THE DISTRIBUTION AND MOLECULAR CHARACTERIZATION OF DISSOLVED DNA IN AQUATIC ENVIRONMENTS by Mary F. DeFlaun A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Marine Science in the University of South Florida December, 1987 Major Professor: Dr. John H Paul
Graduate Council University of South Florida Tampa, Florida CERTIFICATE OF APPROVAL Ph.D. Dissertation This is to certify that the Ph.D. Dissertation of Mary F. DeFlaun with a major in Marine Science has been approved by the Examining Committee on October 30, 1987 as satisfactory for the dissertation requirement for the Ph.D. degree Examining Committee: Major Dr. John H. Paul Meiri8'e?: Dt. Torres Member: Dr. Edward Van Vleet er@gory Stewart
@) Mary F. DeFlaLm 1 987 All Rights Reserved
DEDICATION To my mother, Dorothyanne DeFlaun, who taught me to ask the tough questions and to my father, Austin DeFlaun, who taught me the patience to fin d the answers.
ACKNOWLEDGEMENTS A number of people deserve special thanks for their participation in this research effort. I would like to thank Dr. John Paul, for teaching by example that excellence is its own reward I would also like to thank the members of my committee; Drs. Gregory Stewart, Jose Torres, Ted Van Vleet, Bruce Cochrane and Gabe Vargo for their expert advice and youthful perspective. These men will never grow old. To my friends and family, both willing and unwilling participants in this effort, I gratefully acknowledge your support. In particular I would like to thank Wade Jeffrey for tunes, graphics, and many discussions 'over the wall' ; Andy David for moral support and reptile lore; Denise Bombard for sustenanc e that went beyond her constant supply of coffee; tvlargarita Conkright for proving that the third world can be glamorous; Rosanne Joyce for helping me work out my frustrations on the tennis court; little Marky for surfing tips and tubular vibrations; Michael D'Andrea, minister of transportation, for keeping my wheels on the road and my spirits in the air; Nancy Clark for her perverted sense of humor; Joe Donnelly for the extra personal space; Jim Bannon and Nancy Greenstein for attitude adjustment; Dave Williams for graphics and social commentary; my sisters and brother, t heir existence defies all the laws of genetics; the Rudnick family, who convinced me that if you think enthusiastic, you will be enthusiastic; my parents, who were always so proud of me that I had to rise to their expectations and finally to my husband, Steven Rudnick, he made me do it. ii
TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ABSTRACT CHAPTER 1: INTRODUCTION CHAPTER 2: METHOD FOR DISSOLVED DNA DETERMINATION Intr>oduction Materials and Methods Sampling sites Materials Concentration and Measurement of DNA Verification of the Method Mithramycin Method Results and Discussion Verification of the Method Comparison with Mithramycin Method Concentration of Dissolved DNA in the Environment Freshwater> Samples CHAPTER 3: DISTRIBUTION OF DISSOLVED DNA IN AQUATIC ENVIRONMENTS Introduction Materials and Methods Results Gulf of Mexico Cor>al Reefs Tampa Bay Diel Studies Freshwater Environments Discussion CHAPTER 4: MOLECULAR CHARACTERIZATION OF DISSOLVED DNA Introduction iii v Vi viii 15 15 17 17 17 18 19 20 21 2 1 26 zr 29 32 32 32 37 37 41 44 46 50 51 58 58
Materials and Methods 59 Bacterial Strains and Plasmids 59 Plasmid Isolation 59 Probe Purification 61 Hybridization 62 Sensitivity and Specificity of Probing 64 Molecular Weight Determination and Detection of Genes in Environmental Samples 64 Recovery from ASW and ARW 66 Survival of Plasmid DNA in Seawater 67 Results 67 Molecular Weight of DONA 67 Sensitivity and Specificity of Probing 71 Recovery from ASW and ARW 75 Survival of Plasmid DNA in Seawater 82 Detection of Genes in Environmental Samples 88 Discussion 99 CHAPTER 5: SUMMARY AND CONCLUSIONS LITERATURE CITED APPENDIX 1 : HOECHST 33258 STAINING OF DNA IN AGAROSE GEL ELECTROPHORESIS Introduction Methods Results and Discussion APPENDIX 2: LARGE SCALE PLASMID DNA ISOLATION APPENDIX 3: RECOVERY OF DNA FROM L O W MELTING TEMPERATURE AGAROSE IN A MINIGEL APPARATUS iv 106 109 118 118 119 120 126 128
1 2. 3. 4. LIST OF TABLES Concentration of dissolved DNA in various aquatic environments Dissolved DNA in the CSM and overlying waters Bacterial strains and plasmids. [3H]Thymidine incorporation rates for bacterial strains used in colony hybridization studies. v 29 42 60 75
LIST OF FIGURES 1 Linear regression of the amount of dissolved DNA measured in four volumes of Bayboro Harbor water. 25 2. Locations of the three stations (A, B and C) sampled during the seasonal study in Tampa Bay and the location of Bayboro Harbor (BH) where two of the diel studies were performed 34 3. Maps of the Crystal ( upper panel ) and Alafia (lower panel) rivers and station locations. 35 4. Distribution of dissolved DNA in the southeastern Gulf of Mexico during summer 1984 and 1985. 38 5. Depth profiles for dissolved DNA, salinity and temperature from Cruise 8514. 40 6. Dissolved DNA concentrations in the coral surface microlayer (CSM) and the surface waters in a transect across an Acropora cervicornis patch reef in the Dry Tortugas. 43 7. Salinity (ppt), water temperature (T, C), dissolved DNA (DONA) and dissolved organic carbon (DOC) during a monthly sampling study in Tampa Bay. 45 8 Diel studies performed over a coral reef in the Dry Tortugas and following a buoy in the Gulf of Mexico. 48 9 Diel studies in Bayboro Harbor (Tampa Bay); June 1986 study (top) and August 1986 (bottom). 49 10. Dissolved DNA (DONA) concentration protocol for hybridization. 65 11. Agarose gel electrophoresis of dissolved DNA samples. 69 12. Molecular weight determinations by agarose gel electrophoresis. 70 13. Standard curve of the TK gene detected by [35s] labelled TK on nitrocellulose. 72 14. Specificity of TK probe. 73 15. Two standard curves of TK hybridized to [3H]labelled TK probes with a detection limit of approximately 100 pg of DNA. 76 vi
16. Recovery from ASW. 78 17. Reco very of TK from ASW wit h c35s]TK probes. 79 1 8 Recovery from ARW with [35s]TK probe. 80 19. Variability in recovery of DONA from ASW. 8 1 20. Plasmid survival in seawater -results of dot blots counted by LSC. 83 21. Plasmid survival in seawater -results of gel electro -phoresis and molecular probing of gel filter. 8 4 22. Plasmid survival in seawater #2 -results of gel electrophoresis of the three treatments. 86 23. Plasmid survival in seawater -results of molecular probing of gel filters with [ S]TK. 87 24. Plasmid survival in seawater #2. 89 25. Sensitivity of TK probe and detection of TK in environmenta l samples. 9 1 26. P robing environm ental DONA for the TK gene 93 27. Large v olume of Bayboro Harbor dissolved DNA (10 1) for molecular probing with the DmGST2 gene o n gel filter (B) and dot blot (C). 95 28. Freshwater samples for MW and molecular probing. 96 29. Freshwater samples for MW determination and probing with TK. 97 30. (A) Ethidium bromide stained gel. 121 31. (A) Ethidium bromide stained gel. 123 vii
THE DISTRIBUTION AND MOLECULAR CHARACTERIZATION OF DISSOLVED DNA IN AQUATIC ENVIRONMENTS by Mary F DeFlaun An Abstract Of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Marine Science in the University of South Florida December, 1987 Major Professor: John H. Paul viii
The of dissolved DNA in oceanic, and in southwest Florida and the Gulf of Mexico was by using a method the measurement of dissolved DNA based on the of Hoechst 33258-DNA complexes. Oceanic concentrations of DNA from 0.2 to 19 IJg/1, as a function of distance shore and depth in the column Samples of the surface (Cs-1) collected on in the Tortugas had dissolved DNA 1 8 to 11 7 times that in the ing water concentrations, at stations in Tampa Bay, FL a 15 month followed the seasonal in (Novvalues (6 .05 1.78 IJg/1) were well below the the of the (11.31 + 1.93 lJg/1). Dissolved DNA and dissolved DNA phosphorus in the 0 .11 0 .06% and 6.53 6.48% of the dissolved (DOC) and dissolved ( DOP), vely Diel studies indicated little in dissolved DNA concentrations in while in the were significant, with maximum in nighttime samples Although of dissolved DNA in the Alafia (x = 8.80 + 6.21 1-1g/l; n = 16) than those in the Crystal (x = 4.10 2 32 1-1g/l; n = 10), values as low as 1 .14 1-1g/l in the Alafia. A wide of molecular weights (determined by gel electrophoresis) was found DNA aquatic i x
(< 0.1 to> 36 kb). These results indicate that dissolved DNA is in a size range sufficient to contain gene sequences, which may be important in natural tranformation of microbial populations A model system for probing extracellular DNA from aquatic env ironments was developed using the plasmid pS8TK2 0 containing t he herpes simplex thymidine kinase (TK) gene. Plasmid DNA and the TK gene f ragment added to artifici a l seawater were concentrated and probed with c3 5s]l abelled TK to establis h percent recovery and detection limits for the method. The degradation o f plas mid DNA added to a natural seawater sample was monitored over a 36 h period by probing with the TK gene probe. Intact plasmid was detected f o r up to 4 h and DNA hybridizable to the TK probe was detected for up t o 2 4 h These methods were used to p r obe for the TK gene in environmental samples of extracellular DNA. Hybridization to the TK probe was detected in both freshwater and estuarine samples. Abstract app r oved: X Dr. J H: Paul Major Professor Associate Professor Dept of Marine Science Date df
CHAPTER 1 : INTRODUCTION Dissolved organic matter (DOM) in seawater rarely exceeds a few milligrams of carbon per liter, and of this pool only 10-20% may turn over rapidly enough to be considered biologically labile (Bell, 1984). Although its simpler components have been identified, the high molecular weight compounds in dissolved organic matter are largely uncharacterized (Paul 1983; Carlson et al. 1985b). It is assumed that a portion of the high molecular weight compounds in the DOM come from lysis or excretion by bacterial and phytoplankton cells and inefficient grazing on these cells ('sloppy feeding'; Azam and Cho, 1987). Dissolved carbohydrates amino acids and adenosine triphosphate (ATP) are among the cellular components that have been measured in seawater. Total carbohydrates in seawater have been determined by the MBTH method of Burney and Sieburth ( 1977). Concentrations measured in Narragansett Bay and Rhode Island Sound ranged from 452 to 2023 and accounted for 5 8 to 17.9% of the dissolved organic carbon present. The dissolved carbohydrates wer e largely due to the release of recently fixed photosynthate, demonstrated by a decline in dissolved carbohydrate in dark incubations and an increase in light incubations (Burney, 1986). Dissolved proteins ca nnot be measured directly in seawater due to the heterogeneity of this group therefore, amino acids in the combined (Bada et al. 1982) and free state (Fuhrman and Bell, 1985) are the components most often measured. Combined amino acid concentrations are approximately ten times that of free amino acids
2 (Bada et al., 1982). Dissolved ATP has been measured in a variety of marine and freshwater environments ranging from 65 ng/1 in waters off southern California (Azam and Hodson, 1977) to 3800 ng/1 in a Danish Lake during the decline of a phytoplankton bloom (Riemann, 1979). Production of dissolved organic phosphorus (OOP) compounds occurred at a rate ranging from 2-328 ng P 1-1 h -1 in surface waters at two North Pacific oceanic stations (Orrett and Karl, 1987). Concentrations of DOP were not rapidly increasing i n these waters, indicating rapid uptake or hydrolysis of DOP and its importance as a source of phosphorous for the microbial community Nucleic acids comprise a large organic phosphorus reservoir in bacterial cells (Orrett and Karl 1987), may contribute significantly to the pool of DOP and serve as a useful tracer for dissolved o rganic molecules in aquatic systems Although organic phosphorous compounds have been measured in aquatic systems for many years (Armstrong and Tibbits, 1968; Solorzano and Strickland, 1968; Lean, 1973), relatively few of the compounds that make up this pool have been characterized (Orrett and Karl, 1987). Measurement of deoxyribonucleic acid (DNA) in the oceans was first advocated by Holm-Hansen et al. (1968) as an indicator of microbial biomass. DNA depth profiles were similar to those of particulate organic carbon (POC) and particulate organic nitrogen (PON), but unlike the chlorophyll a profile. Chlorophyll is a commonly used measure of phytoplankton biomass. The ability of over half of the detectable DNA to pass through a glass fiber filter led Holm-Hansen et al. (1968) to conclude that this portion was either soluble, colloidal, or associated with detritus in the less than 2 fraction. This work discredited
3 DNA as a measure of microbial b i omass in the ocean but suggested the existence of a soluble component of DNA in seawater The methodology to detect these molecules in dilute solution, however had not been developed Concentrat i on of purines and pyrimidines in seawater were i nferred by the b i oas s a y technique e m ployed by Belser ( 1 959) Unabl e to measure low concentrations of organic material in solution, he used auxotrop h s to detect o r ga nic micronutrients in seawater which included nucleic acid bases. The conclusion of HolmHansen (1969) that soluble DNA exists in seawater was correct, but for the wrong reason Bacteria were not yet considered to be a significant proportion of the particulate organic carbon in seawater Since that time particulate DNA (PDNA) measurements have proven to be an accurate measure of bacterial biomass i n aquatic systems (Paul and Myers, 1 982; Paul an d Carlson 1984; McCoy and Olsen 1985) Previously developed methods fo r measuring dissolved DNA (DDNA) are unsuited to routine analysis in seawater due either to expense (time and money), sensitivity or specificity. The first method used in seawater to concentrate and measure DDNA involved its adsorption onto in situ precipitated Baso 4 (Pillai and Ganguly Hydrolysis of the nucleic ac i ds was accomplished by heating at 100C for 3 h in 0.02 N HCl. The hyd r o l ysa t e was compared t o calf thymus DNA standards and optimum recove r y and purity was assessed by ultraviolet (UV) spectra at 260 nm. Over a period of six months in Bombay Harbor DDNA concentrations varied between 1 3 -24 1-1 (Pillai and Ganguly 1 972) Minear ( 1972) used gel filtration to look at the molecular weight range of dissol ved organic phosphorus (DOP) compounds extracted from lake water Of the high (>50 ,000) molecular weight fraction,
4 19.6-57 .1% was composed of DNA. Considering DNA to be 8 weight percent -1 phosphorus, this corresponds to 4-30 of DONA 1 . These are minimum values since they were not corrected for recovery which was less than 100%. A correlation coefficient of 0. 966 between DONA and total pigments and 0 970 between DONA and chlorophyll suggests a phytoplankton source for these concentrations. This assumption was tested by Minear (1972) with a culture of Chlamydomonas reinhartii. The molecular weight pattern of the dissolved organic compounds in the culture media was very similar to that found in the lake water with 20-45% of the high molecular weight fraction composed of DNA, corresponding to 34-281 of DONA An independent test was run to confirm that these concentrations were not simply the result of cell lysis during filtration. The soluble materi a l was isolated without filtration by a dual chamber membrane dialysis culture flask. This apparatus enables a culture or water sample to be separated from a sterile volume of water by a 0 .22 membrane. Diffusion of soluble substances especially high molecular weight substances is a lengthy process, but using this system Minear (1972) was able to demonstrate that the extracellular DNA measured in his water and culture samples was not a n artifact of filtration. While developing a high performance liquid chromotography (HPLC) method for the measurement of thymine in the northern Adriatic Sea Breter et al. (1977) found thymine not associated with particulate material. Thymine is a nucleic acid base that makes up, on the average 10% of the weight of DNA. The dissolved thymine concentrations, measured at four depths from the surface to 30 m -1 -1 ranged from 25-75 ng thymine 1 or 250-750 ng DONA 1
The ability of marine bacteria to u t ilize dissolved nucleic acids and the presence of nucleases in the marine environment has b een investigated by Maeda and Taga (1973 1974, 1976 1981) A constant and rather large proportion of heterotrophic bacteria isolated from seawater (Maeda and Taga, 1974) were able to hydr olyze DNA in culture. Seventy -eight of these 88 strains could also hydro l yze RNA. DNA added to sterile seawater was also hydrolyzed indicating that this process occurs in situ. An i nc r ease in growth of a marine Vibrio sp. was demonstrated upon addition of DNA to the culture; co mplete hydrolysis of the DNA occurred w ithin 2 4 hours. In order to demonstrate this process in situ, Maeda and Taga (1973) looked for DNase activity i n seawater and sed iment. Approximately 40% of the DNase activity present in seawater was assoc iated with particulates (either microbes or particles), w ith the remainder in the dissolved fraction. The degradation of DNA in seawater was linear with time for two days the reaction rat e of the enzyme being dependent on su bstrate conce ntration in the range of 1-10 mg DNA Considerable DNase activity was also found in sediments (Maeda and Taga, 1981 ) Extrapolation of these results to 5 en v ironmenta l conditions however, is difficult because the experiments w e r e conducted with concentrations of DNA far exceed ing t hose found in the environment. A seasonal study of DNase activity in Tokyo Bay (Maeda and Taga, 1981 ) inc lud ed measurements of chlorophyll a p articulate DNA and RNA, and seston. DNase activity peaked during summer months, but did not follow profiles of chlorophyll an indicati on that the DNase producers were not phytoplankton Vertical profiles of DNase activity
6 generally showed a maximum at about 5 m rapidl y decreasing to a negligible amount at about 80 m The rapid decrease in the ratio of DNA and RNA to seston with depth suggested to the authors that there is a rapid turnover of the pools of RNA and DNA in seawater An extracellular nuclease purified from a marine Vibrio sp. was shown by Maeda and Taga (1976), to be active both as a DNase and as an RNase. It is activated by magnesium and stabilized by calcium ions in seawater that occur at the optimal concentrations for this enzyme. Increases in enzyme stability were also observed w hen particulates were added to the seawater providing neutral solid supports for the enzyme. Increasing the hydrostatic pressure from 0-1000 atmospheres deactivated the enzyme, but the Vibrio sp was isolated from 150 m (16 atm) so this may not be a factor for this particular species. Bazelyan and Ayzatullin (1979 ) using Michaelis -Menten kinetics, estimated turnover times for bacterial DNA in seawater of 10-20 days in surface waters 1-2 months in intermediate and 3-7 months in cold deep water These estimates were extrapolated from laboratory experiments using high substrate and enzyme concentrations. More realistic estimates have been obtained by adding DNA to seawater samples at ambient concentrations. Using this approach, Paul et al. (1987) measured turnover times of approximately 6 5 h in eutrophic marine environments and 23 h in offshore oligotrophic environments (unpublished data). Dissolved DNA was hydrolyzed by both cell-associated and extracellular nucleases present i n seawater (Paul et al., 1987) Studies of the degradation of dissolved proteins (Hollibaugh and Azam, 1983) and dissolved ATP (Azam and Hodson, 1977) in seawater, however, have shown that cell-associated enzymes are
7 and that dissolved enzymes in not a Dissolved at a of 3%/h in samples which was to be a minimum estimate due to the method used to it (Hollibaugh and Azam, 1983) Dissolved ATP 0 .59 to 15.2%/h (Azam and Hodson, 1 977) and dissolved DNA in 5 was at an of 1 1 .65 47%/h (Pau l e t al., unp ublished data). In addition to column high of DNA not associated with cells have been in sediments (Novitsk y and 1985). A of the DNA in sediments is bound to the sediment themselves and is thus less available by DNases than DNA et al., 1981). T h i s may in much times DNA in sediments and than in the column. Novits k y and ( 1 985) found that >75% of the DNA 36-274 pg g-1 weight sediment) found in coastal sediments a sewage outfall was Using DNA Novitsky (1986) a time of 19.6 days the DNA in sediments This is a estimate since not all of the DNA could be labelled by a Also, DNA was labelled in live cells that subsequently killed, the DNA was not initially which could lengthen the time. The stability of DNA in the sediments may make it a likely place to find ge netic in populations than in the column et al., 1 983) It has been suggested that the of DNA in the is a not only
dispersal of traits, but also for the long term survival of microbial genes ( Reanny et al. 1983). Very little is known about how marine bacteria utilize dissolved nucleic acids. Bacteria are known to possess both extracellular and membrane bound nucleotidases (Ahlers et al., 1978). The extent to which nucleic acids are hydrolyzed before uptake into the cell is also unknown. Evidence suggests that some nucleosides can be transported into the cell intact, whereas others are cleaved to the free base and sugar before uptake ( Pandey, 1984) Bdellovibrio bacteriovorus transports and accumulates intact nucleoside monophosphates (UMP and dTMP) from the cell contents of their prey by two different substratespecific transport systems The ability to take up intact nucleoside monophosphates, however, is considered to be a rare ability among the prokaryotes, most often associated with parasitic bacteria (Ruby et al., 1985). 8 The presence of extracellular DNA in seawater has implications beyond it's potential as a source of nutrients. The persistence of dissolved DNA may be an avenue for genetic exchange between bacteria in aquatic systems The persistence of extracellular DNA in aquatic environments has not previously been considered in predictions of the transferability of DNA from recombinant organisms to native bacteria (Lev in and Stewart, 1977). The existence of DDNA in marine and freshwater and environments, however, and the possibility of transfer into viable organisms (Gealt et al., 1985), further complicates this assessment. Genetic exchange among bacterial populations ca n be effected by the mechanisms of conjugation t ransduct io n and transformation.
