APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1993, p. 712-7170099-22401931030712-06$02.00/0Copyright © 1993, American Society for Microbiology

Abundance of Virus-Sized Non-DNase-Digestible DNA(Coated DNA) in Eutrophic Seawater

A. MARUYAMA,1* M. ODA,2 AND T. HIGASHIHARA'Microbial Resources' and Genetics and Breeding,2 National Institute ofBioscience and Human Technology,

1-1-3 Higashi, Tsukuba, Ibaraki 305, JapanReceived 4 September 1992/Accepted 8 December 1992

Total DNA concentration in 0.2-jim-pore-size Nuclepore filter filtrates (<0.2-jum fraction) of Tokyo Baywater was estimated to be 9 to 19 ng/ml by an immunochemical quantification method. Almost 90% of the DNAin the <0.2-jim fraction was found in the size fractions larger than 3.0 x 105 Da and 0.03 jum, and most wasnot susceptible to DNase digestion, that is, consisted of non-DNase-digestible DNA (coated DNA). A significantamount of DNA was obtained from the <0.2-jim fraction of the seawater by three different methods:polyethylene glycol precipitation, direct ethanol precipitation, and ultrafilter concentration. Gel electrophore-sis analysis of the isolated DNAs showed that they consisted mainly of coated DNAs with a similar molecularsizes (20 to 30 kb [1.3 x 107 to 2.0 x 107 Da). The abundance of the ultramicron virus-sized coated DNA innatural seawater suggests that these DNA-rich particles can be attributed to marine DNA virus assemblagesand that they may be a significant phosphorus reservoir in the environment.

DNA as a genetic material is believed to be present innatural seawater in various kinds of forms: a naked freeform, a free form absorbed by certain detrital particles, a

protein-encapsulated and/or coated form (e.g., most viralDNA), and prokaryotic and eukaryotic cell membrane-coated forms. In seawater, significant amounts of DNA havebeen detected in the particulate fraction larger than 0.2 ,um,showing that the particulate DNA concentration can be a

valuable indicator of total microbial biomass (22, 25). Re-cently, an amount of DNA corresponding to particulateDNA was also detected in the fraction consisting of particlessmaller than 0.2 ,um, the so-called dissolved fraction. De-Flaun et al. (5) detected dissolved DNA (D-DNA) in con-centrations of 0.2 to 5 ,ug/liter in offshore water and 10 to 19,ug/liter in estuary water by ethanol precipitation and fluo-rescence detection with Hoechst 33258 dye (4). Using asimilar dye method, Karl and Bailiff (10) estimated theD-DNA concentrations in fresh water and seawater andsummarized the range of concentrations as 0.56 to 88 ,ug/literin natural aquatic environments. These estimates raise thequestions of why such a large amount of DNA is present inthe <0.2-p,m fraction of natural seawater and whether theabundant DNA in the fraction is a free form easily digestedby DNase or a non-DNase-digestible coated form.

Recently, surprising numbers of virus-like particles (103 to108 particles per ml) were detected in natural seawatersamples (1, 6, 29, 38). This number is very large in compar-ison with the plaque counts (100 to 102 PFU/ml) obtainedwith prokaryotic cells as hosts (8). These findings on theabundance of viral particles in seawater have stimulatedstudies aimed at detection of dominant virus-host systems inmarine environments and their relation to natural microbialmortality (2, 3, 7, 29, 30, 36). Other studies have examinedthe contributions of viral particles to the composition ofmaterial in seawater (27, 40) and the organic-particle flux inthe oceans (30). In addition, an understanding of the detailsof DNA material in natural seawater is essential to evaluatethe scale and the function of marine ecosystems (17, 22-26,

* Corresponding author.

35). However, there is no established method of detectingand characterizing viral DNA in natural aquatic environ-ments, so the contributions of viral DNA to total D-DNAand to total dissolved organic components such as dissolvedorganic carbon, dissolved organic nitrogen, and dissolvedorganic phosphorus (DOP) are poorly understood.

In the present study, we aimed to develop rapid andsensitive methods to detect coated DNA in natural seawater.Enzymatic differentiation between free and coated DNAmaterials, size fractionation of DNA, and highly sensitiveimmunochemical-potentiometric DNA quantification were

carried out with eutrophic seawater samples. We also triedto collect DNA in filtrates of seawater passed through a0.2-,um-pore-size filter (<0.2-p,m fraction) on a large scale toevaluate the approximate molecular size of natural coatedDNA. We found that a significant amount of DNA in the<0.2-pum fraction of seawater is in a non-DNase-digestibleform and that these DNAs were of similar, virus-like sizes.

