, large host population and lower light intensity Biological parameters were more likely to be the controlling factors Because the viral infectivity was sensitive to sunlight, Suttle and Chen

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89 ( 1992) suggested that there should be a strong diet signal in the concentration of infectious viruses. However, no diet rhythm of viral abundance were detected in this study by TEM viral direct counts. No diel signal was found in other viral diel abundance studies (Bratbak et al., 1992, Heldal and Bratbak, 1991), either. These results may be because the sunlight destroys infectivity but does not remove viral particles. Thus, a large proportion of the viruses in seawater observed in TEM are probably not infective. Dissolved DNA has been operationally defined as that which passes a 0.2 pm Nuclepore filter {DeFiaun, et al., 1986). This could therefore include free or soluble DNA, DNA in virus particles, DNA in ultramicrobacteria (Torrela and Morita, 1981 ), and other forms of less than 0.2 pm DNA (ie. membrane vesicles). Because TEM observation of the 0.2 pm filtrates indicated that this fraction contained far too few ultramicrobacteria to enumerate, I therefore deem these an insignificant portion of the dissolved DNA. The abundance of viral populations in aquatic environments suggested that viral DNA may be an important portion of dissolved DNA. Separation of viral DNA from soluble DNA is important for both ecological and genetic reasons (see introduction). BeeBee ( 1991 ) reported the separation of soluble DNA and viral DNA by ultracentrifugation of 0.2 pm filtered seawater at 140 KxG for 4 hours. He simply defined the DNA extracted from the pellet fractions ( > 20 KB molecular weight) as viral DNA and the DNA in the supernatant (molecular weight less than 0.5 Kb) as soluble DNA. By this definition he concluded that viral DNA

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90 was an important portion of dissolved DNA and contributed up to 85% of dissolved DNA in aquatic samples. Our results of differential centrifugation of soluble DNA (ie. calf thymus DNA) or VFF-concentrated dissolved DNA samples indicated that at a centrifugation speed greater than 100 KxG over 60% of the soluble DNA was in the pellet fractions. Reducing the centrifugation speed to 25.8K x G may leave a majority of the soluble DNA in the supernatant. These results may also indicate that there is no easy way to separate soluble DNA and viral DNA by their molecular weight in the dissolved DNA pool (ie. viral particles behave like macromolecular DNA) DeFiaun et al. ( 1987) also reported that the molecular weight of dissolved DNA ranged continuously from 35.2 Kb to 0.12 Kb in the marine envi ronment by agarose gel electrophoresis The properties of soluble DNA (ie calf thymus DNA) and DNA in viral particles (ie. T2 phage) have been demonstrated to be different in terms of staining with Hoechst 33258 before and after ethanol precipitation and susceptibility to nuclease digestion. For example, ethanol precipitation of calf thymus DNA did not change the DNA fluorescence values by the Hoechst staining method; while ethanol precipitat i on of virallysates resulted in a several fold increase in DNA values over that was determined before ethanol precipitation. This viral-like behavior was also found in the concentrated environmental dissolved DNA samples, and more specifically only in the pellet fractions of the samples Since all prior dissolved DNA measurements have been made by ethanol precipitation, this behavior of dissolved DNA had not

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91 been previous l y detected. A tempting hypothesis is that viral DNA released from viral caps i ds by ethanol prec ipitati on facilitates the binding of the Hoechst stain and resulted i n the increase of DNA fluorescence However, these values for the released viral DNA were much greater than the viral DNA content calculated based on total viral numbers in these samples. I have demonstrated that the over-estimation of viral DNA by the Hoechst sta i n is unlikely. Thus, the second hypothesis is that there are other forms of DNA behaving like viral DNA that coexist with viral particles in the pellet fraction. This hypothesis was supported by the DNA determinat i on in the purified phage particles. The conclusion of this study is that viral DNA is usua ll y an insignificant portion of total dissolved DNA pool, be i ng as much as 22% of the dissolved DNA, and i n most cases, less than 5%. A large portion of dissolved DNA is in combi ned forms. DNA-containing membrane vesic les could possibly be an important form of dissolved DNA in these environments. It is reasonable to believe that most phages in aquatic environments are temperate rather than virulent (Freifelder 1987, Bratbak et al., 1990). This study has demonstrated that lysogenic bacterial strains and temperate phage a r e common amongst marine bacterial isolates. We do not, however, know how this applies to the non-cultivatable majority of bacteria in the oceans. These results suggested that temperate phages may not on l y play an important role in balancing phage and bacterial populations, but may also be significant in bacterial gene transfer Transduction has been found more frequently when the

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92 recipient bacterium was a lysogen, probably because of the immunity to superinfection imparted by the resident prophage (Miller et al., 1991). The common occurrence of lysogeny implies the capability for natural transduction. The results presented here provided a basic understanding of viral populations in the marine waters. We have discussed their abundance, distribution and contribution to the dissolved DNA pool. We have also demonstrated that lysogens were common amongst culturable marine bacteria Future studies will focus on the genetic characteristics of viruses and their potential for gene transfer by the process of transduction in natural environments.

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93 REFERENCES CITED Ackermann, H., and M. S. DuBow. 1987. Viruses of prokaryotes. General Properties of Bacteriophages. Flarida: CRC Press, Inc. Ackermann, H., and T. Nguyen. 1983. Sewage coliphages studied by Electron Microscopy. Appl. Environ. Microbial. 45:1049-1059. Adams, M. H. 1959. Bacteriophages. lnterscience publisher. New York. Ahrens, R. 1971. "Untersuchungen sur verbreitung von phagen der gattung agrobacterium in der ostee". Kieler meeresforsh. 27: 102-112. Arderson, T. F. 1948. The activation of the bacterial viruses T 4 by Ltryptophan. J. Bacterial. 55:637-649. Azam, F., T. Fenchel, J. F. Field, J. S. Gray, L.A. Meyer-Reil, and F. Thingstad. 1983. The ecological role of water-column microbes in the sea. Mar. Prog. Ser. 10:257-263. Barkstale, L., and S. B. Arden 1974. Persisting bacteriophage infections, lysogen, and phage conversions. Ann. Rev. Microbiolol. 28:265-271. Baross, J., J. Liston, and R. Y. Morita. 1978a. Incidence of Vibrio parahaemolyticus bacteriophages and other Vibrio bacteriophages in marine samples. Appl. Environ. Microbial. 36:492-499. Baross, J., J. Liston, and R. Y. Morita. 1978b. Ecological relationship between Vibrio parahaemolyticus and agar-digesting Vibrios as evidenced by bacteriophage susceptibility patterns. Appl. Environ. Microbial. 36:500-505. BeeBee, T. J. 1991. Analysis, purification and quantification of extracellular DNA from aquatic environments. Freshwater Biology. 25:525-532. Bergh, 0., K. Y. Borsheim, G. Bratbak, and M. Heldal. 1989. High abundance of viruses found in aquatic environments. Nature. 340:467-4668.

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