9 Conjugation requiring cell-to-cell contact, is plasmid mediated and transduction is bacteriophage mediated; both require the transfer of DNA from a donor to a recipient bacterium. Transformation involves the uptake by bacteria of DNA that is present in solution in their environment (Stewart and Carlson, 1986). All three mechanims for genetic exchange can result in the transfer of either chromosomal and/or plasmid DNA (Stotsky and Babich, 1986). The genes located on plasmids are more likely to encode for the 'peripheral' functions of the cell, or those that enhance survivability during environmental stress, or that confer a nonessential trait. Genes on the chromosome, however, include those needed for essential life functions of the cell and to maintain the gross taxonomic identity of the species (Davey and Reanney, 1 980) Interest in genetic transfer in the environment has been generated by the widesp read transfer of antibiotic resistance, and the ability to degrade xenobiotic compounds. The appar ent ease with which these traits have spread among microbial populations has generated a great deal of interest in the mechanisms of exchange ; there is very little known about how these transfers occur in the environment Many studies have derronstrated the ability of environmental isolates to act as donors or recipients in genetic exchanges with well characterized laboratory strains (Shaw and Cabelli, 1980; Kellog et al., 1981; Alcaide and Garay 1984; Gauthier et al., 1985 ; McPherson and Gealt, 1986). Laboratory studies of this kind however, fail to account for the numerous biotic and abiotic factors that may affect genetic transfer in the environment (Stotsky and Babich, 1986). Other studies of genetic exchange involving environmental isolates have demonstrated
10 tr>ansfer> in situ but in the absence of the r>esident micr>obial population (Mach and Gr>imes, 1982; Gealt et al. 1985). The rrost envir>onmentally significant studies have derronstr>ated the potential for> genetic exchange in the pr>esence of the natur>al micr>obial community Plasmid transduction in Pseudomonas aer>uginosa was derronstrated in the presence of the indigenous microbiota of a freshwater reservoir (Saye et al., 1987) Transconjugation of plasmid DNA with indigenous strains as mobilizers and recipients has been shown to occur in a wastewater treatment facility (Mancini et al. ,1987). Resistance to antibiotics and the ability to degrade xenobiotic compounds, which spread rapidly through microbial communities, are most often found to be encoded by genes found on plasmids (Davey and Reanney, 1980). Plasmids exist in marine bacteria under> a wide variety of environmental conditions, from the rrost polluted (Sizerrore and Colwell 1977; McNicol et al., 1980; Hada and Sizerrore, 1981), to the most pristine water>s (Kobori et al., 1984) In situ plasmid transfer has been implied by laboratory studies that have duplicated aquatic or marine conditions and have effected transfer between bacteria (Mach and Grimes, 1982; Alcaid e and Garay, 1984). The warm temperatures and high salinity conditions typical of tropical oceans were found to enhance plasmid DNA tr>ansfer of mercury resistence markers from marine strains to! coli (Gauthier et al. 1985) In the water column, however, conjugative plasmid tranfer seems unlikely due to the low density of cells and low nutritive status (Curtiss, 1976). In this environment, transformation by extracellular DNA seems a more likely route for gene transfer. Detection of a single gene among a highly varied background of DNA
11 presents some special problems for environmental studies. Identification of a particular gene sequence am::>ng environmental species involves developing a probe for a specific marker and then using the probe to detect that gene sequence (Barkay et al. 1985; Sayler et al., 1985). Colony hybridization techniques are ideal for screening a small number of species, but are inadequate for an entire suite of species from any given environment Although colony hybridization is a highly sensitive method for the detection of single copy genes (Maas, 1983), it has limited usefulness in environmental work because only those bacteria capable of being isolated can be screened, Studies in aquatic environments have indicated that a large percentage of the bacterial species present cannot be cultured under laboratory conditions (Rollins and Colwell, 1 986; Munro et al., 1987). Newer methods for detecting genes in environmental samples involve extracting total bacterial DNA from the sample and probing the total extract for the gene in question (Holben et al., 1987; Jansson et al., 1987). This community genome' approach to gene detection circumvents the problems associated with culturing organisms from environmental samples The detection of viruses in aquatic environments has the same problem of culturabili ty. Methods have been developed for the concentration and detection of hepatitis A virus by both ronoclonal antibody immunological tests (A-ELI SA) (Nasse r and Metcalf, 1987) and b y DNA hybridization tests (Jiang et al., 1986). Sensitivity of detection is approximately an order of magnitude greater by DNA probing than by the A-ELISA technique, but still requires a sample size of 189 liters of water (Jiang et al., 1986) It has been assumed that the ultimate fate of recombinant DNA
( recDNA) sequences is dependent on the survival and growth of the genetically engineered microorganisms (GEMs) after their release in the environment The presence of extracellular DNA in the environment, however, suggests. other possibilities. Under some conditions extracellular DNA may be very susceptible to enzymatic degradation, or it may persist in the absence of an appropriate host. In the latter case genetic information could be transmitted to any suitable host in the environment. Natural transformation appears to be a fairly common trait among bacteria (Stewart and Carlson 1986); therefore, it may be impossible to keep inserted genes isolated within a single bacterial strain once they are introduced into the environment. The potential for extracellular DNA in aquatic environments to participate in the dissemination of genetic information among microbial populations needs to be addressed in the assessment of t h e risk associated with the use of GEMs in the environment As part of a comprehensive study of extracellular DNA in aquatic environments, the objectives of this study were: 1 To verify the assay for dissolved DNA by; a) determining its applicability to a variety of marine environments, b) modification of the method for freshwater samples c) comparison with an alternative method and d) determining the variability between subsamples. 2 To describe the spatial and temporal distribution of extracellular DNA in both marine and freshwater environments to determine the extent and periodicity (seasonal and diel) of its occurrence in aquatic environments 3 To relate concentrations of dissolved DNA to the concentrations of the broader classifications of the dissolved organic matter (DOM) in 12
aquatic environments (dissolved organic carbon [DCC] and dissolved organic phosphorous [DOP] ) in order to determine the significance of dissolved DNA (DDNA) in the DOC and DOP pools. 13 4. To measure the molecular weight range of extracellular DNA from freshwater and marine environments in order to determine whether gene-sized pieces of DNA might exist in the dissolved fraction. The existence of gene -sized pieces of dissolved DNA in aquatic environments would have genetic significance as a route for genetic exchange via transformation. 5 To develop the methodology to probe extracellular DNA from aquatic environments for specific gene sequences The ability to detect genes in this pool of potentially transforming DNA could be used to monitor the fate of recombinant DNA (recDNA) sequences from genetically engineered microorganisms (GEMs) used in environmental applications or any other gene sequence of interest. 6 To follow the hydrolysis of a plasmid-encoded gene from its introduction until it is no longer detectable by molecular probing in order to determine its survival time in seawater. The amount of time that a gene can exist in seawater may determine whether transformation can occur in that environment The thymidine kinase (TK) gene was chosen for both the testing of the molecular probing methods and for the actual probing of environmental dissolved DNA samples Thymidine kinase catalyzes the phosphorylation of thymidine (TdR) to thymidine 5 '-monophosphat e (TMP) in the pyrimidine salvage pathway of DNA synthesis (Saito and Tomioka, 1984). In the de novo pathway, TMP is synthesized directly from deoxyuridine monophosphate by the enzyme thymidylate synthetase
14 (carlson et al., 1985a) Although the distribution of the TK gene among microorganisms is not very well known, certain fungi, algae and cyanobacteria lack this enzyme (Moriarty, 1986). A large prop ortion of marine bacteria are thought to have thymidine kinase because roost of the metabolically active bacteria incorporate radioactive thymidine (Fuhrman and Azam, 1982). Pseudoroonads, however, are unable to incorporate tritiated thymidine, perhaps due to a lack of a thymidine transport system into the cell (Pollard and Moriarty, 1984) or perhaps due to the lack of thymidine kinase activity which has been found in other pseudomonads (Saito and Tomioka, 1984; Carlson et al., 1985a). The thymidine kinase gen e was used as a model DNA sequence with which to test these molecular probing methods beca use as common as it is anong marine bacteria, it would alnost certainly be present in extracellular DNA in seawater
CHAPTER 2: METHOD FOR DISSOLVED DNA DETERMINATION Introduction Approximately 90% of the organic substances in seawater exist as dissolved compounds of which only about 15% have been characterized (MacKinnon, 1981). Uptake studies of the simpler dissolved organic compounds (amino acids, monosaccharides) in different aquatic environments indicate that free-living heterotrophic bacteria are the most active and efficient consumers of these compounds (Azam and Hodson, 1977; Hodson et al., 1981 ; It turriaga and Zsolnay, 1981 ; Ferguson and Sunda, 1984; Murray and Hodson, 1984) However, few measurements of the concentrations of dissolved macrorrolecules have been made, and even less is known about the utilization of these molecules (Ogura, 1977) The problems inherent in measuring the concentration of a complex macromolecule are greater than measuring a simple rronomer. Yet, these measurements are crucial to the study of the cycling of these compounds. 15 As a constituent common to all living cells, DNA is a potential component of the dissolved macromolecular fraction in aquatic environments. Using the diaminobenzoic acid (DABA) method of Kissane and Robins (1958), Minear (1972) found appreciable amounts of dissolved DNA in lyophilized lakewater samples. In this study, 19. 6 to 57.1% of the high-molecular-weight phophorus compounds were DNA, corresponding to 4-30 of dissolved DNA liter1 This technique cannot be applied to marine samples, because seawater cannot be concentrated by lyophilization.
Pillai and Ganguly (1972) used barium sulfate precipitation to concentrate nucleic acids from filtered seawater. After hydrolysis of the precipitate at 100C for 3 h in 0 .02 N HCl, the hydrolysate was compared with calf thymus DNA standards by UV spectra at 260 nm. The 16 phosphate content was compared to that of standard DNA and the presence of deoxyribose in the hydrolysate was demonstrated (Pillai and Ganguly, 1970). By their analysis, the dissolved DNA concentration i n Bombay Harbor varied between 13 and 24 liter-1 over six months These numbers are comparable to those found by Minear (1972) for lakewater. Breter et al. (1977) developed a high-performance liquid chromatography technique for the measurement of thymine in seawater by using a cetyltrimethylammonium bromide precipitation of polyanions. The dissolved thymine concentrations measured at fo u r depths from the 1 surface to 30 m, ranged from 25-75 ng liter or 250 to 750 ng of DNA liter-1 considering DNA to be 10% thymine by weight However, this tedious method is unsuited for routine analysis of DNA in seawater In previous studies a technique was developed for the measurement of particulate (cellular plus detrital) DNA in aquatic environments (Paul and Myers, 1982) Using this technique, the bacterial contribution to particulate DNA was found to be significant (70 -95%) in oceanic environments (Paul and Carlson 1984; Paul et al., 1985) The development of the method for determining extracellular or dissolved DNA in aquatic environments was an extension of these studies. This method is based on the specificity of Hoechst 33258 dye for the A T rich portions of native double -stranded DNA (Latt and Statten, 1976) and is well suited to routine analysis of environmental samples The persistence of measureable concentratio ns of extracellular DNA
17 in aquatic environments is important for several reasons. As a compound rich in nitrogen and phosphorus, DNA could be an important source of microbial nutrition. Dissolved DNA could also be a source of nucleic acid precursors which are energetically expensive for microorganisms to synthesize de novo (Nygaard, 1983). The measurement of extracellular DNA may also be important in light of the environmental u se of genetically engineered mic r oorgan isms, as a means of monitoring DNA in aquatic environments. Materials and Methods Sampling Sites: Preliminary studies on the development of the method were performed on samples from Bayboro Harbor, a eutrophic embayment of Tampa Bay, St. Petersburg, FL. Tampa Bay was sampled at a series of stations located along a salinity gradient that extended from the mouth of the bay to the Alafia River. Sampling sites in the Gulf of Mexico included offshore and coastal environments; these were sampled on four cruises during the summers of 1984 and 1985 (see Chapter 3). Freshwater samples were taken at several sites in southwestern Florida, including Crystal River, a relatively pristine, spring-fed river; the Medard Reservoir, a eutrophic body of water collecting runoff from agricultural and phosphate-mining regions of the Alafia River Basin; and Boyd Hill Nature Park, a thickly vegetated swamp adjoining Lake Maggiore in St. Petersburg. Materials: Hoechst 33258 (bisbenzimide ; 2-[2-( 4-hydroxyphenyl) -2-benzimidazolyl]-6-[1-methyl-4-piperazyl] benzimidazole trihydrochloride), DNase I (bovine pancreas), mithramycin, and DNA
18 (calf thymus, type I) obtained Sigma Chemical Co., St. Louis, Mo. .\ DNA (0.5 )Jg/).Jl) was obtained Bethesda Inc., Md. Elutip-d columns and Schuell, Inc., Keene, N.H., and 20 nucleic acid DuPont-New England Boston, Mass. cellulose dialysis tubing weight cutoff, 12,000 to 14,000) was Scientific McGaw Ill. and of DNA: (100 to 1,000 ml) was passed a combination consisting of a GF/D (Whatman, Inc Clifton, N.J.) and a 0 2 lJffi Pleasanton, CA) a vacuum 150 mm Hg 20 kPa) with the flask in an i ce bath. The DNA in the was by the addition of two volumes of (100%) ethanol. 48 h at 20C, the was collected by with 500-ml tubes in a GS-3 (Ivan Inc., CT) at 6,800 x g 20 min. The was then dialyzed at 4C 48 h against deionized and then 2 4 h against SSC (0.15 M NaCl plus 0.015 M sodium pH 7 .0). samples of the dialysate, 0.5 to 2.0 ml, up to 2.0 ml with sse, and 1 ml of 6 X 107 M Hoechst 33258 in sse was added. The samples to and the was with an Aminco as by Paul and (1982) A 0.1 mg/ml DNA stock solution (A260=2. 0 1983]) was with calf thymus DNA. DNA 0 to 2,000
ng were brought to a final volume of 2 ml with sse. Blanks were prepared by filtering (pore size, 0.2 artificial seawater (ASWJP [Paul and Myers, 1982]) and then dissolved DNA was determined by the protocol outlined above for seawater samples. Freshwater samples were treated similarly, except that 0.1 3 M NaCl, x 103 M cac1 2 2H2o, and .35 x M Mgso4 were added to the filtrate prior to the addition of ethanol to aid the precipitation of dissolved DNA. All measurements were corrected for fluorescence from material other than DNA by DNase I treatment (Paul and Myers, 1982) Samples were also corrected for losses in recovery by the addition of an internal calf thymus DNA standard to replicate samples. From 5-10 of calf thymus DNA standard (spike) per 100 ml of filtrate was sufficient to assess recovery Verification of the Method: A series of experiments were performed to verify the following assumptions inherent in this assay: ( i) that dissolved DNA was not being produced as a result of filtration or subsequent steps in the procedure; (ii) that cells were not passing through the filters, thus contributing to the observed fluorescence; and (iii) that one sample filtered (assayed in triplicate) was a reasonable estimate of the concentration of dissolved DNA. 19 To assess the effect of filtration on dissolved DNA values, 100-ml replicates of Bayboro Harbor water were filtered under various vacuum conditions (25-500 mm Hg, or 3 3 to 66. 6 kPa). One replicate received an internal DNA standard, and both samples were processed for dissolved DNA measurements.
