Seasonal Variations in Virus-Host Populations in Norwegian Coastal Waters: Focusing on the Cyanophage Community Infecting Marine Synechococcus spp

Sample collection.Coastal water samples were collected at a station in Raunefjorden (60°16.2′N, 5°12.5′E), south of Bergen, Norway, from 3 March to 2 November 2004. A total volume of 30 liters was collected from a depth of 2 m nearly every second week from 3 June to 29 June; then on 10, 12, and 19 August; and thereafter every week from 21 September until 12 October. The final sample was obtained on 2 November. The samples were collected using a hand pump connected to a flask. Temperature was measured using an STD SAIV a/s SD 204 with a Sea Point fluorometer (SAIV A/S; Environmental Sensors and Systems, Bergen, Norway).

FCM.The abundances of autotrophic pico- and nanoeukaryotes, Synechococcus bacteria, heterotrophic bacteria, and viruses were determined by FCM. The autotrophs were analyzed on a FACSCalibur or a FACSAria flow cytometer (Becton Dickinson) equipped with lasers supplying 15 mW at 488 nm and 13 mW at 488 nm, respectively, with standard filter setup. Viruses and bacteria were counted in samples fixed with glutaraldehyde for 60 s at a viral event rate of between 100 and 1,000 s⁻¹ using the FACSCalibur. Each sample was diluted at a minimum of two different dilutions from 50- to 200-fold before it was stained with SYBR green I. The flow cytometer instrumentation and the remaining methodology followed the recommendations of Marie et al. (26) and are described in more detail, for a similar study, by Larsen et al. (22).

Concentration of virus communities.Natural viral communities were concentrated from 25 liters of seawater by ultrafiltration. The samples were first filtered through a 0.45-μm low-protein-binding Durapore membrane filter of 142 mm in diameter (Millipore) to remove zooplankton, phytoplankton, and some of the bacteria. The filtered samples were then concentrated to a final volume of 150 to 250 ml, using a 30,000-molecular-weight-cutoff spiral-wound Millipore ultrafiltration cartridge (regenerated cellulose, PLTK Prep/scale TFF, 1 ft²; Millipore Corporation, Bedford, MA). The concentrate was stored in the dark at 4°C until further processing.

One hundred forty-two milliliters of viral concentrate was concentrated further by ultracentrifugation (Beckman L8-M with SW-28 rotor; Beckman GmbH, Germany) for 2 h at 25,000 rpm at 10°C. The viral pellet was resuspended in 400 μl of SM buffer (0.1 M NaCl, 8 mM MgSO₄ · 7H₂O, 50 mM Tris-HCl, 0.005% [wt/vol] glycerol). Two hundred microliters was stored at −20°C for DGGE and quantitative real-time PCR analysis, while 200 μl was used for PFGE analysis.

PFGE.Four virioplankton agarose plugs were made from the 200-μl concentrate, representing 7 liters of water sample (each plug representing 1.75 liters). The samples were run on a 1% (wt/vol) SeaKem GTG agarose (FMC, Rockland, ME) gel in 1× Tris-buffered EDTA (TBE) gel buffer using a Bio-Rad DR-II contour-clamped homogeneous electric field cell (Bio-Rad, Richmond, CA) electrophoresis unit (57). From each sample we used three of the plugs and ran them at three different pulse ramp conditions in order to separate a wide range of viral genome sizes: (i): 1- to 5-s switch time with 20-h run time for separation of small genome sizes (0 to 130 kb); (ii) 8- to 30-s switch time with 20-h run time for separation of medium genome sizes (130 to 300 kb); and (iii) 20- to 40-s switch time with 22-h run time for separation of large genome sizes (300 to 600 kb). A molecular size standard (lambda ladder and 5-kb ladder [Bio-Rad, Richmond, CA]) was employed on each side of the gel. Further details of the procedure may be found in the work of Larsen et al. (22). The gels were visualized and saved as computer files using the Fujifilm imaging system LAS-3000. The banding patterns were analyzed using a computer program, GEL2K (Svein Norland, Department of Microbiology, University of Bergen, Norway), which calculates the molecular weight of the different DNA fragments and the intensity of each of the DNA fragments and determines the presence or absence of bands. The intensity of a peak/band is calculated as the integrated pixel value (in the peak) over its background value. Clustering of the viral profiles was based on the simple matching algorithm, while the dendrogram was drawn using the complete link method.