MATERUILS AND METHODS

Natural seawater samples were obtained from TakeshibaWharf, Tokyo, Japan, in the northern part of Tokyo Bay(35°39'N, 139°46'E), in June and July 1991. After removal oflarge particles and organisms with an XX13 plankton net(mesh opening size, 100 ,um), surface seawater samples wereaseptically filtered with Nuclepore filters (0.2-,um pore size)under a vacuum (<200 mm Hg [1 mm Hg = 133.322 Pa]).The filtrates were stored on ice and used as samples for DNAanalysis a few hours later. Surface seawater samples werealso filtered with Nuclepore filters (0.4-, 0.1-, and 0.03-jimpore sizes), a Millipore Millex-HV filter (0.45-jim pore size),and Millipore ultrafiltration apparatuses (Molcut L; normalmolecular weight limits [NMWLs], 10,000, 30,000, 100,000,and 300,000), and the filtrates obtained were used for DNAanalysis. Total bacterial cells were directly counted with an

epifluorescence microscope as described previously (17) byusing a fluorescent dye, 4',6-diamidino-2-phenylindole(DAPI) (28).The DNA concentration in the seawater samples was

determined by the immunochemical-potentiometric method

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(11) with single-stranded DNA (ssDNA)-binding proteins(SSB) and monoclonal anti-DNA antibodies. Portions of theseawater filtrates stored on ice were treated with DNase(37°C, 30 min) to degrade free DNA and to provide non-

DNase-digestible DNA (coated DNA) samples. Samples nottreated with DNase were regarded as total DNA samples.DNase I (type II; Sigma) was used for the treatment at a

concentration of 10 p.g/ml (ca. 20 Kunitz units/ml), and thereaction temperature and time were determined so as toallow the complete digestion of A phage DNA HindlIldigests (Takara, Kyoto, Japan) but not to affect the viabilityof A cIts857Sam7 phage (Stratagene, La Jolla, Calif.). Allsamples were then incubated with a mixture of proteinase K(pH 7.5; final concentration, 50 p.g/ml; Wako, Osaka, Japan),EDTA (pH 8.0; final concentration, 80 to 100 mM; Dojin,Kumamoto, Japan), and sodium dodecyl sulfate (SDS; finalconcentration, 0.2%; Wako) at 56°C for 1 h, since completedestruction of DNase and disruption of DNA-coating mate-rials such as viral proteins were indispensable for furtherquantitative analysis (11, 33).

After serial dilution with NT buffer (150 mM NaCl, 20 mMTris-Cl, 0.05% NaN3, 1 mM EDTA [pH 7.5]), DNA-contain-ing samples were heated at >95°C for 15 min. The resultingssDNA in the samples was conjugated with SSB and urease-bound anti-DNA antibodies on a membrane. The urease

activity of the membrane-SSB-ssDNA-urease-bound anti-DNA antibody complex was then measured by a siliconsensor-based potentiometer system (Threshold; MolecularDevices, Menlo Park, Calif.) according to the directions inthe Threshold manual. Denatured calf thymus DNA (type I)was used for standard ssDNA in the range of 2 to 200 pg, andan amount-signal calibration curve was made by Thresholdcomputer analysis. All of the reagents were purchased fromMolecular Devices and Wako and used under aseptic condi-tions. Samples from the same origin were analyzed at thesame time to eliminate experimental error. Duplicate analy-sis was carried out for every sample. The DNA concentra-tion was further corrected by the recovery rate of 50 pg ofssDNA added to samples before the immunochemical reac-tion. The free DNA concentration was obtained by subtract-ing the coated DNA concentration from the total DNAconcentration.For large-scale isolation of DNA materials from seawater

filtrates, polyethylene glycol (PEG; molecular weight, 8,000;Sigma) at various concentrations and NaCl (final concentra-tion, 0.3 to 0.5 M) were added to 200 to 500 ml of theseawater filtrates, and the mixtures were stored at 4°Covernight. After centrifugation at 12,000 x g for 15 min at4°C, precipitates were resolved in TE buffer (25 mM Tris-Cl,1 mM EDTA [pH 8.0]). Some portions of the precipitatedsamples were submitted to DNase treatment (37°C, 30 min,ca. 10 to 16 Kunitz units/ml), and all samples were treatedwith EDTA (final concentration, 20 mM) and SDS (finalconcentration, 0.2%) at 65°C for 1 h. The samples were thenextracted with phenol, and nucleic acids were precipitatedwith ethanol (33). The purified DNA samples were loadedinto sample wells of a 0.7% agarose gel (type I; Sigma) (theamounts of the loading samples were measured so as to beequal in initial extraction volume), and the gel was electro-phoresed at 10 V/cm in TBE buffer (45 mM Tris-borate, 1mM EDTA [pH 8.0]). DNA fragments in the gel were thenstained with ethidium bromide (0.5 p.g/ml; Sigma) and pho-tographed under a UV transilluminator with Polaroid type667 film.