20 To investigate the of (MacDonnel and Hood, 1982) to the dissolved DNA signal, dissolved DNA was in both 0.1 and It is unlikely that a significant of the of less than 0.2 would also pass a 0.1 Two 1 00-ml subsamples passed a combination GF/D and and a second set of 100 ml subsamples was passed a combination of GF/D and 0 .1 both at 150 mm Hg (20 kPa) The ty of the method was tested in two A single sample was by the and in volumes (50, 100, 150, and 200 ml). The second involved nine 1 00-ml volumes of the same sample and adding a DNA spike to one subsample to assess All of these samples as above dissolved DNA quantification. To the effect of on the amount of dissolved DNA 100-ml samples of ethanol and at -20C The dissolved DNA was in one sample each week the following weeks. Method: To the Hoechst assay with method of DNA quantification, of dissolved DNA was Samples with ethanol, and dialyzed as above, except that the sse in the final dialysate was with 0.2 M NaCl, 2 0 mM (pH 7 .3-7.5), 1 0 mM EDTA (low salt TE). The dialysate was then and by passage an Elutip-d column to the
instructions provided by the manufacturer The 400 eluate from the column was then precipitated with two volumes of ethanol at -20C for 48 h, and the precipitate was collected by centrifugation i n an Eppendorf microcentrifuge (Brinkmann Instrume nts, Inc., Westbury NY) for 10 min. The pe llet was suspended in an app ropriate v olume of TE buffer (Maniatis et al., 1 982). The method used for the mithramycin assay was a modification of 21 the method of Hill and Whatley (1975), with a final concentration of 10 of mithramycin per ml and 10 mM MgC12 Standards were prepared (0 to 10 with 0 1 mg/ml cal f thymus D N A and TE buffer (pH 7 4) was added to a final volume of 2 0 ml. Concentrated extracts of dissolved DNA were also brought to a final volume of 2 0 ml with TE buffer. The stain was prepared by adding 1 2 mg of the mithramyci n composite (2.5% mithramycin) to 10 ml of TE buffer containing 30 mM A 1-ml portion of the stain was added to each sample and to the standards. The fluorescence was measured after 5 min at an excitation of 430 nm and an emission of 570 nm. Results and Discussion Verification of the Method: Ethanol precipitation of ASWJP followed by dialysis of the precipitate provided blanks for the method These blanks yielded f l uorescence values identical to that of sse buffer alone Therefore, DNA or non-DNA fluorescence was not introduced by the techniques used to isolate and concentrate dissolved DNA. Recovery of calf thymus DNA standards added to artificial seawater was 85-95% (data not shown). The average recovery for DNA internal
22 standards added to filtered natural water sample for estuarine and nearshore environments averaged 93.4 + 6.5%, while offshore recoveries averaged 72. 3 .:!:_ 7 .8%. Lower recoveries in offshore samples are probably related to the length of storage and to manipulation of large volumes of precipitate. Percent recovery and DNase information w as used in calculations of dissolved DNA values such that DONA = (C/Vs) x (Vd/Vf) x %R-1 x %DNase, where DONA is dissolved DNA in micrograms per liter, C is the amount of DNA measured, Vs is the volume analyzed, Vf if the volume filtered, Vd is the volume of the dialysate, %R is the percent recovery of the internal standard, and %DNase is the percentage of DNA that was degradable Microbial cell lysis during filtration was investigated by filtration of a seawater sample at a range of pressures. Dissolved DNA values for samples filtered at 25, 75, and 150 rnrn Hg were not significantly different, while values for samples filtered at 250 and 500 rnrn Hg were significantly higher. This experiment was performed on two occasions In the first experiment, samples filtered at 25, 75, and 150 rnrn Hg had an average value of 11.75 .:!:_ 0.17 DONA while samples filtered at 250 and 500 rnrn Hg averaged 15.30 .:!:_ 0 .31 or approximately 30% higher. In the second experiment the mean value for samples filtered at 25, 75 and 150 rnrn Hg was 17.10 .:!:_ 1.36 and that for samples filtered at 250 and 500 rnrn Hg was 25.15 .:!:_1 .17 or 47% greater. These results indicate that cell lysis was not contributing to the fluorescence at a vacuum of 150 rnrn Hg or less but may have been a factor in samples filtered at 250 and 500 rnrn Hg. Thus, all filtrations for dissolved DNA were performed at 150 rnrn Hg. Independent verification that cell lysis was not occurring at these
23 pressures is provided by a study that looked for dissolved chlorophyll by filtration (DeFlaun et al., 1986). Less than 1% of the particulate chlorophyll was found in the dissolved fraction, therefore, it seems unlikely that dissolved DNA is an artifact of filtration under the low vacuum used in thi s study. Filterable bacteria or ultramicrobacteria have been found to pass through pore sizes of 0 .45 vm (Taboret al. 1981), 0.3 vm (Torella and Morita, 1981) and, most recently 0 2 vm (MacDonnel and Hood, 1982) The possibility that very small cells were passing through the 0.2 vm filters was examined by filtering replicate samples through 0.1 vm Nuclepore filters. Dissolved DNA values for the 0 1 vm filtered samples were not significantly different (Student s t test; significance level, 5%) and had actual values that were slightly higher than those obtained from the 0.2 filtered samples. If filterable cells were contributing to the observed fluorescence, the
FL. Her numbers at 3 stations ranged from 6 x 106 to 0.1% of the total colony forming units (CFUs) present. Considering that the MPN method probably underestimates bacterial numbers by 3 orders of magnitude, multiplying MPNs by 1000 yields concentrations of 24 ultramicrobacteria ranging from 1 0-6000 per ml. Assuming a value of 5.66 fg DNA cell-1 (Paul et al., 1 985), this would equa l 5.66 x 105 to 3.4 x 103 DNA liter-1 which is still orders of magnitude lower than the lowest DONA values measured These calculations and the data from the 0 1 samples indicate that filterable bacteria are not contributing to the observed fluorescence. DNA encapsulated in cells and within viral coats are probably resistant to the DNase treatment performed on replicate samples and, therefore, any fluorescence they contribute would be subtracted in the calculation of DDNA concentration. The amount of DNA as a function of volume filtered is shown in Fig 1 The concentration of dissolved DNA obtained in March 1986 from these four volumes collected in Bayboro Harbor was 18.32 + 1. 78 and the correlation coefficient for the linear regression was 0 997 These results indicate that dissolved DNA concentrations are not a function of the volum e of water filtered. The variability among samples of the same volume was calculated from the eight 100-ml subsamples of Bayboro Harbor water collected in January 1986. Means for each subsample were calculated from triplicate measurements. The overall m ean for the eight samples after correction for percent recovery and percent DNase degradable was 6 .72 0.35 of DNA per liter (x = 95% CI) with an average coefficient of variation of 6.19%. When comparing any two subsamples, the coefficient of
c( z c 4 3 1 2 5 0 100 200 Volume Filtered (ml) F i g 1 Linear regressi on of the amount of dissolved DNA measured in fou r volumes of Bayboro Harbo r water. The correlati on coefficient (r) = 0 997 Error bars ar e x + sd.
26 variation ranged from 0 2 to 14%, indicating an acceptable degree of . reproducibility betwee n subsamples Water samples filtered and stored in ethanol at -20c showed no loss in DNA over the first 2 w eeks Over weeks 3 and 4, however, there was a 12% loss in DNA (12.17 versus 10.69 of DNA per liter). If possible, samples were processed within 2 weeks of collection. Evidence that what was being measured in these extracts was indeed DNA was provided by a study of the fluorescence spectra of the extracts compared to that of calf thymus DNA with and without added Hoechst 33258 (DeFlaun et al., 1986). The observed shifts in spectra when the dye was added to the extract were characteristic of the binding of Hoechst 33258 to DNA (Latt and Statten, 1976) and identical to the shifts observed for calf thymus DNA. Comparison with Mithramycin Method: Purified DNA and dissolved DNA in concentrated extracts were quantified by the mithramycin and Hoechst 33258 methods. A commercially available preparation of phage ). DNA (0.5 yielded values of 0.48 and 0.44 by the Hoechst 33258 and the mithramycin assay, respectively. For dissolved DNA extracts from Bayboro Harbor and Tampa Bay, however results of the m ithramycin assay indicated the presence of 4.5 to 11 times (data not shown) the amount measured by the Hoechst assay. The extract was further purified by passing the dialysate through a NENsorb-20 cartridge, which removes interfering substances (protein, salt, low-molecular weight materials) which may not be removed by Elutip-d columns This cartridge can be used in place of a phenol-chloroform extraction step in the purification of DNA. Even after this purification, values for
dissolved DNA in concentrates of Bayboro Harbor water by the mithramycin assay were still 3 times higher than those obtained by the Hoechs t 33258 method ( 1 20 versus 40 ng/ J,ll) The differing 27 specificities of the two fluorochromes may cause some differences. Mithrarnycin preferentially binds to G-C rich regions of double-stranded DNA (Williams et al., 1980) and Hoechst 33258 preferentially binds to A-T rich regions of double-stranded DNA (Latt and Statten, 1976). It seems unlikely, but it is possible, that dissolved DNA base composition would vary that much from those of calf thymus and phage A DNA standard. It seems more likely that there were substances in the seawater concentrates that were not removed by the purification techniques that increased in fluorescence in the presence of mithramycin but not Hoechst 33258. Elutip-d concentrated extracts of Tampa Bay water have also been analyzed by a modification of the DABA method for DNA determination (DeFlaun et al., 1986) Again, DONA values by the DABA method were greater (average, 1 75 x greater) than those obtained by the Hoechst method, however, DABA is known to be a less specific indicator of DNA than Hoechst. DABA reacts with aldehydes possessing an unsubstituted a carbon a property of deoxyribose, and will detect single-stranded DNA (Szafarz et al., 1981). Another possible explanation is that the Hoechst 33258 method underestimates dissolved DNA for some unknown reason. This is unlikely, however, since >90% recovery of added internal standards routinely occurs in this analysis. Indicating that >90% of the DONA that was present was being measured. Concentrations of Dissolved DNA in the Environment: Dissolved DNA was
28 measured in a wide variety of aquatic environments The volume of water filtered depended upon the expected concentration of dissolved DNA. Estuarine and coastal samples required only 100 rnl of filtered water. For surface samples at offshore oligotrophic stations 300 rnl was filtered, while for deep water m) samples, as much as liter was filtered. The assay required at least 100 ng of DNA in the 2 ml of dialysate (50 ng/ml) for a reliable measurement. All samples were checked for fluorescence before the addition of stain. Normally this fluorescence was not found in estuarine samples however, it was present in about 25% of the Gulf of Mexico samples The fluorescence was treated as a blank value and subtracted from the fluorescence value after the stain was added The percentage of the fluorescence that was degradable by DNase I treatment ranged (n = 101 ) from 24 to 100%, with an average value for all samples of 75 19%. The portion of the fluorescence not degraded may be attributable to encapsulation of DNA in viruses, binding of DNA in some way which makes it unavailable to the enzyme, or simply a nonspecific binding of Hoechst 33258 dye which increases its fluorescence. The ranges of dissolved DNA concentrations for different envi r onments are summarized in Table 1 Dissolved DNA concentrations decreased with distance offshore and depth in the water column Estuarine values were the highest measured; the 44 vg/liter value was measured in May at a station on the Alafia River, which drains an agricultural region with an active phosphate mining industry. Offshore oligotrophic stations had values for dissolved DNA in surface waters of 5 vg/1 or less, with samples deeper than 100 m having values below 1 vg/1.
Table 1 : Concentration of dissolved DNA in various aquatic environments. Environment Avg DNA concentration (range) (IJg/liter) Marine Estuarine 14.52 (6-44) Coastal (5-15) Offshore surface 1.75 (0.5-5.0) 500 to 1 ,500 m (0.2-0.5) Freshwater Medard Reservoir 6 .97 Crystal River 1.74 Boyd Hill n 46 10 21 5 Freshwater Samples: Ethanol precipitation of DNA from solution is facilitated by the presence of inorganic salts (Maniatis et al., 1982) The ambient levels of inorganic salts in freshwater environments (salinity, to 2 ppt) were insufficient to allow recovery of internal DNA standards. When sodium chloride was added at concentrations of 0 2 or 0.5 M, the recoveries of the internal calf thymus DNA spike were low (57 and 46%, respectivel y) To determine the optimal concentration of salts for the precipitati on of DNA, 1 0 1-1g of calf thymus DNA and three d ifferent salt concentrations were added to 100-ml volumes of deionized water The l owest concentration of salts consisted of 0 .13 M NaCl, 5 4 3 2 x 10M cac1 2 2H2o, and x M MgS04 (concentration of salts in 30% ASWJP). This concentration was doub l ed and tripled for the other two spiked deionized water sa mples. The best recovery of the DNA sp ike (90%) was obtained at the lowest salt concentration; therefore, 29
this protocol was used for the freshwater samples Of the freshwater samples, those from the eutrophic Medard Reservoir and Boyd Hill had values comparable to those of coastal and estuarine marine samples while the Crystal River sample value was similar to those for samples found at offshore marine stations ( Table 1 ) The dissolved DNA concentrations estimated by Pillai and Ganguly (1972) in Bombay Harbor are similar to those found in these estuarine samples (Tabl e 1). 30 Freeze-drying was also investigated as a means of concentrating samples (Minear 1972) from freshwater environments This method was inappropriate for several reasons: (i) recovery of the added DNA spike was lower than with ethanol precipitation (64 versus 90%), (ii) there was a high fluorescence blank signal associated with the lyophilized samples before the stain was added and (iii) ethanol precipitation was faster than freeze drying The concentrations measured by Minear (1972) seem reasonable when compared to the concentrations measured in this study, even though they were not correcte d for recovery Recovery of total phosphorus compounds ranged from 60-90% and was probably even less for the DNA-containing high molecular weight fraction. Correcting the values of Minear ( 1972) for recovery yielded values higher than those measured in this study There are several possible reasons for these higher va l ues. Minear (1972) used 0 .45 pore size filters, which may have allowed more filterable bacteria to be measured as dissolved DNA (Tabor et al., 1981) Also DABA measures single-stranded as well as double-stranded DNA (Szafarz et al., 1981 ) Estimates made by Breter et al., (1977) of dissolved DNA in seawater based on nonparticulate polyanionic thymine concentrations are low compared with the values measured in this study for surface water
The extensive manipulations required to concentrate samples to a final volume of 25 for high-performance liquid chro matography analysis makes this an extremely tedious method. Also, the measurement of thymine is not a direct measurement of native double-stranded DNA. The method developed in this study for the determination of dissolved DNA in aquatic environments is simple and well suited to routine analysis. Hoechst 33258 is much more sensitive than other DNA-specific fluorochromes, allowing the measurement of DNA with a minimal amount of concentration. 31
CHAPTER 3: DISTRIBUTION OF DISSOLVED DNA IN AQUATIC ENVIRONMENTS Introduction Deoxyribonucleic acid (DNA) is a constituent of all living cells and its presence in the dissolved organic matter (DOM) of aquatic environments is widespread. Although measurements of dissolved DNA have been made in both marine and freshwater environments (Pillai and Ganguly, 1972; Minear, 1972; Breter et al., 1977), a comprehensive study of the distribution of extracellular DNA has not been done. The purpose of this study was to measure dissolved DNA in a variety of marine and freshwater environments, and to relate its concentration to the activity and biomass of autotrophic and heterotrophic microbial populations. Correlations with these parameters could indicate the source of extracellular DNA in these environments. Seasonal and diel studies were also performed in order to determine whether there were any significant temporal variations in the concentration of dissolved DNA. Materials and Methods 32 Samples were taken on four cruises in the southeastern Gulf of Mexico: Cruises 8408, 8414, and 8518 aboard the RV Bellows in May 1984, August 1984, and June 1985 respectively, and Cruise 8514 aboard the RV Suncoaster, in August 1985. All samples were of surface water (< 10m) except where noted, and were collected with 30 1 Niskin bottles. Temperature and salinity were determined with a Neil Brown Smart CTD. All sample processing was initiated within 1 h of collection, and all
33 filtrations were performed at a vacuum 150 mm Hg to minimize cell lysis. Samples of water overlying the coral reefs of the Dry Tortugas were collected in 8 1 Niskin bottles by skin or SCUBA divers. Samples of the coral surface microlayer (CSM) on the reefs were taken with sterile or ethanol-rinsed 50 ml plastic syringes (no needle), also by skin or SCUBA divers. A complete description of the stations sampled in the Dry Tortugas and a map of the transect area is given in Paul et al. ( 1986). Sampling in Tampa Bay occurred monthly during a 15 month period in 1985 and 1986. Samples were taken at three stations located along a salinity gradient from the mouth of the bay to the Alafia River (Fig. 2) Two freshwater environments were also sampled on a seasonal basis during the winter, spring and summer of 1987. Four stations were sampled along the entire length of the relatively pristine Crystal River and eight stations on the eutrophic Alafia River, which drains an agricultural and phos phate mining region (Fig. 3). A large proportion of the samples taken at the Alafia River were colored and/or fluorescent due to tannins, humics and other organic matter in the water This color/fluorescence interfered with dissolved DNA determinations by the Hoechst method. Therefore, these samples were subjected to further post-dialysis purification by passage over a NENsorb (DuPont -NEN) DNA purification column according to the manufacturers directions, except that diethylamine replaced triethylamine in Solution A. Summer samples from the Alafia River were so discolored that NENsorb purification was insufficient. Post-dialysis samples were extracted with phenol/chloroform to eliminate fluorescence or excessive quenching Samples for dissolved DNA (100 to 1000 ml) were filtered, ethanol
r ... N MMM M WI 0 10 KILOMETERS 34 Fig. 2. Locations of the three stations (A, B and C) sampled during the seasonal study in Tampa Bay and the location of Bayboro Harbor (BH) where two of the diel studies were perfonned.