DGGE.PCR-DGGE analysis was performed using the primers CPS1 and CPS8 to amplify a 592-bp product of the g20 gene (59) with a 40-mer GC clamp in the 5′ end of the CPS8 primer. The 50-μl reaction mixture contained the following: sterile distilled water, PCR buffer (10× PCR buffer B; Promega, Madison, WI), deoxynucleoside triphosphates (200 nM each), primers (0.5 μM each), 1.5 mM MgCl, 2.5 U Taq DNA polymerase (Promega), and template amplicon (1 to 2 ng). We performed PCR amplification according to the protocol of Zhong et al. (59) and checked the 592-bp amplicon for length and purity on a 1.5% agarose gel. The DGGE was performed with a Dcode 16/16-cm gel system (Bio-Rad, Hertfordshire, United Kingdom). We used a gradient of DNA denaturant ranging from 20 to 45% and loaded the gel with 20 μl of PCR product together with 15 μl of a standard on each side. The standard consisted of a 1:1:1 mixture of PCR products using the phages S-PM2, S-BnM1, and S-WHM1 as template. The gels were run at 60°C at 60 V for 20 h in 1× TAE buffer (40 mM Tris [pH 7.4], 20 mM sodium acetate, 1 mM EDTA) and stained with SYBR green II (Molecular Probes, OR) before they were visualized and saved as computer files using the Fujifilm imaging system LAS-3000. The presence and absence of the different viral populations were recorded using the GEL2K software program (Svein Norland, Department of Microbiology, University of Bergen). Clustering of the DGGE profiles was based on the simple matching algorithm, while the dendrogram was drawn by applying the complete link method.

Sequencing DGGE and PFGE bands.DGGE bands to be sequenced were excised from the gel and eluted in 20 μl sterile distilled water overnight at 4°C. Two microliters of eluted DNA served as template in a PCR with the same primers (CPS1 and CPS8) and conditions as described above. PCR products were purified using the Microcon-PCR spin column (Millipore). PFGE bands to be sequenced were excised from the gel and frozen at −20°C before the DNA was extracted from the gel using the GeneClean Turbo kit (Bio 101) for extractions of large DNA fragments from the agarose gel, following the manufacturer's instructions, yielding approximately 10 ng/μl of DNA. The genomic DNA required for sequencing and PCR was produced by the GenomiPhi DNA amplification kit (Amersham Biosciences, Buckinghamshire, United Kingdom) according to the manufacturer's instructions, yielding between 4 and 17 μg of DNA. This DNA was used for PCR with the CPS1/CPS2 primers (18).

The different PCR products were sequenced by cycle sequencing according to the protocol from Perkin-Elmer (Foster City, CA), using the forward primers as a sequencing primer. Sequences were obtained on an ABI PRISM 3700 sequence analyzer (Perkin-Elmer). Analysis of DNA sequences was carried out by alignment to the closest relative in the GenBank database using TBLAST (2). Alignments were performed using CLUSTALX (51). Maximum parsimony and neighbor-joining (NJ) analysis were conducted on a nucleotide data set by using the PAUP* 4.0 b10 program (48). Supports for clades were estimated by means of bootstrap analysis, as implemented in PAUP* using 1,000 replicates. The trees were viewed using the TreeView program and rooted with the gp20 gene of Escherichia coli phage T4 as an outgroup.