In the case of DNA isolation without PEG, EDTA (finalconcentration, 80 mM) and SDS (final concentration, 0.2%)

TABLE 1. Size fractionation and immunochemical-potentiometricquantification of DNA materials in Tokyo Bay water

Relative content (%) in water sampleSize fraction (filter)a obtained onb:

6 June 10 June 4 July

<0.45 p,m (HV) 168.9 NT<0.4 p.m (NP) NT 167.7<0.2 ,um (NP) 100.0 100.0 100.0<0.1 pm (NP) 83.5 35.3 73.2<0.03 p.m (NP) NT NT 10.0NMWL <300,000 (M) 7.6 NT 0.6NMWL <100,000 (M) 7.0 NT 0.2NMWL <30,000 (M) 2.4 NT NTNMWL <10,000 (M) <2.4 NT NT

a Filters: NP, Nuclepore; HV, Millipore Millex-HV; M, Millipore MolcutL.

b All samples were obtained in 1991. The D-DNA concentrations (innanograms per milliliter) in the <0.2-p.m fraction (Nuclepore filter) were 13.0on 6 June, 18.9 on 10 June, and 9.2 on 4 July. The bacterial counts (in cells permilliliter) in the >0.2-pm fraction (Nuclepore filter) were 8.2 x 106 on 6 June,5.9 x 106 on 10 June, and 4.9 x 106 on 4 July. NT, not tested.

were directly added to seawater filtrates (direct ethanolprecipitation) or ultrafilter concentrates (ultrafilter concen-tration), and the mixtures were incubated for 30 min at 65°C.An amount of ethanol equal to twice the volume of themixture was then added. After centrifugation, the precipi-tates were resolved in TE buffer containing 150 mM NaCland applied to a Whatman DEAE-cellulose column (2- to3-ml bed volume). After the column was washed with 10 mlof TE buffer containing 150 mM NaCl, adsorbed materialswere eluted with TE buffer containing 1 M NaCl, and thennucleic acids were precipitated with ethanol. A Molcutultrafiltration filter (NMWL, 300,000) was used for theconcentration of DNA materials in the <0.2-p.m fraction ofthe seawater. Retained materials on the filter were resus-pended in TE buffer and further purified by phenol extrac-tion and ethanol precipitation. The final samples obtained bythree isolation methods (PEG precipitation, direct ethanolprecipitation, and ultrafilter concentration) were applied tothe gel for electrophoresis. RNase treatment was carried outat 37°C for 30 min with RNase A (final concentration, 22p.g/ml; Sigma). A phage DNA EcoRI and HindIII digestswere obtained from Nippon Gene (Toyama, Japan).

RESULTS AND DISCUSSION

The DNA concentration in the <0.2-p.m fraction (D-DNA)of Tokyo Bay water was determined to be in the range of 9to 19 ng/ml by the immunochemical method (Table 1). Thesevalues corresponded to the upper part of the range ofseawater D-DNA concentrations estimated by the dyemethod (5, 23). Bacterial cell number was also high in theTokyo Bay water, on the order of 106 cells per ml (Table 1);this result indicated that the environment was still eutrophic(34).

Size fractionation experiments showed that almost 90% ofthe D-DNA was present in the >300,000-Da fraction or inthe fraction consisting of material larger than the 0.03-p.mfilter pore size (Table 1). In addition, most of the DNA in the<0.2-p.m fraction of the seawater was resistant to DNasedigestion (Fig. 1), as was the DNA in the <0.4-p.m and<0.1-p.m fractions. These results indicated that a consider-able amount of DNA in the 0.03-p.m (or 300,000-Da) to

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DNA content (%7.)

50 100 150

FIG. 1. Relative contents of DNase-digestible DNA (free DNA)and non-DNase-digestible DNA (coated DNA) in different sizefractions of Tokyo Bay water.

0.2-,um fraction, almost 80% of the total D-DNA, was in thenon-DNase-digestible coated form.