35 .......... Gulf of Mexico ... ._...... Fig. 3 Maps of t h e Crystal ( upper panel ) and A l afia ( lower pan e l ) rivers an d stati on locations < ) (2 ) on the Crystal River map is the location of the station 2 sampled i n Januar y 1987
precipitated and stored at -20C for 48 hours or until they could be processed in the laboratory as described previously. Precipitation of DNA in freshwater samples was aided by the addition of salts (see Chapter 2) Dissolved organic carbon (DOC) determinations were made acco r ding to the persulfate oxidation method of Menzel and Vaccaro ( 1 964) as modified by Federicks and Sackett (1970). Samples (2 to 9 ml) were filtered through precombusted Whatman GFF filters into precombusted 10 ml ampules. The samples were acidified by the addition of 0 2 m1 of 10% phosphoric acid and 0.2 g of potassium persulfate was added. The samples were then brought up to a total of 10 ml with deionized, distilled water (DDW). After purging for approximately 10 min with a stream of oxygen, the ampules were sealed in a propane 0 2 flame with precautions taken to exclude flame generated co2 from the sample Oxidation of the organic matter was accomplished by heating the sealed vials to 120C for 4 hours in a pressure vessel (-10 psi). Standards were prepared from dilutions of potassium hydrogen phthalate (KHP) ranging in organic carbon (OC) concentration from 0-3 mg OC/1. KHP is a suitable standard for DOC analyses because it can be made in a very pure form and because it is not hygroscopic it can be weighed precisely. Samples and standards were analysed on an Oceanography International Total Organic Carbon Analyzer which uses N 2 as the carrier gas and a non-dispersive infrared detector to measure the Linear regression analysis of the standards after correction for reagent and DDW blanks, was used to calculate the concentration of organic carbon in each vial. Samples for dissolved organic phosphorous (DOP) were filtered 36
37 through precombusted GFF filters and analysed by the method of SOlarzano and Sharp (1980). DOP analyses were kindly performed by Dr. Gabriel Vargo. A number of other microbial parameters were measured during t hese field studies in order to determine what factors control the distribution of dissolved DNA. These included the bacterial parameters of particulate DNA, bacterial direct counts, bacterial activity as measured by thymidine incorporation and the phytoplankton parameters of and primary productivity. Details on t he methods used to measure these parameters can be found in Paul et al. (1985) and DeFlaun et al. (1987). Four studies were conducted to determine if diel variations occur in dissolved DNA concentrations and the other m icrobial parameters. During the first diel study, the waters above a coral reef were sampled every 4 h over a 28 h period. A second diel study in oligotrophic Gulf waters was performed using a 4 h sampling interval over a 52 h period while following a buoy. Two diel studies were performed in Bayboro Harbor (Tampa Bay) with the same sampling scheme over a 52 h period. Results Gulf of Mexico: Concentrations of dissolved DNA in t h e Gulf of Mexico are shown in Fig. 4. Data for two consecutive summers has been plotted in this figure. Little variability was found over large areas i n offshore waters with similar distributions found on all four cruised, allowing the data from two consecutive summers to be combined. Lowest values of DDNA wer e found in the southeastern Gulf (0.2 to 1 .9 an area that is strongly influenced by intrusions of o ligotrophic Loop
I I I I / 2-5 / / ' ""' Gulf of Mexico <2 \ \ 38 \ 26 \ \ \ ":-. \ ..... ..... .. \ \ . \ ' ) ,. I < 2 Keys I 85W 84 83 82 Fig 4. Distribution of dissolved DNA in the so u t heastern Gulf of Mexico during summer 1984 and 1985 ( + ) Stations from Cruise 8408; ( ) Cruise 8414; Cruise 8518 ; ( ) Cruise 8514 : small asterisk ind icates location of station where deep profil e was taken
39 Current water onto the Florida She lf. Higher concentrations (2 to 5 were found north of this area, at a latitude of approximately 27 N. Dissolved DNA concentrations increased shoreward with plumes of higher concentrations (10 to 19 near the mouth of Tampa Bay and Charlotte Harbor (Fig. 4). Elevated concentrations ( 1 0 to 1 5 were also found in Florida Bay, which is influenced by the highly productive Ten Thousand Islands estuarine ecosystem. Dissolved DNA and the other microbial biomass and activity parameters were m easured in a vertical profile at an offshore station (indicated in Fig. 4) on Cruise 8514 (1985). The results of this profile appear in Fig 5 DONA values were maximal in the surface waters and decreased sharply through the thermocline Concentrations remained relatively constant(< 0.3 from 300 to 1500 m, the greatest depth sampled A similar dissolve d DNA profile was found during Cruise 840 8 the previous summer. The dissolved DNA profile (Fig. 5) was very similar to the ones for bacterial direct counts and particulate DNA, but unlike the profile for chlorophyll a which had a strong maximum at 100m (DeFlaun et al., 1987) Of the four cruises, the most parameters were measured on 8514, therefore, a correlation coefficient matrix was c onstructed for this data (DeFlaun et al., 1987) Correlation ana lysis of all the parameters indicated that dissolved DNA correlated best with bacterial direct counts (r = 0 .9421), followe d by particulate DNA ( r = 0 8133), and then the log of thymidine incorporation (bacterial activity; r = 0.7844) The poorest correlation was obtained with (r = 0.4986). Stepwise or forward multiple linear regression analysis of this data set with the Regress II statistical software by Human Systems
JJ9 DNA/l 1 2 3 5 Temperature ec) 15 25 4 0 I I I 100 U) ... CD G) 300 E :I: t-Q. w a 500 1500 32 34 36 Salinity ( ppt) Fig. 5. Depth dissolved DNA, salinity (e) and (*) 8514. ""' 0
Dynamics, Inc. (Northridge CA) indicated that the only independent variable necessary to describe the dependent variable ( DDNA) was bacterial direct counts (r = 0.94 p < 0.001). Dissolved DNA also correlated best with bacterial direct counts in the < 1 fraction (r 0 839) Correlation analysis of the cumulative data set for the 3 cruises 8403, 8518, and 8514 (excluding primary produc tion measurements) indicated that dissolved DNA correlated best with particulate DNA, followed by the log of thymidine incorporation and bacterial direct counts Individual analysis of eac h of these cruises indicated that these 3 parameters yielded the highest correlation coefficients with dissolved DNA. Chlorophyl l yielded the lowest correlation coefficient with dissolved DNA for these data sets. 41 Coral Reefs: Samples taken of the water overlying the reefs and of the mucus on the surface of the corals (coral surface microlayer; CSM) showed an enrichment in the CSM for all o f the microbial parameters measured (Paul et al., 1986). Concentrat ions of dissolved DNA in the waters above the reef were typical for an offshore, oligotrophic environment, ranging from 0 .63 to 1 .95 DDNA/1 (Tab l e 2) while concentrations in the CSM were characteristic of a eutrophic e nvironment ranging from 2 .4 5 to 10. 5 Enrichment in the CSM for other microbial parameters measured (particulate DNA, primary productivity, bacterial activity) ranged from 3 .34 to 280 times the concentration in the overlying water. Bacte rial direct counts were not significantly different from overlying waters (M/W = 0.93 to 7 .4 6 ; Paul et al., 1986).
Table 2 : Dissolved DNA in the CSM and overlying waters. Mucus: value obtai ned in the CSM; Water : value obtained in the overlying water; M/W the ratio of these values DRY TORTUGAS Loggerhead Key 8 a,b 8 c,d 8 e,f 8 g h IV c,d Garden Key 9 a,b KEY WEST Sand Key Dissolved DNA Mucus Water 4.66 10.50 2 .91 7.43 3.59 ().lg/1) 1.32 0 0.-74 1.17 1.95 M/W 3 .53 11 4 .04 4 .34 1.84 42 A transect was conducted across an Acropora cervicornis patch reef to see if there was any difference between the water overlying the reef and the surrounding surface waters (Fig 6) The DDNA concentration over the reef was slightly, but not significantly lower than the surrounding water, while the concentration in the CSM was f our times higher. Little variatio n was noted for the other parameters measured in the transect, and all except for bacterial direct counts showed significant enrichment in the CSM (Paul et al., 1986). During Cruise 8514 in August 1985 samples of the CSM and the overlying water of the Montrastrea annuleris heads w es t of Loggerhead Key were taken at noon and midnight. For all parameters except bacterial direct counts, CSM values at midnight exceeded those at noon (Paul et al. 1986). Dissolved DNA values in the CSM were almost twice as high at midnight as at noon (4.65 vs 2 .45 ).lg/1). Values in the
0 tn .. G) ;10 E 2 IVE IVD IVB,C IVA CSM lili DONA water Fig. 6. Dissolved DNA concentrations in the coral surface microlayer (CSM) and the surface waters in a transect across an Acropora cervicornis patch reef in the Dry Tortugas. ""' w
overlying water were not significantly different in the noon and midnight samples for all parameters, but the actual concentration for dissolved DNA was slightly higher at midnight. 44 Tampa Bay: The seasonal study conducted on Tampa Bay over a 15 month period included the same suite of microbia l parameters measured o n the cruises in the Gulf of Mexico with the additions of dissolved organic carbon and dissolved organic phosphorus. The results of this study on Tampa Bay are depicted in Figure 7 The water temperature throughout the 15 month period ranged from a low of 14 C in the winter to a high of approximately 30 C during the summer months with greater variation in both temperature and salinity occurring with d istance from the mouth of the estuary. At the Alafia River station (station C; Fig. 2) salinity varied from 7 to 28. 9 ppt, station B in the middle of the estuary ranged from 25 to 31. 4 and station A, at the mouth of the bay, had the highest salinity ranging from 29.5 to 34. 7 ppt. Dissolved DNA concentrations followed the trend of water temperatures with the lowest values during the winter and the highest in the summer at all three stations. All of the microbial parameters measured including dissolved DNA, were generally highest at station C near the mouth of the Ala fia River, lowest at the mouth of the estuary station A and intermediate at the mid-estuary station B. There was, however no statistically significant difference between the means for dissolved DNA concentrations at these 3 stations. Values for dissolved DNA concentrations at station C averaged 10.85 5.57 (range 1 8 to 18.9 those at station B averaged 8 .90 3.28 (range 4.1 to 1 5 and t he average for station A was 6.90 2.78 ( r ange
2. 2 to 1 0 4 JJg/1) The seasonal tr-end in dissolved DNA was ver-y similarto that of par-ticulate DNA at station B (r= 0.90) and station C (r= 0 72) but did not follow each otherclosely at station A (r= 0 22) at the mouth of the estuar-y Cor-r-elation analysis of the data fr-om all thr-ee stati ons indicated that in both the whole water-(r0 72) and the less than 1 J.lffi fr-action (r= 0 70) dissol ved DNA cor-r-elated best with par-ticulate DNA. 46 Dissolved or-ganic car-bon (DOC) did not exhibit much seasonal var-iation at station A (r-ange 1 2 to 2 .83 mg/ 1) and station B (r-ange 2 .46 to 4 .26 mg/1), but exhibited ver-y lar-ge peaks in the spr-ing of both year-s at station C (r-ange 3 4 1 to 1 5.37 mg/1; Fig 7) Consider-ing DNA to be 39% car-bon dissolved DNA car-bon r-epr-esented a n aver-age of 0 .11 0 .06% (w/w) of the DOC at all 3 stations. At station A, dissol ved DNA car-bon was an aver-age of 0.15 0 .08% of the DOC, at station B, 0.10 0 .04% and at station C, high in the estuar-y dissolved DNA car-bon aver-aged only 0.07 + 0.04% of the total DOC. Station A at the mouth of the estuar-y, had a significantly (p < 0 01) higherper-cent of its DOC as DNA car-bon than stations B or-C. Disso l ved or-ganic phosphor-us (DOP) was measur-ed at these thr-ee stations during 5 months of the study per-iod fr-om Januar-y 1986 to May 1986. Con sider-ing DNA to be 10% phosphor-us dissolved DNA phosphor-us r-anged fr-om 0.46 to 22.7% of the total DOP measur-ed. No r-eal tr-end in DOP concentr-ations by season orby station could be detected in this small numberof samples Diel Studies: On Cr-uise 8518 in June 1985, a diel study was per-formed in the water-s over-lying an Acropor-a cer-vicornis patch reef in the Dry
Tortugas. The only notable variation in dissolved DNA concentrations was an increase at 0400 h (Fig. 8). All of the other parameters measured remained relatively constant over the 28 h period, except for a decrease in thymidine incorporation (bacterial activity) which also occurred at 0400 h (Paul et al., 1 986) In an attempt to sample a single water mass, a buoy was followed in the offshore Gulf of Mexico for 52 h. All of the samples had low concentrations of dissolved DNA characteristic of an oligotrophic environment (Fig 8), but there was a trend of increasing concentratio ns throughout the night and decreasing concentrations during the daylight hours. The highest values were at 0400 h and 0800 h on the first day and 0400 h on the second. The lowest values o n these two days were both at 2000 h. The first diel study conducted in Bayboro Harbor in June 1986 (Fig 9), showed a very strong diel periodicity in dissolved DNA concentrations with minimum values during daylight hours and stron g peaks at midnight on both days Dissolved organic carbon values did not seem to vary in a ny significant pattern and stayed within a fairly narrow range except for one large peak that occur r ed at 0800 h on the second day. The second diel performed in Bayboro Harbor in August 1986 ( Fig. 9) also had peaks in dissol ved DNA conce ntrations that occ urred at midnight on both days but these were not as pronounced as those that occurred in the earlier study. Again DOC concentrations did not appear to follow any discernable pattern. During the last 8 h of this study, a large input of warm freshwater doubled the dissolved organic carbon concentration and decreased the dissolved DNA concentration to 47
6 5 t I \ I l -4 I ........... c( z I Q f \ c 3 ,t, = :1.. 2 1 24 12 24 12 Time of Day Fig. 8 Diel studies a in the ( ) and following a buoy in the Gulf of Mexico < + ) Black night samples. 48
10 g 8 Q Q 8 E Q. 28 t8 6 0 0 0 Q 4 e 2 I ............... ---A-----.. 24 12 24 12 Time of Day 18 30 8 0" 20-; Cl) 12 ....... 9 .: z 0 8 0 Q =l 3 30 8 ...: Ill 20 (/) 49 Fig. 9 Diel studies in ( Tampa Bay) ; June 1986 study (top) and August 1986 (bottom). B lack nigh.ttime sampling. as in Fig. 8 Temp. (. .); Salinity( . ) ; DOC(. ) with deviation (sd) ooNA 1 ___ ) with sd
50 almost a fifth of its value the influx. The and Alafia sampled three seasons i n the season and summer wet season (July) o f 1987. Station 1 on the Crystal River (Fig. 3) was sampled at the main at the of the Thi s station had the lowest value for dissolved DNA at all four stations on those dates 0 .63 to 3.19 x = 1 .74 1 .31 Stations 2, 3 and 4, which extend along the length of the had values dissolved DNA ranging 2 .83 to 8 .02 ( x 5 .11 1 .87 There was no apparent correlation between dissolved DNA values and (season) with salinity (distance of internal DNA in Crystal R iver samples 83.5 6.5% ( n = 10) DOC values also lowest at station 1 on each sampling date = 0.33 to 1 .22 mg/1; x = 0 .83 mg/1) but with salinity downriver (r = 0.76; p < 0 005). DOC values for stations 2, 3 and 4 ranged 1 .02 to 2 .90 mg/1 (x = 1 .95 + 0.66 mg/1) For all of the measured on the River, dissolved DNA best with (r = 0 82; n = 10) and the log of = 0.69). The eutrophic Alafia had a much larger of values for dissolved DNA (1.14 to 25.56 x = 8.80 than the in this study Values as high as 44 of dissolved DNA have been in the Alafia (DeFlaun et al. 1986) of DNA in Alafia samples were low due to the steps required to these samples (x = 31. 1 + 22. 6 %; n =
51 21). DNase degradable fluorescence in all of the freshwater samples averaged 87.5 .: 25.3% (n = 31). Again, there was no seasonal correlation of dissolved DNA with water temperature. DOC values in the Alafia ranged from 3.2 to 17.43 mg/1 and had the highest correlation with dissolved DNA of all of the parameters measured during the three seasons (r = 0.44; p < 0.05). During the two sampling periods before the summer rainy season, particulate DNA values had a very high correlation with chlorophyll values (r = 0.98) and dissolved DNA correlated best with the log of primary productivity (r = 0.534) and particulate DNA (r = 0.533). When data for both rivers were combined, for all the parameters measured, dissolved DNA correlated best with DOC values (r 0.57; p < 0.001) Dissolved DNA carbon represented a significantly larger percentage of the DOC in the Crystal River samples (x = 0.12 + 0.10%) than in the Alafia River samples (x = 0.04 0.03%; p < 0.01), perhaps due to a greater contribution by terrestrial DOC in the Alafia. Discussion The concentrations of dissolved DNA in the Gulf of Mexico are highest in coastal waters, decreasing with distance from shore and depth in the water column. Nearly identical values were found during successive years at the same station or nearby stations. Therefore, the distribution of concentrations depicted in Figure 4 are probably representative of summer concentrations in this portion of the Gulf of Mexico for these two years. This distribution may not be representative of other seasons or previous years. The depth profile for dissolved DNA resembles the profiles for bacterial direct counts