Estimation of abundance of cyanophages.The abundance of cyanophages was estimated using real-time PCR with the primers CPS1 and CPS2 amplifying a 165-bp product of the g20 gene. The assay was run directly on the viral concentrates without any DNA extraction step (18, 56). For the detection of the CPS1/CPS2 product, a SYBR green I real-time PCR assay was used (Molecular Probes, Eugene, OR). The 20-μl master mix contained the following: Mastermix solution (2× Mastermix, DyNAmoHS; Finnzymes, Espoo, Finland), primers (0.5 μM each), sterile distilled water, and template amplicon (1-μl viral sample). The PCR was optimized using the cyanophage S-BnM1 as template. Sterile water was used as a negative control. The optimized cycling protocol was as follows. After an initial denaturation step at 95°C for 15 min, the reaction mixture was run for 35 cycles at 95°C for 10 s, 53.5°C for 30 s, and 72°C for 30 s, followed by 72°C for 10 min. Thereafter, a melting curve from 72 to 95°C with an increase of 0.2°C per s was run in order to check the specificity of the PCR. The assays were run on an MJ Research Opticon real-time PCR machine (Bio-Rad). Each sample was assayed at three different concentrations and repeated three times. Concentration values were obtained using serial dilutions from 1 to 10⁵ phages μl⁻¹ of S-BnM1 containing one copy of the g20 gene. The serial dilutions were made fresh every time, and the number of phages in the lysate was determined by epifluorescence microscopy at a 1,000× magnification, after staining with a 400-fold dilution of SYBR green I (Molecular Probes) as described by Noble and Fuhrman (31).

Nucleotide sequence accession numbers.The nucleotide sequences reported in this paper have been submitted to GenBank (DQ354990 to DQ355010).

Succession of viruses, bacteria, and algae.In general, the abundance of Synechococcus, cyanophages, total bacteria, and viruses increased throughout the sampling period (Fig. 1A and B). Total viral numbers rose from 6.0 × 10⁶ ml⁻¹ in the spring to 2.0 × 10⁷ ml⁻¹ in the early fall, before declining to 1.4 × 10⁷ ml⁻¹ in the late fall (Fig. 1). The total bacterial number, which was 5.6 × 10⁵ ml⁻¹ on the first sampling day, peaked three times during the sampling period (Fig. 1A): in April (9 × 10⁵ ml⁻¹), July (1.6 × 10⁶ ml⁻¹), and October (1.8 × 10⁶ ml⁻¹). Cyanophage abundance ranged from <5.0 × 10 ml⁻¹ in early spring to 7.2 × 10³ ml⁻¹ in late fall (Fig. 1B) and revealed two distinct peaks: a minor one on 25 May and a major one on 12 October. Synechococcus also peaked twice, with a minor peak on 15 June (3.5 × 10⁴ cells ml⁻¹) and a major one on 21 September (7.3 × 10⁴ cells ml⁻¹) (Fig. 1B).

• Open in new tab
• Download powerpoint

FIG. 1.

Development of bacteria and viruses (A) and algae belonging to the nano- and picoeukaryote groups (B) as measured by FCM. (C) Synechococcus (measured by FCM) and cyanophages (measured in real time using primers against the g20 gene). Panel C also shows water temperature measured at the sampling site in Raunefjorden, Norway, from March to November 2004.

Autotrophic pico- and nanoeukaryotes varied substantially throughout the sampling period. The autotrophic picoeukaryotes reached highest concentrations during spring and summer (13 April, 2.1 × 10⁴ cells ml⁻¹, and 15 June, 2.0 × 10⁴ cells ml⁻¹) (Fig. 1C) but peaked three times in the course of the fall as well, with the highest cell number on 6 October (1.4 × 10⁴ cells ml⁻¹). The autotrophic nanoeukaryotes peaked three times during the sampling period, twice in spring (13 April, 9.6 × 10³ cells ml⁻¹, and 11 May, 1.4 × 10⁴ cells ml⁻¹) and once in summer (19 August; 8.2 × 10³ cells ml⁻¹) (Fig. 1C). The abundance subsequently decreased and fluctuated around 1.6 × 10³ cells ml⁻¹ for the rest of the period. The water temperature rose from 5.2 to 15.4°C from March to early September before falling again to 9.8°C (Fig. 1C).