In the samples collected directly by PEG precipitationfrom the seawater filtrates, ethidium bromide-stainable ma-terials appeared as almost a single clear band (Fig. 2). Thesematerials were completely degraded by DNase treatment inthe final step of the purification but not by RNase, and theamount of the materials obtained corresponded approxi-mately to the DNA concentration of the original sample.These results clearly showed that the PEG method wasuseful in collecting natural DNA materials from seawater.Most of the DNAs collected had molecular sizes of 20 to 30kilobase pairs (kb) (13 x 106 to 20 x 106 Da). In addition, 20-

to 30-kb DNA was resistant to DNase treatment before theaddition of the surfactant SDS, indicating that the DNAoriginated from certain coated DNA materials. The amountof non-DNase-digestible DNA was estimated to be almostequal to that of total DNA obtained, and more non-DNase-digestible DNA was recovered in the <0.4-p.m and <0.2-,umfractions than in the <0.1-,um fraction (Fig. 2).As shown in Fig. 3, higher PEG concentrations resulted in

higher recovery of DNA from the seawater samples, al-though the collection-and-purification step was technicallydifficult with high concentrations of PEG. In addition, mostof the PEG-precipitated DNA appeared to be in the coatedform. Since the PEG precipitation method has been used asa standard method for concentrating viruses from culturebroth on a large scale (33, 41) and it is known that PEGstimulates the precipitation of certain free DNAs (12), thePEG-precipitated DNA seems to reflect the natural DNAprofile and to include all of the DNA in the higher concen-trations. Very similar results were obtained by direct ethanolprecipitation, which may be more comprehensive for naturalDNA samples than the PEG method, and by ultrafilterconcentration (Fig. 3). These results ensured that a majorportion of the DNA in the seawater filtrates was in theultramicron-sized non-DNase-digestible form. That there isa large quantity of ultramicron-sized coated DNA in seawa-ter seems reasonable, since the level of DNase activity ineutrophic environments is remarkably high (14, 15) andalmost all viral particles in natural seawater are larger than30 nm (1, 6). The present estimate of the molecular sizedistribution of DNA in the <0.2-,um fraction of seawater is inthe range of 0.12 to 35.2 kb reported by DeFlaun et al. (5),while the remarkable abundance of the coated DNA of 20 to30 kb has not previously been reported.

Since, in addition to the particle sizes, the estimatedcoated DNA sizes are in the range reported for DNA virusesto date, it is very likely that the coated DNA in seawaterconsists mainly of viral DNA. From the approximate DNA

M 1 1D 2 2D 2d 2r 4 4D C CD M

(kb)

21.23

5.15 & 4.984273.52

- 2.02i 1t91

1.581.380.950.83

FIG. 2. Gel electrophoresis of DNA materials isolated by 10% PEG precipitation from the <0.1-,um (lanes 1 and 1D), <0.2-pm (lanes 2,2D, 2d, and 2r), and <0.4-pLm (lanes 4 and 4D) fractions of Tokyo Bay water (sample taken on 10 June 1991). Samples were treated withDNase before addition of EDTA and SDS (D) or with DNase (d) or RNase (r) immediately before electrophoresis. Lanes C and CD, reagentcontrol; lanes M, X phage DNA EcoRI and Hindlll digests.

Size fraction 0(Pm) -

<0.4 - I

<0.2 Cad T Free

< 0.1 L10 June, 1991

<0.2

< 0.1

<0.03 4 July, 1991

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MMlih Et F - 3 2 1 MA M2EtF 4* 3 Z T PAM

(kb)

- 23.13. 9.426-656-- 4.36

:- 2.32- 2.03

FIG. 3. Gel electrophoresis of DNA materials isolated by 5% (lanes 1 and 1*), 10% (lanes 2 and 2*), 25% (lanes 3 and 3*), or 50% (lane4) PEG precipitation, direct ethanol precipitation (lanes Et and Et*), and ultrafilter concentration (lanes F and F*) from the <0.2-p.m fractionof Tokyo Bay water (sample taken on 18 July 1991). *, samples treated with DNase before addition of EDTA and SDS. Lanes Ml, A phageDNA EcoRI and HindIII digests; lanes M2, A phage DNA HindIll digests; lane -, no sample addition.

size of 25 kb, the DNA content in a coated DNA particle issimply calculated as about 0.03 fg particle-', which is lessthan the previous estimation of 0.08 fg particle-' for abacteriophage (2). On the basis of the coated DNA concen-tration measured, the approximate number of coated DNAparticles in the water of Tokyo Bay is therefore estimated tobe 3 x 108 to 6 x 108 particles ml-'. This estimate is slightlyhigher than the estimated virus number, 0.9 x 108 to 1.5 x108 particles ml-', which was calculated from the presentbacterial count by using the virus/bacterium ratio of 18observed in a Japanese eutrophic estuary (6). However, thetwo estimates could be equivalent if we adopt the virus/bacterium ratio of approximately 50 (1) or 70 (7) reported foraquatic environments.