and par'ticulate DNA, r:'eflecting the high cor:'r'elation between these par'ameter:' s in offshor:'e water's. Pr:'esumably, par'ticulate DNA is the sour:'ce of dissolved DNA, since cell-free DNA synthesis in seawater' is not known to occur:'. It has previously been shown that most of the particulate DNA in offshor:'e envir'onments (71 to 99%) is associated with bacter'ioplankton (Paul and Carlson 1984; Paul et al., 1987). The pr:'oduction of dissolved DNA by actively growing heterotr:'ophic bacterioplankton has also been demonstr:'ated (Paul et al., 1987). The corr:'elations found between dissolved DNA and bacter:'ial counts, par:'ticulate DNA and thymidine incor:'por:'ation (DeFlaun et al., 1987) agr:'ee with these pr'evious observations, and imply bacterioplankton as a significant sour:'ce of dissolved DNA in offshor:'e envir'onments. Grazing, cell lysis and/or:' active excretion (Hara and Ueda, 1981; Stewart and Carlson, 1 986) are possible mechanisms for:' the production of extr:'acellular:' DNA by bacteria. The lack of COr:'r:'elation between dissolved DNA and phytoplankton par:'ameters (chlorophyll a and pr'imary pr:'oductivity) and the inability to demonstr'ate dissolved DNA pr:'oduction in incubations of natural phytoplankton populations (Paul et al., 1987) implies a lesser:' !:'ole for:' phytoplankton in dissolved DNA pr:'oduction, at least in offshor:'e envir:'onments However, Minear:' (1972) conclusively demonstrated the production of extracellular' DNA by a Chlamydomonas species in diffusion chamber:' studies. An alter:'nate explanation for' the lack of correlation with phytoplankton parameters is that phytoplankton show extr:'eme patchiness with r:'espect to space and time, responding to transient changes in light quality, quantity, and nutrients. Dissolved DNA may 52
be similar to particulate, or bacterial direct counts (Paul et al., 1985) in that it represents a steady-state pool that is relatively invariant over larger areas and time intervals, even though there may be short-term dynamics within the pool. Dissolved DNA, although relatively constant in terms of concentration, is rapidl y hydrolyzed at near ambient concentrations by both extracellular and cell associated enzymes (Paul et al. 1987). However, turnover times may be greater in sediment (Novitsky, 1986) where DNA may be protected from nuclease digestion (Aardema et al., 1983). The water overlying the coral reefs in the Dry Tortugas is similar to other oceanic oligotrophic environments that were sampled. Levels of dissolved DNA were low with little significant variability spatiallY o r temporally The coral surface microlayer (CSM), however, is a much more dynamic environment as evidenced by much higher concentrations and greater variability in dissolved DNA and other microbial parameters. Increased coral feeding activity and polyp openings at night may increase mucus production and consequently microbial production and dissolved D N A concentrations. Previous studies have demonstrated a positive correlation between bacterial biomass and activity parameters and dissolved DNA concentrations (DeFlaun et al. 1 987) and this relationship appears to hol d in the CSM. The system, however, appears to be a closed one. The enrichment in the CSM does not appear to affect the productivity of the overlying water c olumn which has the characteristics of an oligotrophic environment. Tampa Bay samples had dissolved DNA concentrations that correlated with particulate DNA and water temperature A l though bacterioplankton make up the greater portion of particulate DNA in oceanic environments 53
(71 to 91 %) phytoplankton DNA becomes a substantial percentage in estuarine and freshwater environments (Paul and Carlson, 1 984). This would indicate that phytoplankton DNA may make a substantially higher contribution to the pool of dissolved DNA in the estuary than in the ocean Minear (1982), found that actively growing pure cultures of Chlamydomonas reinhardtii incubated for 12-14 days produced 34-38 of extracellular DNA and long term cultures (81 days) produced as much as 281 Maximum values for dissolved DNA during the summer coinc ided with peaks in bacterial activity (Paul et al., submitted). Increased bacterial activity has been significantly correlated with increased dissolved DNA values in offshore environments (DeFlaun et al., 1987) and may be a factor in the estuarine environment also. Dissolved DNA peaks lagged behind those of dissolved organic carbon (DOC) and the spring phytoplankton blooms, perhaps due to the release of high molecular weight dissolved organic matter (DOM; >50,000 daltons) known to occur during the scenescence of phytoplankton blooms (Sondergaard and Schierup, 1982). The first peak in DOC at station C coincided with the spring phytoplankton bloom in 1985, while a second DOC peak in March 1986 preceded the chlorophyll maximum, which occurred during April. Phytoplankton excrete a large proportion (10 to 50%; Larsson and Hagstrom, 1982) of the carbon fixed by photosynthesis as dissolved organic compounds. Whether the increases observed at station C are the result of phytoplankton bloom conditions or allochthonous input from terrestrial sources, is unclear. Similar increases in chlorophyll a and primary productivity in the spring at stations A and B did not result in appreciable increases in DOC. 54
Dissolved DNA phosphorous was a much larger percentage of the dissolved organic phosphorous (DOP) (x = 6.53 + 6.48%) than dissolved DNA carbon was of the DOC (x = 0.11 + 0.06%) at these stations. The amount of DOP that was dissolved DNA phosphorous in Tampa Bay was very similar to that found by Minear (1972) in his study of three lakes (x = 5 .19 3.18%), even though DOP made up a much larger percentage of the total soluble phosphorous in the lake samples than those from Tampa Bay (Paul et al., submitted). Dissolved DNA carbon represented a smaller percentage of total DOC at station C than at station A This may be attributable to a greater input of terrestrial DOC in the upper estuary. Diel periodicity was not apparent for most of the microbial parameters measured in the studies performed in the oligotrophic waters of the Gulf of Mexico (Pau l et al., 1986 and unpublished data), although there was a tendency for dissolved DNA values to increase throughout the night and decrease during daylight hours. Studies performed in Bayboro Harbor, however, had a pronounced diel periodicity in most of the parameters measured, especially in the June study when freshwater input was not a factor. Although bacterial numbers did not fluctuate significantly, bacterial activity peaked at 20:00 h each night, four hours before the maximum dissolved DNA values. Even though primary productivity was measured in the lab under constant illumination it was highest during daylight hours (Pau l et al., submitted). The dissolved DNA peak may be a result of a lag in extracellular DNA production after a surge in bacterial DNA synthesis, or it may be in response to the release of DOM by phytoplankton. Sondergaard and Schierup (1982), found that although the absolute 55
amount of DOM released in the light was greater than that released in the dark cycle, large molecules (>10 ,000 daltons) comprised only 1 to 4% of the light release, while the high MW fraction was 20% of the dark release. Tidal influences may have contributed to the periodicity in this data. Maximum values for dissolved DNA and primary productivity occurred on alternate high tides and maximum values for thymidine incorporation occurred on every low tide. Whether the pattern observed in these parameters was due to changes in the microbial populations with the tides is not known. An increased contribution by phytoplankton in estuarine and freshwater environments to the pool of dissolved DNA is suggested by the data from the Crystal and Alafia Rivers. Although correlations between dissol ved DNA and the other parameters i n freshwater samples were generally low, higher correlations betwee n dissolved DNA and phytoplankton, rather than bacterial parameters indicate that the major source of dissolved DNA in fresh and marine waters may differ. Paul and carlson (1984) found up to 50% of the particulate DNA in the Potomac River to be attributable to phytoplankton Like oceanic environments, most of the bacteria in the Alafia and Crystal rivers are in the <1 fraction (x = 77 12%) and most of the chlorophyll is in the >1 fraction (x = 88 + 11%). Unlike offshore environments however, an appreciable amount of the particulate D N A in the rivers is in the >1 (11 to 79%; Paul et al., unpublished data). This and correlations with chlorophyll primary productivity and DOC point to phytoplankton as a major source of dissolved DNA in freshwater environments Further dissolved DNA production studies with natural phytoplankton populations (Paul et al. 1987) in freshwater 56
environments may be able to demonstrate this more conclusively. Dissolved DNA is a ubiquitous component of the DOM in oceanic, estuarine and freshwater environments. Usually, the more eutrophic the environment, the greater the concentration of dissolved DNA. Exceptions occurred in several of the Alafia River samples There were dissolved DNA values of <5 in samples that had h igh values for all of the other parameters measured. Similar conditions also occurred after the input of warm freshwater into Bayboro Harbor during the August 1986 diel study. In the last 2 samples of the diel every other parameter increased dramatically while the concentration of dissolved DNA dropped below the level of any other concentration measured during the diel. In an estuarine environment, dissolved DNA exhibited both diel periodicity and a seasonal periodicity that was related to both bacterial and phytoplankton cycles. Correlations of dissolved DNA with other microbial biomass and activity parameters indicate bacteria as its' major source of in oceanic environments, with phytoplankton becoming more important as in estuaries and rivers. 57
58 CHAPTER 4: MOLECULAR CHARACTERIZATION OF DISSOLVED DNA Intr-oduction The genetic impor-tance of extr-acellularDNA in aquatic envir-onments is r-elated to its molecularweight. The pr-esence of gene-sized fr-agments of dissolved DNA r-epor-ted in this study suggested moleculartechniques as a means to pr-ovide useful infor'ffiation about this extr-acellulargenetic pool The method developed in this study for-conce ntr-ating and pr-obing extr-acellularDNA was used to investigate the r-esidence time of a plasmid encoded gene in seawater-. The stability of extr-acellulargenes in this envir-onment is a deter-mining factorin its potential forgenetic exchange Molecularpr-obing techniques wer-e also developed in or-derto detect a par-ticulargene sequence fr-om arrong the natur-ally occur-r-ing extr-acellularDNA. Identification of genes in the envir-onment is impor-tant for-sever-al r-easons The spr-ead of disease by vir-al and bacter-ial agents i n shellfish and r-ecr-eational water-s has become a majorhealth concer-n. A test for-Vibr-io vulnificans, a cause of wound infections and sceptacemia fr-om eating r-aw oytser-s has been devised using a DNA pr-obe forits species specific cytotoxin-herrolysin gene (Mor-r-is et al., 1987). A eDNA pr-obe has also been developed forthe detection in estuar-ine water-s of hepatitis A vir'US (HAV), which is also tr-ansmitted in waterand shellfish (Jiang et al., 1986). The concer-n overthe r-elease of GEMs in agr-icultur-al applications has led to the development of methods forthe detection of genes in soil (Holben et al. 1987; Jansson et al., 1987) In addition to detection and monitor-ing of
potentially harmful genetic sequences any ecologically important sequences could be detected. For example, molecular probing of dissolved DNA with highly conserved eukaryotic and prokaryotic sequences could be used to determine its source(s). Materials and Methods Bacterial Strains and Plasmids: The bacterial strains and plasmids used for the preparation of probes and for colony hybridization are listed in Table 3 Escherichia coli HB 101, maintained on Luria-Bertani (LB) medium with the appropriate selective antibiotic, was used for the propagation of plasmids. Plasmid pSS-NI was the vector into which the herpes simplex virus thymidine kinase gene was 59 inserted to form pS8TK2. 0 (M. Diekmann, pers. comm. ) JM 300 was a wild type, thymidine kinase minus strain of Pseudomonas stutzeri, maintained on minimal succinate media (carlson et al., 1983) and used as a negative control in thymidine incorporation and colony hybridization experiments All other strains were maintained on LB medium with the appropriate antibiotic (Table 3). Plasmid Isolation: Plasmids were amplified coli HB101 by adding 5 ml of saturated overnight culture to 500 ml of M9 media (Maniatis et al., 1982) supplemented with 0 .5% casamino acids, 2 thiamine and 50 ampicillin. These cultures were then incubated at 37C and shaken at 200 rpm. At an optical density (OD; A= 550) of 0 1 2 0 ng/ml of uridine was added When the culture reached an OD550 of 0.7, chloramphenicol was added to a final concentration of 170 and the incubation continued for 16-24 h in the dark. Cell lysis was achieved
60 Table 3: Bacterial strains and plasmids. Strain Plasmid Phenotype Source Selected E. coli HB101 pSSTK2.0 P,C Amp Paul Berg ---Marianne Diekmann E. coli HB101 pSS-NI c Amp Stanford Uni v. --Stanford, CA Pseudomonas stutzeri JM 300* wild type Gregory Stewart Biology Dept. E. coli C600 Univ. of s Florida Jl, lf550* pKT230 c Kana Tampa, FL E. coli HB101 JL lf002* pGQ3 P,C Kana Gregory Stewart E. coli C600* R388 c Trimeth Stephen Cuskey --US EPA Pseudomonas Gulf Breeze FL aeruginosa PA01* pR02317 Amp Stephen Cuskey E coli JM101 Bs+DmGSt2 c Amp Bruce Cochrane ---Biology Dept USF Tampa, FL Strains used in colony hybridization study. Thymidine kinase gene on P (plasmid) or C (chromosome)
by a modification of the alkaline lysis in Maniatis et al. --(1982) followed by a and (detailed in Appendix 2) The pellets in TE (10 mM 1.0 mM EDTA pH 7 6) and the high weight RNA was by tat ion with 2 5 M ammonium acetate. A final ethanol and of the pellet in TE used to the plasmid to an The 2 0 kilobase (kb; 1 base (bp) = 632 daltons [Lewin, 1 987]) thymidine kinase gene was obtained by a Hind III/Bam HI (Bethesda double digest of the plasmid pS13TK2.0. The 0.8 kb glutathione S gene (DmGST2) was obtained by EcoRI digestion of the Bs+ DmGST2 plasmid The the by 61 a 1% low-melting gel Biotechnologies, Inc., New Haven, CT) with Hoechst 33258 as the DNA stain (DeFlaun and Paul, 1986) The band was cut the gel and the DNA the by a developed use with an Elutip-d column and Schuell Technical Bulletin #206 Keen e NH; detail ed in Appendix 3) The DNA was then labelled by nick (Bethesda kit, MD) with [ 3H]dNTPs most with [ 3 5 s]dCTP and [ 3 5s]dGTP (1 000-1500 Ci/mmol; DuPont-NEN, Boston, MA) to a final specific activity of 2 to 8 x 108 of DNA. was by the in 10% acid (TCA)
62 subsamples Unincorporated nucleotides were removed by the spun column procedure described in Maniatis et al. ( 1 982) The probe was denatured by boiling for 5 min and cooled immediately on ice. This was followed by a second spun column purification run at 0C, 1600 g (4500 rpm) in a refrigerated centrifuge. This additional spun colurrn removed low molecular weight radiolabelled impurities and eliminated a large amount of background originally encountered with this probe Hybridization: Charge modified nylon membranes (Zeta-Probe; BioRad, Richmond, CA) were prewashed for 30 min at room temperature in 0.4 M Tris-HCl (pH 7.5). Prehybridization was in 1% sodium dodecyl sulfate (SDS), 10X Denhardt s solution (per liter: 2 g Ficoll, 2 g polyvinylpyrrolidone, 2 g bovine serum album i n [BSA; Pentax Fraction V]) 1 M NaCl, 10 mM dithiothreitol (DTT) and 10% dextran sulfate (100 )..ll/cm2 of filter) in a heat sealed plastic bag for 3 h at 65C. Dextran sulfate was ommitted in later experiments. When probing with the TK gene 150 vg/ml of the vector pS8-NI was added to the prehybridization solution to eliminate hybridization due to vector contamination of the probe DNA. The DmGST2 gene was a eDNA clone of the glutathione S-transferase gene from Drosophila melanogaster cloned in the vector Bluescribe (Stratagene, Inc ) and includes the poly T tail, therefore, 100 mg/ml of poly A was added in the prehybridization step. After 3 h of prehybridization, 100 vg/ml of denatured salmon sperm DNA and denatured probe were added Hybridization was continued for 18 hat 65C. The filters were washed two times in 2X SSPE (1X SSPE is 0 2 M NaCl, 10 mM NaH2Po4 1 mM EDTA [pH 10 mM DTT for 15 min each at room temperature; two times in 2X SSPE, 2% SDS for 45
63 min each at 65e; two t imes i n 0.1 X SSPE for 30 min each at room temperature and one time in unbuffered 3mM Tris base for 15 min at room temperature. The filter s were then air dried, sprayed lightly 3X with En3Hance (DuPont-NEN) and exposed to Kodak XAR film at 20e overnight. Filters to be rehybridized were taken directly from the last wash, covered with plastic wrap and then exposed to the XAR film. Hybridized probe was removed by washing the Zeta Probe membrane in two changes of 0 .1X sse, 0.5% SDS, 10 mM DTT at 95e overnight. The effectiveness of the washing procedure was checked by overnight exposure to XAR film. For dot blots, the radioactivity of the hybridized probe was quantified by cutting out the dots, placing them in a vial and adding 10 ml of Econofluor (DuPont-NEN). The radioactivity was measured in a liquid scintillation counte r For probing on nitrocellulose, filters were prewashed at 42e fo r 3 h in 50 mM Tris-Hel (pH 8 .0), 1 M Nael 1 mM EDTA and 0.1% SDS. Prehybridization was in 50% formamide, 5X Denhardt s 5X SSPE, 0 .1% SDS, 10 mM DTT and 100 vg/ml denatured salmon sperm DNA for 4 h at 42e Hybridization was in the same solution with the addition of the probe for 36 h at 42e The filters were washed at room temperat ure in 2X sse, 0 .1% SDS and 2 0 mM DTT, four times for 10 min each Thi s was followed by three washes for 1 5 h each in 1X sse and 0 .1% SDS at 68e (Maniat i s et al. 1982) The filters were then air dried, sprayed with En3Hance and exposed to XAR film as before. For quantification, dot blots were cut out, dissol ved in 1 ml ethyl acetate and measured by liqui d scintillation counting (LSe) with 10 ml of Aquasol (DuPont-NEN) counting fluid.