Seasonal changes within the virioplankton community.The virioplankton PFGE banding pattern revealed 29 viral populations of different genome sizes ranging from 26 to 500 kb (Fig. 2A). The most abundant populations were those of 31, 38, 44, 46, 67, 75, 95, 140, 200, and 240 kb. All samples comprised viral populations in the size range from 26 to 240 kb. One population with a size of 95 kb was present in all samples. From 13 April to 21 September we also detected viral populations with sizes from 260 to 500 kb. On average 13 PFGE bands were detected in each sample, with the lowest number of bands (five) in the sample collected on 12 August and the highest (18) on 29 June.

• Open in new tab
• Download powerpoint

FIG. 2.

(A) Schematic outline of the relative abundance (indicated by the size of the dots) of viral populations determined by PFGE. Viral populations are defined by genome size, and the outline is based on three different electrophoresis runs for each viral concentrate. (B) Cluster analysis of the total viral populations from individual samples. The dendrogram is constructed from a binary matrix of similarity values, using a distance calculation algorithm based on the absence or presence of bands. Clustering is based on the simple matching algorithm, while the dendrogram was drawn using the complete link method.

The cluster analysis resulted in a dendrogram consisting of two main groups (I and II). Samples from March to 21 September clustered in one group (II), whereas samples from late September to November clustered together in the other (I). Group II consisted of three subgroups: (i) samples from April and May; (ii) samples from June and August; and (iii) samples from March, 12 August, and 21 September (Fig. 2B).

Cyanophage diversity.Fifteen out of 25 PFGE bands gave a positive signal with the CPS1 and CPS2 primers when DNA extracted from the bands was used in a PCR on products amplified with the GenomiPhi DNA amplification kit (Table 1). These PFGE bands ranged from 26 to 380 kb. Between 2 and 13 bands were detected when PCR products amplified with the CPS1 and CPS8 primers were run on a DGGE gel (data not shown). A dendrogram based on the fingerprints grouped the samples into two main groups (I and II) (Fig. 3). Group II comprises samples from 2 March to 15 June, while group I consists of samples from 12 August until the end of the sampling period (Fig. 3).

• Open in new tab
• Download powerpoint

FIG. 3.

Cluster analysis of the DGGE profiles of amplified g20 gene fragments from individual samples. The dendrogram is constructed from a binary matrix of similarity values, using a distance calculation algorithm based on the absence or presence of bands. Clustering is based on the simple matching algorithm, while the dendrogram was drawn using the complete link method.

Phylogenetic analysis of the g20 gene was carried out in PAUP* using both maximum parsimony and an NJ analysis. The two methods gave similar results, and we present those emerging from the NJ analysis (Fig. 4). All g20 sequences amplified from the DGGE/PFGE bands cluster within the cultured Synechococcus phage group (40, 59) (Fig. 4). One group displays highest similarity with the cyanophage S-BnM1 and consists of sequences from four DGGE bands (D-7a, D-9b, D-10b, and D-11b). Another group comprises sequences from three DGGE bands (D-9a, D-11a, and D-5b) and clusters within clade III of the cultured phage of Synechococcus (CPS) group. These two groups are composed of DNA sequences originating from DGGE bands which dominated the samples throughout the entire experimental period, with the exception of band D-5b, which originated from 25 May. All sequences from the PFGE bands, which were in the size range from 26 to 380 kb (P-S6, P-S7, P-5b, P-3b, P-6b, P-7b, P-6c2, P-4c2, P-5c2, P-S8, P-S2, and P-S5) fell within CPS group II, showing closest homology to the cyanophages S-RIM20 and S-RIM17. Sequences from three DGGE bands (D-6b, D-7b, and D-8b) clustered within clade I, with highest homology to S-PM2. These sequences originated from DGGE bands that appeared only in samples collected in the fall.

• Open in new tab
• Download powerpoint

FIG. 4.

Phylogenetic analysis using neighbor-joining analysis (PAUP*) of sequences from DGGE and PFGE bands and representative g20 sequences retrieved from the GenBank (NCBI) database. Groups I, II, and III refer to the cyanophage classification by Zhong et al. (59). Boldface indicates g20 sequences from GenBank. Bootstrap values were generated with 1,000 replicates; values of <50 are not shown. The scale bar represents 0.1 substitution per site.