Recently, Romanowski et al. (32) showed that DNA whichadsorbed to particles such as sea sand is more resistant toDNase digestion than free DNA in seawater. As ultramicronparticles other than viral particles appear to be abundant inseawater (40), DNA-adsorbed ultramicron particles may benon-DNase-digestible DNA particles. They are also attrib-utable to histone-DNA conjugates originating from marineeukaryotes and to DNA-containing phospholipid-membra-nous vesicles which are thought to be derived from bacteri-ovorous marine flagellates (21). In addition, so-called "ultra-microbacteria" which are known to be less than about 0.3p.m in diameter (37, 39) and/or to pass through a 0.2-,um filter(9, 13) could be an origin of such ultramicron-sized coatedDNA materials in seawater. However, the contribution ofthese nonviral DNA particles to the coated DNA in eutro-phic seawater seems to be less important, since it has beenshown that the number of DAPI-stainable ultramicron-sizedparticles in seawater usually does not exceed 1.6 times theviral population (6). Although the extent of the viral contri-bution to the total D-DNA might vary depending on thelocation or the eutrophication level (27), the present andprevious (6) results allow us to presume that the ultramicron-

sized coated DNA particles in the eutrophic marine environ-ment consist mainly of DNA viruses.

Nucleic acids such as DNA have been considered to beamong the major organic phosphorus compounds in naturalaquatic environments as well as in algal cells (20). Minear(19) reported that a significant portion of the high-molecular-mass fraction (>50,000 Da) of DOP (previously consideredto be the <0.45-p.m fraction) in lake water could be attrib-uted to DNA. A relatively high contribution of nucleicacid-like fractions to total DOP fractions was also found inseawater (18). These results suggest that nucleic acids couldbe a good nutrient source in natural aquatic environments.The approximate C/N/P atomic ratio of DNA components

can be estimated as 10:4:1 if the guanine-plus-cytosine(G+C) content in DNA is 50%, that is, if C39H55N15031P4 isa basic unit. Since the C/N/P ratio of the overall particulateorganic materials in natural seawater is approximately 106:16:1 (31), DNA-rich particles such as DNA viruses could bea significant pool of phosphorus, rather than nitrogen andcarbon, in these environments. In addition, the concentra-tion of D-DNA in the seawater was quite high (9 to 19 ng/ml),so the phosphorus content of the DNA (G+C content =50%) was calculated as 0.8 to 1.7 ng/ml. As the concentrationof DOP in Tokyo Bay surface water is ordinarily 0.02 to 0.04mg/liter in the summer (16), the contribution of phosphorusin D-DNA could be assumed as - 10% of DOP. This estimateis not inconsistent with the previous result of gel chromato-graphic characterization of DOP in Tokyo Bay water (18).However, it is remarkable that a significant portion of thephosphorus in D-DNA could be nutritionally less utilizablebecause of being insusceptible to phosphatase as well asDNase and that the turnover of viral particles (mostly DNAviruses) in marine environments could be so fast (1-3, 7, 29,30).

In conclusion, non-DNase-digestible DNA (coated DNA)was dominant in eutrophic seawater; almost 90% of the

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DNA in the <0.2-,um fraction was resistant to DNasedigestion. The major coated DNA in the seawater had amolecular size of 20 to 30 kb, which is the size of many DNAviruses. DNA could be a significant reservoir of phosphorusrather than nitrogen and carbon in the <0.2-,um fraction ofthe seawater. A major portion of the coated DNA in eutro-phic seawater may be attributed to marine DNA virusassemblages. Further studies of the size, amount, and ge-netic composition of the coated DNA in seawater will help toelucidate microbial community succession and gene transfermechanisms in marine environments.

ACKNOWLEDGMENTS

We thank M. Maeda (National Research Institute of Aquaculture)and 0. Matsuda (Hiroshima University) for stimulating discussions.

This work was supported by a Marine Biotechnology Projectgrant from the Ministry of International Trade and Industry ofJapan.

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