Sensitivity and Specificity of Probing: To establish the sensitivity of the probing technique and to quantify the amount of DNA hybridized, standard curves were constructed by dot blotting a series of concentrations of the plasmid pS8TK2 .0 or the TK 2 0 onto nitrocellulose or nylon membrane. These filters were transferred to 64 blot paper soaked in i) 10% SDS for 5 min; ii) 1.5 M NaCl, 0 5 M NaOH for 15 min and; iii) twice in 1.5 M NaCl, 0.5 M Tris (pH 8.0) for 5 min each. The filters were then vacuum dried at 80C for 2 h and stored at -20C until hybrization. Colony lifts were subjected to the same series of treatments before hybridization. [3H] Thymidine incorporation into a trichloroacetic acid (TCA) insoluble fraction was measured by the method of Fuhrman and Azam (1982) and was used to verify results obtained by hybridization to the thymidine kinase gene probe. Molecular Weight Determination and Detection of Genes in Environmental Samples: Large volume samples ( 1 to 10 l) for rrolecular weight determinations were filtered, concentrated and purified by the Elutip-d protocol outlined in Chapter 2 and Figure 10. The rrolecular weight range of the dissolved DNA was determined by electrophoresing the sample in a 1% agarose gel with Hind III A DNA and BstN1 pBR322 DNA molecular weight standards. Gels were stained and photographed as described in Appendix 1. Several of the samples concentrated by the Elutipd columns were subjected to RNase and DNase treatment. F o r DNase treatment, the pH of the eluate was adjusted to 5.0 by addition of 6 5 of 0 .25 M acetic acid. Five of 2 M MgS04 and 100 of 2 mg/ml DNase I (Sigma
DDNA CONCENTRATION FOR HYBRIDIZATION SEAWATER FILTRATION 0. 2 NUCLEPORE 2 VOLUMES ETOH (-20 C, 48 HRS) CENTRIFUGATION DIALYSIS48 DI H2 0 24 low salt TE and 0.4 ml t AGAROSE GEL SOUTHERN BLOT HYBRIDIZATION ETOH PRECIPITATION 5-15 TE 1 t NYLON MEMBRANE DOT BLOT HYBRIDIZATION Fig 10. Dissolved DNA (DONA) 65
66 Chemical Co., St. Louis, MO) in 0 02 M sodium acetate buffer (pH 5.0; Paul and Myers, 1982) was added and the mixture was incubated for 2 h at room temperature RNase treatment consisted of adding DNase-free RNase to the 400 Elutipd eluate at a final concentration of 10 and incubating at room temperature for 1 h (Maniatis et al., 1982). After incubation, the RNase and DNase treated samples were precipitated with 2 volumes of ethanol and prepared for electrophoresis as described above Once the gels were photographed the DNA in the gels was denatured and t ransferred to nylon or nitrocellulose membranes by Southern transfer (Maniatis et al., 1982), vacuum dried at 80C for 2 h and hybridized by the protocol described above. Recovery from ASW and ARW: Plasmid pSSTK2.0 or the p urified TK 2 0 kb fragment was added to 100 ml volumes of sterilized artificial seawater (ASW; M NaCl, M MgS04 M CaC12 ) or to sterilized artificial river water (ARW; 8.5 mM NaCl, 50 NaH2Po4 5 mM Tris, pH 8 .0) at various concentrations. Calf thymus DNA (10 ng/rnl) was added to simulate endogenous dissolved DNA concentrations and ARW samples also received 1 / 1 0 volume of 10X salts for precipitation (see Chapter 2) Controls included ASW or ARW with added calf thymus DNA and ASW or ARW with no added DNA. These samples were ethanol precipitated, dialyzed and concentrated with Elutip-d colu nns. A final ethanol precipitation was used to reduce the total volume to 15 ).11. Five microliters were dot blotted and 10 ).11 were electrophoresed in a 1% agarose gel and then transferred to nylon membrane by Southern blot (Maniatis et al. 1982) The gel filters and dot blots were then hybridized with the TK 2 0 probe. The amount of radioactivity in the
67 dot blots was measured by liquid scintillation counting. The variability in recovery of the added DNA was assessed by adding 3 ng/ml of pSSTK2.0 to 6-100 rnl aliquots of ASW. These samples were then processed and analyzed as described above Survival of Plasmid DNA in Seawater: Plasmid pS8TK2.0 (50 ng/ml and 15 ng/ml) was added to seawater in polymethylpentene flasks and incubated at environmental temperature on a slow shaker for up to 48 h. One ht.mdred rnl subsamples were taken as a function of time, filtered through a 0.2 Nuclepore filter, ethanol precipitated, dialyzed and concentrated with an Elutip-d column. Controls consisted of an equal volume of autoclaved, sterile filtered seawater with the same concentration of pSSTK2.0 plasmid added and an equal volume of seawater with the same concentration of calf thymus DNA added Seawater and sterile seawater controls with no added DNA were processed and analyzed by the same protocol. Ethanol precipitation of the Elutip-d eluate was used to bring the volume to 15 in TE. One third was blotted onto nitrocellulose and two thirds of the volume was electrophoresed on a 1% agarose gel, photographed and then transferred to nylon filters. After hybridization with the TK probe the gel filters were autoradiographed and the radioactivity in the dot blots was measured by liquid scintillation counting. Results Molecular Weight of DDNA: The molecular weight range for dissolved DNA from various environments is depicted in a series of p hotographs of
agarose gels in Figs. 11 and 12 Figure 11, gel 1, shows untreated (lane B), DNase (lane C), and RNase ( l ane D) digested extracts of Bayboro Harbor water The fluorescent ma.terial in this gel was completely digested by DNase, but not by RNase. These samples co ntained DNA with a heterogeneous ran ge of molec ular weights Apparent molecular weights, predicted by linear regression analysis of the log molecular weight versus the d istance migrated, ranged from 150 basepairs (bp) t o ap p roximately 35 kb. 68 A concentrated extract from a sampl ing site in Charlotte Harbor (Fig 4) with molecular weights ranging from 120 bp to 25.6 kb appears in lane B of ge l 2. This pattern is similar to the one from Bayboro Harbor with some very high molecular weight fragments and a preponderance of low molecu lar weight material. Bands within the smear indicate concentrati ons of DNA at certain molecular weights. There is one band at approximately 1 2 5 kb and another at 3 .31 kb. Gel 3 in Fig. 11 contains dissolved DNA concentrated from a sample taken above a coral reef in the Dry Tortugas Lanes B and C contain replicate samples except that the concentrate in lane C was RNase-treated prior to electrophoresis. The dissolved DNA in this sample appears to have a smaller range of molecular weights than the estuarine samples extending from 240 bp to 14.27 kb and does not have a high concentration of DNA in any one area of the smear Figure 12 has several examples of molecular weight ranges from both eutrophic and oligotrophic environments Gel 1 contains a Bayboro Harbor dissolved DNA concentrate ranging in molecular weight from less than 500 bp to 22. 9 kb, with a concentration of DNA at the high MW end of the range Gel 2 lane B is dissolved DNA from oligotrophic waters
23.1-9.4-6.6-4.4-2.3-2D0.56Fig 11. Agarose ge l electrophores i s of d issolved DNA samples Gel 1 (lanes B C and D), contain an untreated, DNase treated and RNase treate d sample from Bayboro Harbor G e l 2 ( l ane B), contains a sample from Charlotte Harbor Gel 3 (lanes B & C), contains an untreat e d a n d RNase treated sample from water overlying a coral reef in the Dry Tortu gas Lane A in a l l ge l s contains Hind III cut A DNA with molec u l a r wei ghts in k ilobase pairs. 0"1 \D
23.1-9 .4_ 6 .6-4.4-2 .32.0-0.56_ Fig 1 2 A 8 A 8 A 8 c D E weight by gel Gel 1 (lane B) i s a dissolved DNA Lane B in both ge l 2 and gel 3 samples the Gulf of Mexi co Lane D (ge l 3) i s a dissolved DNA mucus samples collected on the in the Lan e A (and E in gel 3) Hind III cut A DNA a n d l ane C (gel 3) i s B stN1 pBR322. -1.86 _1.06 -o.93 -0.38 -0-12 -.1 0
in the Gulf of Mexico. The molecular weight ranges from <120 bp to 12.6 kb. Gel 3, lane B, is dissolved DNA concentrated from another Gulf of Mexico sample with a molecular weight range from 120 bp to 19 kb. Lane D is a coral mucus sample taken in the Dry Tortugas. This sample has a very narrow MW range from <120 bp to 1 kb. Freshwater samples o f extracellular DNA from the Crystal and Alafia rivers also had a wide range o f molecular weights (see 'Dete ction of Genes in Environmental Samples'). Sensitivity and Specificity of Probing: Results from LSC of dot blots indicated that as little as 2 pg of DNA could be detected by [35s] labelled TK 2.0 (Fig. 13) Standard curves of TK were linear to at least 1 which was the highest concentration measured (data not shown, r = 0.993), allowing a wide range of DNA concentrations to be quantified. 71 Three methods of hybridization were tested with colony lifts of 7 bacterial strains (Table 3) to determine the specificity of the probe and the method which gave the least amount of background hybridization. [3H]Thyrnidine incorporation was used to verify the results of these hybridizations. The method for hybridization using nylon membrane (see Methods) compared favorably with that for nitrocellulose (Fig. 14), except that when dextran sulfate was added the amount of nonspecific binding of probe increased, which could be interpreted as false positives. All subsequent hybridizations were performed without dextran sulfate. For [ 3 5s] labelled probes the addition of dithiothreitol ( 1-10 rnM) in the hybridizations and washes also decreased non-specific binding of probe.
5 r = 0.999 4 2 1 0.3 1 2 3 LOG PG DNA Fig. 1 3 Standard cu rve of the TK gene detected by [35s] labelled TK on nitrocellulose. Limit of detection is approximately 2 pg. A, b, c a n d d ( A ) are the four dot blots of Bayboro Harbor DONA (from -330 m l of water each ) that hybridized to the TK probe. 72
A 8 fig. 14. Specificity of TK probe A = nitrocellulose membrane; B = nylon membrane without dextran sulfate added ; C = nylon membr ane with dextr an sulfate added Plate 1 in a l l cases had E coli pKT230 in the uppe r left and JL 4062 in the right. Plate 2 E coli R388 upper l eft, JM 300 lower left and PA01 i n the lower right. Plate 3 E coli pSSTK2. 0 upper left, E coli pSS-NI upper right and PA01 again i n the lower left quadrant. 73
74 The that exhibited the amount of coli with plasmids pKT230 and pS6TK2.0 (Fig. 14). Positive obtained all but the two pseudomonad (JM 300 and PA01). These consistent with those the [3H]thymidine (Table 4) of c35s] labelled TK 2.0 to pBR322 weight was on some of the gel This was to contamination of the since pS6-NI is a pBR322 (M. Diekmann, comm. ) When for the TK 2 0 and pS6-NI with TK 2.0, 100 pg of pS6-NI could be detected. This was only a little than one of magnitude than the detection limit of the hybridized to itself. Addition of 150 ug/ml of cold to the this nonspecific binding to a point where the of pSS-NI had to be a t least four orders of magnitude higher than the concentration of TK 2 .0 it could be detected on an (data not shown). In without added cold the radioactivitY in dot blots of pSS-NI were an average of 73% of the radioactivity in an equivalent concentration of pS6TK2 .0. With the addition of cold vector, the radioactivity of a pSB-NI dot blots dropped to an of 1.2% of that in the dot blots of pS8TK2.0.
Tabl e 4: C3H]Thymidine incorporation rates fo r bacterial strains used in colony hybridizati on studies. Bacterial strain and associated plasmid nmle/1/h E. coli HB101 pS6TK2. 0 264 .62 3.63 --P. aeruginosa PA01 pR02317 3.07 + 1.14 P stutzeri JM 300 2 70 + 0.28 E. coli HB101 --JL4062 pGQ3 378.35 + 5.10 E. coli C600 R388 333.05 + 8 .15 ---E coli C600 JL4060 pKT230 403.25 + 100.43 Recovery from ASW and ARW: Preliminary experiments in recovering DNA 75 added to ASW were probed with pS6TK2 .0 radio labelled with all four [3H]dNTPs. The specific activity of these probes was approximately 9 3 x 107 DNA o r 42 DNA. This probe was able to detect 100 pg of DNA in dot blots counted by LSC (Fig. 15) In the first gene tracking experiment there were 6-100 rnl aliquots of sterile ASW with i) no DNA added; ii) 100 ng/ml calf thymus DNA; iii) 1 ng/rnl pS6TK2.0; iv) 10 ng/rnl pS6TK2 0; v) 100 ng/ml pS6TK2.0 and vi) 1 vg/ml pSSTK2. 0 All four concentrations of plasmid were easily detectable in the dot blots (Fig 15) and in the autoradiogram of the gel filter (Fig 16) There was a greater loss in recovery in the 100 ng/rnl treatment than in the others, Which was apparent in both the amount of radioactivity in the dot blots (Fig 15) and in the lighter
7 6 E 5 Q. m 0 4 -3 2 1 2 3 r=0.994 r=0.997 4 5 6 log pg DNA 76 7 8 Fig. 15. Two standard curves of TK hybridized to [3H]labelled TK probes with a detection limit of approximately 100 pg of DNA. < ) Results of dot blots3 counted by LSC in gene recovery experiment probed with [ H]TK.
77 image on the autoradiogram (Fig. 16). . With c35sJ labelled probes the greater specific activity allowed the detection of much lower concentrations of DNA in seawater. The TK 2.0 fragment was added to 100 ml sterile ASW in concentrations of 500 fg/ml, 1 pg/ml 10 pg/ml, 100 pg/ml and 1 ng/ml. Calf thymus DNA was added (10 ng/ml) to simulate endogenous DONA concentrations present in a natural sample. Again, two thirds of the final concentrate was electrophoresed in an agarose gel and one third was dot blotted. In the gel photograph, only the 100 pg/ml and 1 ng/ml concentrations were visible. On the autoradiograph of the gel, however, all five concentrations of TK 2.0 were detected (Fig. 17) The lowest concentration added was also detected in the dot blots, although there was very little difference in the amount of radioactivity detected in the two lowest concentrations. This experiment was repeated in ARW, except that whole plasmid pSSTK2.0 was added at concentrations of 500 fg/ml, 1 pg / ml, 5 pg/ml 10 pg/ml 100 pg/ml and 1 ng/ml. The TK ge n e comprises 2 kb of a 6 kb plasmid so these concentrations are equal to 167 fg / ml, 333 fg/ml, 1 .67 pg/ml, 3.3 pg/ml, 33 pg/ml and 333 pg/ml of the TK 2 0 fragment. Only the highest concentration was visible in the gel (Fig. 18A), but in the autoradiograph of the gel (Fig. 188) the three h i g hest concentrations were visible. A l l six concentrations of p lasmid were detected in the autoradiograph (not all visible in photograph), and LSC of the dot b lots. Recovery of added DNA averaged 32 1 24.5%. Variability in the recovery of DNA from ASW by these methods was assessed by both gel electrophoresis and LSC of dot blots (F i g 19). . The dot blots were fairly uniform in size and dens ity, but there
1 2 3 4 5 6 7 8 1 2 3 4 5 6 F i g 1 6 Recovery from ASW Aga3ose gel a n d autoradiograph of experi ment probed w ith [ H]TK Lane 1 -!lind III >.DNA; 2 -pSSTK2 0 positive control. Lanes 3-8 are the 6 treatments; ASW w ith 3) n o added DNA, 11) 1 00 calf thymus DNA, 5) 1 ng/ml pSBTK2. 0 6) 1 0 ng/ml p S!3TK2. 0 7) 1 00 ng/ml p SSTK2. 0 anci 8) 1 IJg/ml pSBTK2. 0 The sarnple in eac11 lane represents approximately 6 6 rnl of the 100 ml treatrnen t 7 8 --.J 00
1 2 3 4 5 6 7 8 12345678 F i g 17. R e co very of TK from ASW with [35s]TK probes TK fragme n t was added t o 100 ml A SWin concentrations of 500 fg/ml (lane 4 ) 1 pg/ml (5) 10 pg/ml (6), 100 pg/rnl (7), an d 1 ng/ml (8). Lan e 3 i s ASW with no added TK DNA. Each sample electrophoresed represents 2/3 or -66 m l of a 100 ml sample Detection limit i s 500 fg/ml or -33 pg of DNA. ........ \0
J I a .J I' "f a at o; A 8 1 2 3 4 5 6 7 II ... c Fig. 18. Recovery from ARW with c35s]TK probe Plasmid pS8TK2. 0 was added to ARW in concentrations of 0, 500 fg/ml, 1 pg/ml, 5 pg/rnl 10 pg/ml, 100 pg/ml and 1 ng/ml (lanes 4-10). Hind III A DNA i n lane 1 and a pS8TK2. 0 positive control in lane 2 One third of the extract was dot b lotted in columns 1 7 in C with a standard curve (0-1000 pg) in columns 1-6 above. Detection limits were 3.3 pg/rnl on the ge l autoradiograph and 167 fg/ml by dot blots. 80
1 2 3 4 5 6 7 8 a b c 1 2 3 4 5 6 7 Fig 19. Variability in recovery of DDNA from ASW. Plasmid pS8TK2. 0 (30 ng/ml) was concentrated from 6 100 ml aliquots of ASW according to the protocol in Fig. 11. The final volume was divided 2/3, 1/3 between the gel (lanes 3-8) and dot blots (row C, 1-6). The coefficient of variation was 19.6%. Standard curve (rows a & B, 1-7) O, 5 10, 100, 500 and 1000 pg of TK DNA. 8 1
82 appeared to be less than the average amount of plasmid in lane 3 of the gel and more than average in lane 8. This result was confirmed by LSC of the dot blots. The samples in lanes 4-7 were all within 14% of the average amount of radioactivity in the dot blots, while the sample in lane 3 was 20% lower and the sample in lane 8 was 30% higher. The coefficient of variation (CV) for all six samples was 19.6%. Survival of Plasmid DNA in Seawater: In a preliminary experiment, 50 ng/ml of plasmid pS8TK2 .0 were added to seawater and 100 rnl samples were taken over a period of 48 h. Except for a slight decrease at 2 h (Fig. 20 )., there was a fairly constant level of radioactivity i n the dot blots up to 8 h At the 24 h timepoint, however, the radioactivity or the amount of hybr idizable DNA had decreased to nearl y a third of what it had been at 8 h. By 48 h, the amount of radioactivity remaining w a s indistinguishable from filter blanks. Although the plasmid was not very distinct in the gel photograph (Fig. 21), the decrease in the molecular weight (MW) of the DNA over 48 h was obvious The degradation of the plasmid with time is evident in the autoradiograph of the gel. From 0 to 4 h, the two bands of the plasmid were still distinguishable, but at the 8 h timepoint the bands were gone and a larger quantity of low molecular weight DNA was present. At 24 h most of the high molec ular weight DNA was gone and by 48 h there is no hybridizable DNA left on the filter and none apparent on the gel. In the second plasmid survival experiment, 15 ng/rnl of pS8TK2. 0 was added to seawater and to an equal volume of autoclaved, sterile-filtered seawater. A third treatment consisted of seawater with 15
D. c <0 2 0 ,... 1 0 2 4 6 Hours 8 24 48 Fig 20. Plasmid survival in seawater -results of dot blots counted by LSC. Plasmid pS8TK2. 0 (50 ng/ml) was added to seawater and samples were taken over a period of 48 h. The TK gene was detected by molecular probing (X) w
A B 0 2 4 8 24 48 A B 0 2 4 8 24 48 Fig 2 1 Plasmid surviva l in of gel and of gel Most of the plasmid had been by the ambient population by 24 h. Lan e A i s the pS8TK2. 0 pos itive and lane B i s the BamH1 cut pS8TK2.0. (X) .p.
85 ng/ml of calf thymus DNA added. At each timepoint, duplicate 100 m1 samples were divided equally and filtered through four 0.2 Nuclepore filters, prewashed with sterile 10 calf thymus DNA in ASW. This method of sampling decreased the amount of time that it took to filter the samples. Four 25 ml aliquots were pooled and the samples treated as previously described. Figure s 22 and 23 are the ge l pictures and autoradiograms from these three treatments. Treatment A, sterile seawater with added plasmid and treatment B, seawater with added plasmid were sampled at 0 2, 4, 8, 24 and 36 h. Treatment C, seawater with calf thymus DNA added was sampled at the 0 8 and 36 h timepoints only therefore, both replicates appear on a single gel and autoradiogram The sterile seawater treatment shows the intact plasmid in all samples, both in the gel and in the autoradiogram. The plasmid DNA recovered from seawater was smeared in comparison to the plasmid standard added directly to the gel. In treatment B distinct plasmid bands were not obvious in the gel photograph The autoradiogram, however, shows a progressive degradation of the plasmid and decrease in the molecular weight of the DNA. This degradation had already begun by the first timepoint, even though the addition of plasmid, and filtration of the sample took less than 5 min. Plasmid bands can be seen up to the 4 h timepoint, but there was a dramatic decrease in the molecular weight of the DNA by the 8 h timepoint. The 24 and 36 h samples had only small amounts of very low molecular weight DNA remaining. In treatment C, the calf thymus DNA added to seawater appears as a smear on the gel photograph. On the autoradiogram, the only DNA that hybridized to the probe was the
A tk c 0 8 36 c 0 8 36tk 8 c Fig. 22. Plasmid in #2 of gel of the (A) pSBTK2. 0 (15 ng/ml) added to (B) p SBTK2. 0 (15 ng / ml) added to seawater ; A & 8 both sample d a t 0 2 4, 8, 24 and 36 h. (C) calf thymus DNA ( 1 5 n g/ml) added to and sampled at 0 8, and 36 h; both o n the same gel Lanes tk and C the p SSTk2. 0 positive control and t h e 00 0'1
t k c 0 2 4 8 24 36 t k c 0 2 4 8 24 36 tkC0836 C0836tk A B c Fig. 2 3. Plasmid in of of gel with [ S ]TK. and samples as in Fig 23. The s i milar to t h ose found in the 1st with most of the p lasmi d by populations by 24 h In s eawater (A), the plasmi d was not degra ded a t all over the 36 h s a mpling 00 -...!
88 pS8TK2.0 standard in lanes 2 and 12. Figure 24 shows the results of the dot blots measured by LSC represented both as the percent of the time (t) = 0 sample and as the number of count s minus the control blanks These results support those seen in the autoradiograms of the gels. When plasmid was added to sterile seawater, the amount of hybridizable DNA maintained a fairly constant level over the 36 h sampling period. The anount of radioactivity in unaltered seawater, however, started to decrease after the 4 h timepoint and continued to decline throughout the 36 h experiment. Levels o f hybridizable DNA in this treatment were lowe r than in the sterile seawater treatment at the 0 timepo int even though the same amount of plasmid was added to both treatments. This result was supported by evidence in the autoradiogram that plasmid degradation had already begun by t=O. Calf thymus DNA controls (treatment C), were relatively constant and always less than 1% of the radioactivity in treatment A (Fig. 24) Results from treatment A dot blots indicate that the overall recovery of DNA after the many steps of the concentration protocol (Fig. 10) ranged from 46 to 75% (x = 61.5 + 10.5%). Detection of Genes in Environmental Samples: Six-one liter Bayboro Harbor (BH) samples were concentrated to a final volume of approximately 15 )11 in TE. Two thirds of the volume was electrophoresed in an agarose gel and one third was dot blotted onto nitrocellulose. After transfer of the gel to nylon, both filters were probed with TK, autoradiographed and the dot blots counted by LSC. There was no hybridization apparent on the Southern transfer of the gel
0 II .... .... 100 z w () a: 50 :! a. c <0 0 3 2 1 ...... / / / __ .... ---------'.. 0 2 4 ' 6 8 HOURS \ \ ' \ \ ---24 36 89 Fig 24. Plasmid survival in seawater #2. Results of LSC of dot blots presented as: 1) Top panel-the radioactivity in each sample (two replicates) as a percent of the radioactivity in the t 0 sample. Treatment A sterile seawater + pSSTK2. 0 ( . ) Treatment Bseawater + pSSTK2. 0 ( .). 2) Bottom panel -the amount of radioactivity_in-each sample minus the seawater control. Treatment A < ) treatment B ( A ) and treatment C-seawater+ calf thymus DNA ( e )
90 (data not shown), but four out of the six dot blots of Bayboro Harbor extract showed up faintly on the autoradiograph (may not be visible on photograph; Fig. 25) and were detectable by LSC (Fig. 13). The amount of DNA hybridized to the TK probe in these four samples ranged from 1 6 to 6 2 pg. In order to obtain a better signal by autoradiography, 4600 ml of BH water were concentrated and dot blotted onto nitrocellulose, along with a dot blot of 1 of calf thymus DNA to detect non-specific binding. The BH sample hybridized to the TK probe (Fig 25) while the calf thymus DNA did not. Ten liters of BH water were concentrated and divided with two-thirds of the extract electrophoresed on an agarose gel and one-third dot blotted for hybr idization (Fig. 26) The gel (Fig 26A) had an appreciable amount of DNA remaining in the well and the DNA smear extended from the well to the bottom of the gel. The TK probe hybridized to Bayboro Harbor water DDNA both in the gel and in the dot blot (Fig. 26 Band C). Although the smear of dissolved DNA extended the full length of the gel, only DNA in the high molecular weight end of the range, from the well to approximately 5 kb (lane 2 ) hybridized to the probe. These filters were washed extensively ( see Meth ods) and then probed with the DmGST2 gene The TK probe was not completely removed by the washing procedure from the pS8TK2.0 positive control and the pBR322 MW standards on the gel, but it was removed from the environmental samples which were the targets of the reprobing. The DmGST2 probe was used to determine whether the hybridization observed with the TK probe and environmental DNA was a specific binding
91 1 2 4 6 7 a b c d 1 2 3 4 5 BH CT Fig 25. Sensitivity of TK probe and detecti on of TK in environmental samples Top panel, rows a-c (column s 1 -8), are dot blots on nitrocellulose o f 0 2 5 10, 50, 1 00 500 and 1000 pg of TK fragment Row d (columns 1 6) are dot blots of 6 Bayboro Harbor DDNA concentrates. Rows 1 3 4 and 6 are the samples blotted in Fig 12. Each dot represents approximately 330 m l of seawater. Bottom panel, 1 6 are dot blots of 0 10, 50, 100, 500, and 1000 pg of TK DNA. BH is a 4600 ml concentrate of Bayboro Harbor water. CT is 1 of calf thymus DNA.
fig. 26. Probing environmental DDNA for the TK gene. (A) Agarose gel electrophoresis of approximately 6.6 1 of concentrated Bayboro Harbor (BH) water in lane 4. Hind III cut A DNA MW standards in lane 1, pS8TK2.0 in lan e 2 and BstN1 cut pBR322 in lane 5. (B) Autoradiograph of the Southern transfer of this gel after hybridization to the TK probe Note hybridization to pBR322 caused by vector c o ntamination of the probe (C) Standard curve of TK in columns 1-6 in concentrations of 5, 10, 50, 100, 500, and 1000 pg. BH is the dot blot of Bayboro Harbor DDNA representing about 3.3 1 of water.
23.1-9 .4_ 6 .6-4 .3_ 1 2 3 4 5 1 BH 93 -1.85 _1.06 -0.93 _0.38 -0.12 A B 3 4 5 6
94 of TK sequences to each other, or whether it was a non-specific attachment of probe to small fragments of DNA. The DmGST2 gene was used because it would not be found in a pool of extracellular DNA from aquatic environments. The BH water dissolved DNA did not exhibit any hybridization with the DmGST2 p r obe, even when films were exposed four times longer than the autoradiographs resulting from the probing with TK (Fig. 27). The only hybridization that resulted from the probing with the DmGST2 gene was with a 50 ng dot blot of DmGST2 t ha t served as a positive control (Fig. 27C). The length of expos ure was increased for the DrnGST2 probed filters because the J:MGST2 fragment had incroporated a lower percentage of radioactive dNTPs and was four times less radioactive than the TK probe. The results of molecular weight determinations and probing of freshwater samples for TK are in figures 28 and 29. The gel in figure 28 contains both Crystal and Alafia river samples. Station 1 on the Crystal River, sampled in April 1987 (lane 2), had dissolved DNA with an apparent mlecular weight range of 290 bp to 17. 8 kb. DNA concentrated from station 4 water on the same date (lane 3) ranged from approximately 200 bp to 19.4 kb. Station 2 from July 1987 (lane 4) had . dissolved DNA that extended from the well to about 200 bp, with a band of DNA concentrated at approximately 18 kb. The two Alafia River samples on this gel were from the July 1987 sampling date. Lane 6 contains station 4 dissolved DNA which extends from the well to the bottom of the gel, with a band of DNA at approximately 17.8 kb. Station 7 (lane 7 ) had a much smaller range from <120 bp to 2 .45 kb, although probing data (see below) indicated DNA in the upper mlecular weight end of the range.
9 5 1 2 3 4 5 1 2 3 4 5 23.19 .4_ 6.8= 4.3' .. 2.3_ 2 .0-1.85 .. _1.06 -0.93 -0.38 -0.12 A B 1 2 3 4 5 6 DmGST2 c BH Fig 27. Large volume of Bayboro Harbor dissolved DNA ( 1 0 l) for molecular probing with the DmGST2 gene on gel filter (B) and dot blot (C). These filters were reprobed with DmGST2 after original probing with the TK gene (Fig. 26) DmGST2 positive control (panel C) was the only one that hybridized with this probe
aa.1_ .... 4.3-2.3-a.C>-o :ae_ -1 1 _1.08 -o.ea -0.38 -o.1 a 123456 78 ..w Fi g 28. Freshwater samples for MW and molecular probing Molecular weight s tandards are; 1 -Hind III A and 8 -BstN1 pBR322. Dis s olved DNA samples are: 2 Crystal River (CR) 4/87 station 1; 3 CR 4/87 station 4 ; 4 CR 7/87 station 2 ; 5 n o sampl e ; 6 -Alafia R iver (AR) 7/87 station 4 ; 7 -AR 7/87 station 7 1.0 0'\
23.1-9 .4_ 6.6_ 4 .3-2 .3_ 2 .0-0 .561 2 3 4 5 6 -1.85 _1.06 -0.93 -0.12 1 2 3 4 5 6 Fig 29. Freshwater samples for MW determination and probing with TK. Molecular weight stan da r ds in lanes 1 & 6 as in Fig 28 lanes 1 & 8 Lanes: 2-AR 2 /87 station 4; 3 -AR 2 /87 station 8 ; 4 -AR 4/87 s t ation 4; 5 -AR 4/87 station 7 1.0
98 The DNA in this gel was transferred to a nylon filter and probed with TK (Fig. 28). The DNA in every extract exhibited hybridization to the TK probe (may not be visible on photograph). Stations 1 4 and 2 (lanes 2, 3 and 4) of the Crystal River (CR) samples and station 4 (lane 6) of the Alafia River (AR) samples have light bands at approximately 2 kb. Station 4 CR, also hybridized to the probe in a smear below the 2 kb band. Station 7 AR, hybridized to the probe in two darker bands in the high molecular weight end of the gel. Comparison to MW standards indicated apparent molecular weights for these bands of 11.04 and 4.36 kb. Figure 29 shows the molecular weight ranges of four Alafia River samples from the February and April 1987 sampling dates. Lane 2 contains station 4 dissolved DNA with an apparent MW range of 180 bp to 22.5 kb. Station 8 (lane 3) has a smear from 129 bp to 20.2 kb with a considerable amount in the well. During April water from stations 4 and 7 was concentrated for molecular weight determinations (lanes 4 and 5). Very little DNA was visible in the gel. There are two possible explanations for this result. Either not enough extracellular DNA was present in these samples to determine an accurate molecular weight range, or the DNA in this sample was extensively hydrolyzed and most of it was below 2 kb in size. Figure 29 also includes the autoradiograph of the Southern transfer of this gel after hybridization with the TK probe. All of the DDNA extracts hybridized to the probe in bands which were approximately 2 kb in size. The station 8 sample (lane 3) also hybridized to the probe below this 2 kb band
Discussion One of the primary considerations when developing a method for detection of genes in environmental samples is sensitivity. Traditionally, [ 32P]labelled probes have been used to obtain the required specific activity, but their short half-life and inherent 99 health risks make them inconvenient and dangerous to work with. The technique of labelling DNA with two [35 s]dNTPs yielded probes with very high specific activity and sensitivity. The [35s]TK probe was able to detect 2 pg in dot blots by both autoradiography and LSC. Recovery experiments indicated that less than 33 pg could be detected in Southern transfers of DNA recovered from ASW. This estimate is an upper limit since 33 pg is the amount of DNA added to the ASW, and does not take into account losses due to recovery in the concentration procedure and in the transfer of DNA from the gel to the nylon filter. The ability to quantify particular gene sequences concentrated from aquatic environments allows the magnitude and variability of recovery to be determined. This information is useful in the application of these methods for the detection of genes which may be present in very low concentrations in aquatic environments. The thymidine kinase gene was chosen for this study because [3H]thymidine incorporation could be used to independently verify the presence of the TK gene in culture organisms. Also, a large proportion of aquatic bacteria incorporate thymidine (Moriarty, 1986), therefore, one would expect the thymidine kinase gene to be present in a pool of aquatic extracellular DNA. The viral thymidine kinase gene used in this study has approximately 70% sequence similarity with the E. coli thymidine ---kinase gene (G. Stewart, pers. comm.), therefore, one could be used to
100 detect the other by hybridization. The recovery experiments in ASW and ARW indicated detection limits of as little as 500 fg/ml of added DNA in a 66 ml sample electrophoresed in an agarose gel and transferred to nylon membrane. In dot blots, as little as 167 fg/ml from a 33 ml sample was detectable. These concentrations are approximately 20,000 to 60,000 times less than the total dissolved DNA concentration in an average nearshore sample. This degree of sensitivity is sufficient for a fairly common gene in the small volume samples used in this study. Fuhrman and Azam (1982) demonstrated by autoradiography that the percentage of metabolically active heterotrophic bacteria in seawater samples was not statistically different from the number of cells that take up thymidine. If a large proportion of the heterotrophic bacteria in seawater incorporate thymidine into DNA via the salvage pathway, then the thymidine kinase gene should be fairly cornnon aiJDng marine bacteria. In order to estimate the contribution that the TK gene might make to the total pool of dissolved DNA, a few assumptions must be made: 1) That bacteria produce most of the DNA that makes up the extracellular DNA pool. 2) That extracellular DNA is representative of the 'community genome', i.e. that all bacterial genes are represented in the proportion that they occur in the cell; or that fractionation does not occur upon release from the cell or upon hydrolysis after release. 3) That all thymidine kinase genes have appreciable (>70%) sequences similarity with the TK probe used. Using a conservative estimate of 50% of marine bacteria in a eutrophic marine environment (2 .5 x 109 cells/1; W .H. Jeffrey, pers. comm.) being TK+, with an . . average chromosome size of 3.8 x 103 kb (Glass 1982), having the size
101 of an E. coli thymidine kinase gene (1 .4 kb; Carlson et al., 1985), and ------2 copies of the TK gene in each cell; then the TK gene would be 0 .037% of the total extracellular DNA. In an average eutrophic environment with 10 )..lg DDNA/liter, there would be 3.78 ng/l of TK DNA. A 35% recovery from 1 liter of seawater would result in 1 .29 ng of TK DNA, which is more than 600 times the limit of detection of the TK probe in dot blots. Even if only 10% of the indigenous bacteria possessed a single gene for thymidine kinase 0 .0037% of the ambient dissolved DNA would be TK or 368 pg per liter. After concentration there would be approximately 129 pg available for probing which is still 60X the detection limit. Therefore, TK should be detectable in relatively small volumes of seawater. The major objective in developing this method was to develop a model system for the detection of genes in aquatic environments and a means to quantify the target DNA. Highly concentrated extracts of dissolved DNA from eutrophic environments were usually brown in color due to humic acids and other organic materials. The influence of humic acid on hybridizations was tested by Jiang et al., (1986), while developing a method to probe for hepatitis A virus (HAV) in estuarine samples. At humic acid concentrations of 100 mg/liter, or more than twice what is normally found in estuarine environments, humic acids did not adversely affect hybridization tests. Hybridization to the TK gene probe was detected in dot blots of seawater concentrated from as little as 33 ml of Bayboro Harbor water, but more reliably from 3 3 l of sample Approximately 6 liters of Bayboro Harbor water produced a strong hybridization signal with the TK probe in a Southern transfer. Hybridization was detected in Southern
102 blots of 1500 ml samples from freshwater environments with dissolved DNA concentrations comparable to those found in Bayboro Harbor. Detection of less common DNA sequences would require larger samples, and/or a probe with 100% sequence similarity to its target DNA. Recent technology involving the use of single stranded DNA probes generated by a primer extension reaction (W. Holben, pers. comrn.) would increase sensitivity by avoiding the competitive hybridization of the double stranded DNA probes. Problems encountered with the specificity of probes prepared by the methods used in this study involve contamination by vector and chromosomal DNA, and nonspecific hybridization of probes of very high specific activity with random oligonucleotides or short DNA sequences. The results of reprobing the Bayboro Harbor samples with the DrnGST2 gene suggest that the hybridization obse rved with the TK probe was not due to the hybridization of random short-sequences of DNA. It is possible that contamination by pBR322 vector sequences was responsible for the hybridization observed in environmental samples. Although TK sequences would be detected with at least four orders of magnitude greater sensitivity, very high con centrations of pSS-NI could still be detected in dot blots even after the addition of 150 lJg/ml of unlabelled vector to the hybridizations. The probability that pBR322 sequences would be present in much higher concentrations than TK sequences in environmental samples is low, but must be considered. A wide molecular weight range (<0. 1 to >36 kb) was found for dissolved DNA from various marine environments, as might be expected for a heterogeneous and highly hydrolyzed mixture of DNA. In many samples there appeared to be some very high molecular weight material
103 that in the well, and often the DNA extended to the end of the gel, below the lowest weight (1972) high weight DNA lake as that which 50,000 daltons as by gel on Sephadex G-75. The with using gel to the weight of dissolved DNA is that if is an insufficient amount of DNA in the gel, only those of the ge l with a high of DNA will be visible. This may lead to of the weight of the DNA. These weight analyses, have that dissolved DNA and is sufficiently enough to encode gene sequences, assuming an gene as 1.5 kb (Zubay, 1983). In fact, dissolved DNA contains plasmid-sized pieces of DNA, since plasmids in weight 1 to 200 kb (Maniatis et al., 1982) That dissolved DNA is this is in view of the ubiquitous of bacteda (Maeda and Taga, 1973) and DNase activity in (Maeda and Taga, 1974; Paul et al., 1987). The of gene-sized pieces of DNA in aquatic environments the question of is the by which DNA is taken up by competent cells, into the genome an element, and the gene and 1986). Native, high weight DNA is (Notani and Setlow 1974). et al. (1983) found 15 kb-sized pieces of DNA to be optimal of Pseudomonas The size of DNA Bacillus subtilis was found to be 10 kb (Smith et
104 al., 1981 ) Concino (1981; cited in Goodgal, 1982) found no transformation with 0.3 kb fragrrents but low levels at 0.45 kb DNA. The molecular weight range of the extracellular DNA reported in this study is clearly in the range required for transformation. Only a few samples had dissolved DNA that appeared to have relatively small m lecular weight ranges, and as discussed above, this may be an artifact of the method. The process of transformation may have evolved from the utilization of DNA as a nutrient source, since many transformable bacteria can use the constituents of DNA as carbon, nitrogen, and energy sources (Stewart and Carlson, 1986 ) Previous studies have demonstrated the uptake of radioactivity from [3H]DNA by natural populations of bacteria ( Paul et al., 1987) It is not known whether DNA or its components were taken up (after hydrolysis) by these microbial populations. Natural transformation by extracellular DNA is one mechanism whereby microbial populations could exchange genetic information in aquatic environments (Stewart and Carlson, 1986). The residence time of plasmid DNA in seawater found in this study would allow this type of exchange to occur. Although the onset of degradation was imrrediate, intact plasmid was detected for at least 4 h and DNA hybridizable to the TK probe was still present at 24 h. Phillips et al. ( 1987 ) found that covalently closed plasmid DNA added to the untreated influent waters of a wastewater treatment plant converted to open circular or linear form within 10 minutes. The rate of disappearance of the pS8TK2 .0 plasmid DNA varied between sampling periods, but at the initial, fastest rate when DNA was added to Bayboro Harbor water at approximately ambient concentration ( 15 it was hydrolyzed at a
105 rate of 1 57 J.l.g/lh. When a high concentration c 7 x ambient ) of plasmid DNA was added ( 50 ]Jg/1), the rate was 2 ]Jg/l h These results are very similar to those obtained by Paul et al. (1987) in their study of the hydrolysis of [ 3HJ labelled E coli B DNA in seawater. When ---c3HJDNA was added at an ambient concentration it was hydrolysed at a rate of 1 .4 ]Jg/lh and when added to a final concentration of 56.07 ]Jg/1, the rate of hydrolysis was 2 036 ]Jg/l h (unpublished data), indicating a rapid degradation of dissolved DNA by resident microbial populations. Bazelyan and Ayzatullin (1979), calculated a much slower rate of hydrolysis of 1 to 2 ]Jg/l day or 0.04 to 0 08 ]Jg/l h but had added DNA in milligram per liter concentrati ons and inexplicably DNase to 0 5 ]Jg/1. The process of natural transformation mediated by dissolved DNA may represent on e mechanism for the flux of genetic information through aquatic microbial populations. Knowledge of s uch a process is important due to the use of genetically engineered organ isms in the environment. The presence of transformable bacteria among marine microbial populations (G. Stewart pers. comm.) and the persistence of extracellular DNA demonstrated in this study, make studies of this kind imperative. Extracellular DNA is probably representative of the community genome' in aquatic environments. Most aquatic microorganisms probably contribute to this pool by either death, cell lysis or acti ve excretion. The ability to monitor this pool of potentially transforming genetic material is very important to a technology whose future lies in its ability to assess its own risk to the environment.
106 CHAPTER 5 : SUMMARY AND CONCLUSIONS Dissolved DNA was found in all of the and sampled Oceanic of dissolved DNA the in the Gulf of Mexico 0.2 to 19 with the lowest in Dissolved DNA concentrntions with depth in the colurrn maintaining a constant minimum value 300 to 15 00 m of dissolved DNA low in the the in the but (1 8 to 11.7 times) in the a seasonal study, in the Tampa Bay dissolved DNA 1 8 to 18.9 and with Dissolved DNA was only a small (x = 0 .1%) of the total dissolved (DOC) in the a 15 month Dissolved DNA 6 .5% of the total dissolved in the Although diel of DNA was minimal in studies, was a distinct of maximum DONA in the at night. The Alafia had a of of DONA a seven month = 1 1 to 25. 5 The had a of the same (0 6 to 8 Dissolved DNA in the 0.12% of the total DOC in to the Alafia values which 0 .04%. The wide of weights dissolved DNA
107 from a variety of aquatic environments (120 bp 35 kb), indicated that the fragments of DNA were of a size sufficient to encode for genes gene sequences and plasmids. A concentration and molecular probing protocol was developed for DNA dissolved in aquatic environments The thymidine kinase gene, contained on the plasmid pSSTK2 0 was used in this model system for detecting genes in environmental samples The TK gene DNA, dissolved in ar-tificial seawater, was detected at concentrations ranging fr-om 20,000 to 60, 000 times less than the total dissolved DNA found in environmental samples. Plasmid pS8TK2.0 DNA added to seawater was rapidly degraded. Intact plasmid bands were detected 4 h after the addition of plasmid and low molecular weight DNA hybridizable to the TK probe was detected after 24 h By 36 h the plasmid DNA was no longer detectable in the dissolved fraction. Hybridization to the TK gene probe was detected in both marine and freshwater dissolved DNA samples The results of this study r-aise many questions. The distribution and seasonal variation of dissolved DNA concentrations found in subtropical aquatic environments may be very different from those in other climatic regimes. The determination of molecular weight ranges of dissolved DNA by agarose gel electrophoresis is unable to resolve DNA fragments of sizes greater than 60kb Alternatives such as gel chromatography or field inversion gel electrophoresis (FIGE) may be useful in determ ining the size distribution of larger DDNA fragments. The degradation of circular plasmid DNA in seawaterwas examined however, linear forms are probably more prevalent and the rate of linear DNA degradation should be determined The results of molecular probing of dissolved DNA extracts indicate that these techniques may
108 have many applications in aquatic environments Definitive results, however require gene probes with greater specificity and sensitivity than was achieved with the thymidine kinase probe. Molecular probing with genes conserved within certain groups of micr oo rganisms may be used to determine the source of DNA dissolved in aquatic environments as well as providing taxonomic information. Othe r application include monitoring genes introduced into these systems
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APPENDIX 1 : HOECHST 33258 STAINING OF DNA IN AGAROSE GEL ELECTROPHORESIS Introduction 118 Agarose gel electrophoresis has become a widely used technique for resolving DNA molecules by molecular weight and configuration. Most often DNA is visualized in these gels by staining with the fluorochrome ethidium bromide. Upon intercalation with double stranded regions of nucleic acids, ethidium bromide emits a bright orange fluorescence under ultraviolet illumination. At low concentrations, ethidium bromide in the gel and the electrophoresis buffer allows the visualization of DNA and RNA both during and immediately after the run, and does not require destaining (Sharp et al., 1973). Although a popular tool, the powerful mutagenic and probable carcinogenic properties of ethidium bromide make it dangerous for routine use (Strauss, 1971). Researchers who make frequent use of ethidium bromide risk toxic exposure both from inhalation of the powdered form and absorption through the skin of the powdered or dissolved forms. Other dyes that have been used for agarose gels include acridine orange (McMaster and Carmichael, 1 977), 4' 6 diamidino-2-phenylindole (DAPI) (Kapuscinski and Yanagi, 1979), and Hoechst 33258 (Hyman and James, 1983). The non-intercalating dyes, DAPI and Hoechst 33258, are not as toxic as ethidium bromide or acridine orange However, all three of these alternative stains were much less convenient to use because the methods proposed involved tedious destaining procedures.
119 Appendix 1: con't In developing a method for visualization of dissolved DNA extracts in agarose gels, the logical choice of stain was one whose properties were most familiar and one which was used for other DNA assays in this study Hoechst 33258 is a weakly fluorescent dye that increases in fluorescence upon binding to the A-T rich portions of double-stranded DNA (Lat t and Stat ten, 1976; Steiner and Sternberg, 1979). This non-intercalati ve mode of dye binding is relatively non-toxic and non-mutagenic, allowing its use as a vital stain for the determination of the DNA content of viable cells (Arndt-Jovin and Jovin 1977; Maryanski et al., 1980). The method developed for Hoechst 33258 is as expedient and simple to use as the ethidium bromide technique with the distinct advantage of being relatively non-toxic. Methods Bacteriophage A DNA, Hind III digest was purchased from Sigma Chemical Co. (St. Louis, MO), wheat germ ribonucleic acid was from Calbiochem (La Jolla, CA) and the plasmid pBR325, both EcoRI cut and unc u t plasmid were kindly provided by Dr. Lee Weber (University of South Florida, Tampa). The electrophoresis buffer system used was 0.04 M Tris-acetate0.002 M EDTA (TAE) (pH 7 6) The loading buffer consisted of 0.25% bromphenol blue and 40% sucrose in deionized distilled water. A 1 .5 x 104 M stock solution of Hoechst 33258 (Calbiochem) was prepared weekly in deionized distilled water and stored at 4C in a foil wrapped bottle. One percent agarose gels (Type 1 : Low EEO, Sigma) were
120 Appendix 1: con t prepared in TAE buffer containing either 5 x 108 M Hoechst 33258 or 2 5 x 10-6M ethi dium bromide. The electrophoresis buffer consisted of 1x TAE with either 2.5 x 10-6M ethidium bromi de or 5 x 10-8 M Hoechst 33258 Samples prepared in 0 5 ml minifuge tubes were brought to a final volume of 9 with 1x TAE and 2 loading buffer and spun briefly in a microfuge to facilitate loading. The BioRad Mini-sub gels were run at 6 V/cm until the bottom of the marker dye was approximately 1-2 em from the bottom of the gel. Gels were illuminated with two 15 W General Electric blacklights and photographe d with a H o n eywel l Pentax Spotmatic F 35 camera equipped with an Osah i 50 mm macro lens. A Tiffen G15 orange filter was used for ethidium bromide gels and a Corning Glass 3 -70 filter (yell o w-orange) for Hoechst 33258 gels. Results and Discussion Figure 30 shows the results of identical agarose gel electrophoresis runs using ethidium bromide (gel A) and Hoechst 33258 (gel B). Seve ral species of DNA and RNA (see legend) were used to illustrate the similarities and differences i n staining by these two dyes. DNA and RNA fluoresce orange on ethidium bromide gels. DNA fluoresces light b lue on Hoechst 33258 gel s whil e RNA fluoresces yellow. This color difference aids in interpretation of bands on the gel. Similar band patterns indicate that the relative migration rates of the different species of DNA were not altered by the different modes of binding of Hoechst 33258 and ethidium bromide
121 A B 1 2 3 4 1 2 3 4 F i g 30. (A) Ethidium stained gel. (B) Hoechst 33258 stain ed gel. Both gels contain: l a n e 1, Hind III cut ADNA (320 ng); lane 2 wheat kRNA (2000 ng); lane 3 plasmid pBR325 DNA (350 ng); lane 4 EcoRI cut pBR 325 DNA (200 ng)
122 Appendix 1: con't The bands on the Hoechst gel did not fluoresce as intensely as those on the ethidium bromide gel suggesting that more DNA might be needed to detect bands with this dye. The sensitivity of this method was tested by runnin g a series of concentrations of Hindiii digested A DNA on both an ethidium bromide and a Hoechst 33258 stained gel (Fig. 31 ). Both staining methods showed a detection limit of 1 ng of DNA per band. The Hoechst stained gel showed greater resolution over the range of DNA concentrations, decreasing the problems associated with overloading. The sensitivity of this method could probably be improved upon using a stronger UV source than the one available for this study. Hoechst 33258 has been found to be highly specific for DNA, with little interfer e n ce by RNA in fluorometric determinations (Cesarone, et al. 1979; LaBaraca and Paigen, 1980; P aul and Myers, 1982), while ethidium bromide stains both DNA and RNA. Although not as bright as on ethidium bromide gels, RNA was visible on the Hoechst stained gels. In order to confirm that this fluorescence was indeed due to RNA, the samples were treated with DNase-free RNase (Maniatis et al., 1982) prior to electrophoresis. The disappearance of the bands a t the bottom of lanes 2 and 3 (Fig. 30) which contain wheat germ RNA and a crude extract of plasmid pBR325 DNA confirmed that these were indeed due to RNA (results not shown). The simplicity of this method and the relative safety of Hoechst 33258 as opposed to ethidium bromide make this dye a viable alternative for routine resolution of DNA in agarose ge l electrophoresis. The presence of the dye in the gel and the electrophoresis buffer allows
123 f I .. o-: . I 1 I . 1 . . . "-' "" c .:. 0 ..... J :'.; 0 .>..) ..... t'l ""!:: 0 ,. 1 I 1 --t ; __ I J111 II "') ;.... c "J ""'\ """' ',') (V) c :> 0 c .:..> :f) -. 0 t... 0 .Q [:J I I II 6 ........ ;? ::.... 1 ) J t II : . i . ) . 4 =-...... Q t'l c :..') -,-'C t... ...:. ':..) Q) () c c ..... "0 ""' -.... .;..) c '/) -' 0 -""J .:'\ 'l.' .:.> rY'I -:; c ..... ,... <:: 5 <;... .D :') .::. :: -::--:...) _::; < "0 '"" z -< ...... -< :: ......, ...:. .-Y) '..!) ::.,_.
1 2 4 Appendix 1 : con t for immediate visualization of the bands as well as the ability to check the bands during the run. Although this is a standard procedure for ethidium bromide gels, previously described methods for Hoechst 33258 (Hyman and James, 1 983) DAPI (Kapuscinski and Yanagi 1 979), and acridine orange (McMaster and Carmichael 1 977), required lengthy destaining steps in order to visualize bands in the gel. The method of Hyman and James (1983) for Hoechst 33258 used a dye concentration of 0 1 lJg/ml which resulted in overstaining and background fluorescence of the gel itself which necessitated 3 h of destaining. Our method used Hoechst 33258 at concentrations of 3 .12 x 10-8 g/ml. This concentration is adequate for visualization of DNA bands at concentrations equivalent to those used on ethidium bromide gels, yet is not high enough to cause the gel itsel f to fluoresce. Most ethidium bromide gels are run at concentrations of 0 .5-1 0 lJg/ml which is approximately 25-50 times the molar concentration of Hoechst 33258 used in this method. In addition to further reducing the possibility of toxicity, this very low concentration of stain may not interfere w ith further manipu lations of the DNA. If this is the case, then the use of Hoechst 33258 in gels, cesium chloride gradients and other DNA visualization methods may eliminate the steps needed to remove the ethidium bromide in these procedures. Cytotoxicity studies have shown that short incubations with Hoechst 33258 do not affect cell viability (Arndt-Jovin and Jovin, 1977) However, aberrant mitosis in cells exposed for long periods of time (Hilw i g cited in Arndt-Jovin and Jov i n 1977) and slight
125 Appendix 1: con't abnormalities in the chromosomes of cultured mammalian cells held in high concentrations of Hoechst 33258 (Pimpinelli et al., 1976 ) have been observed Therefore, direct contact should be avoided when handling this dye This method f o r Hoechst 33258 staining of agarose gels satisfies the requirements for an alternative to ethidium bromide: (i) it is as simple to use employing pre-existing equipment; (ii) it has equal or greater specificity for DNA; (iii) and most importantly it is less dangerous to laboratory personnel and the environment.
* APPENDIX 2: LARGE SCALE PLASMID DNA ISOLATION 1. Divide a 500 ml saturated overnight between 2-500 ml centrifuge tubes, cool in an ice bath. Spin at 5,000 rpm in a Sorvall centrifuge for 10 min at 4C. 126 2. Decant and resuspend the pellets in 25 ml TE. Transfer to 2-38 ml Oak Ridge tubes. Centrifuge for 10 min, 4,000 rpm, 4C. Decant leaving the pellets as dry as possible. 3. Resuspend the pellets in 5 ml GTE buffer containing 4 mg/ml lysozyme added immediately before use-vortex well. Store for 5 min at room temperature. GTE 50 mM glucose 10 mM EDTA 25 mM Tris, pH 8 0 4. Add to each tube 10 ml freshly prepared solution of 0 2 N NaOH, 1% SDS. Invert the tube rapidly 3x -store on ice exactly 5 min. 5. Add to each tube 7 5 ml ice cold potassium acetate solution-vortex for 1 0 sec Store on ice for 10 min. Centrifuge 10 min, 12,000 4oc. rpm, Potassium acetate solution 60 ml of 5 M potassium acetate 11.5 ml glacial acetic acid 28:5 ml distilled water 6. Decant into polypropylene tubes containing 5 ml redistilled phenol Vortex 30 sec. Add 5 ml chloroform. Vortex 30 sec. Centrifuge 5 min at 10,000 rpm.
Appendix 2: con't 7 Transfer -20 ml of the upper phase to a fresh polypropylene tube. Add 10 ml chloroform and vortex for 30 sec. Centrifuge 5 min at 10,000 rpm. 8. Transfer the upper phase to a fresh polypropylene tube and add 12 ml of isopropanol Mix and let stand at room temperature for at least 10 min (can be much longer). Centrifuge for 15 min, 12,000 rpm at room temperature 9. Dissolve each pellet in 2.5 ml TE. Vortex and keep on ice until dissolved. Transfer to a 15 ml Corex tube and add 0 5 vol of 7.5 M ammonium acetate. Mix and hold on ice for at least 20 min (can be several hours) Centrifuge 10 min, 12,000 rpm, 4C. 10. Transfer the supernatant to another Corex tube and add 2 vols of ethanol. Store at -20C overnight. 11. Centrifuge 10 min at 12,000 rpm, 4C. Decant supernatant and bring pellet up in 0.5 ml TE. Pool and quantify by Hoechst 33258 method. Method provided by Dr. Lee Weber.
APPENDIX 3: RECOVERY OF DNA FROM LOW MELTING TEMPERATURE AGAROSE IN A MINIGEL APPARATUS 1. Dissolve 0.25 g LMT agarose in ml DI Dissolve in a boiling water bath. 128 2. Cool to -40C, add 2.5 ml 10x TAE and 250 8.3 x 10-3M Hoechst 33258. Cool and pour into gel mold with a preparative comb. 3. Place the partially set gel, the minigel apparatus and 250 ml 1x TAE containing 2.5 ml of 8 .3 x 10-3 M Hoechst at 4C for -30 min. 4. Electrophoresis is performed with the minigel apparatus immersed in an i ce bath at no more than 5 V/cm. 5. Once the band of has been cut it out of the gel and place it in a 38 rnl Oak Ridge tube with -10x volume ( 15-20 ml) of low salt TE. 6. Melt the gel with the Oak Ridge tube immersed in a 65C water bath for 30 min. periodically to make that the gel is completely dissolved in the TE. 7. the sample, low salt TE and high salt TE in a 37C water bath. 8. Pass 2 ml of the warm high salt TE over an Elutip-d column. Work quickly so that the solutions or the column do not cool. back solution up the column-one way only. 9. Pass 5 m1 of the low salt TE over the column and then force the DNA solution over the column at the rate of 2 drop/sec. Follow the sample with 3 ml of low salt TE.
129 Appendix 3 : con't 10. Elute the DNA with 0 4 ml of the warm high salt solution into a 1.5 ml microfuge tube Repeat the elution. Add 2 vols of ethanol to each tube and store at -20C overnight. 11. Spin the samples for 15 min in a rnicrofuge and resuspend the pellets in an appropriate v olume of TE.