Microbes Environ. Vol. 22, No. 3, 207–213, 2007
http://wwwsoc.nii.ac.jp/jsme2/
Development and Application of Quantitative Detection of Cyanophages Phylogenetically Related to Cyanophage Ma-LMM01 Infecting Microcystis aeruginosa in Fresh Water YUKARI TAKASHIMA1, TAKASHI YOSHIDA1*, MITSUHIRO YOSHIDA1, YOKO SHIRAI2, YUJI TOMARU2, YOSHITAKE TAKAO2, SHINGO HIROISHI1, and KEIZO NAGASAKI2 1
Department of Marine Bioscience, Fukui Prefectural University, 1–1 Gakuencho, Obama, Fukui 917–0003, Japan 2 Harmful Algal Bloom Division, National Research Institute of Fisheries and Environments of Inland Sea, Fisheries Research Agency, 2–17–5 Maruishi, Hatsukaichi, Hiroshima 739–0452, Japan (Received December 14, 2006—Accepted April 5, 2007)
To develop a real-time PCR method for quantification of the abundance of cyanophages infecting Microcystis aeruginosa in aquatic environments, we characterized three cyanophage clones infecting M. aeruginosa, and compared them to the cyanophage Ma-LMM01 which was isolated previously. The clones were similar to MaLMM01 in morphological features and genome size. Further, the nucleotide sequences of the putative genes coding for the alpha- and beta-subunits of ribonucleotide reductase and the sheath protein from the three isolates were identical to those of Ma-LMM01. The isolates were closely related to Ma-LMM01 and designated MaLMM01-type phages. We designed a real-time PCR primer set to amplify a conserved region of the gene encoding the sheath protein, and quantified Ma-LMM01-type phages in environmental samples. The phages were detected when Microcystis blooms occurred, however, the amino acid sequence deduced from the nucleotide sequence of the PCR products was relatively diverse. This will be a useful tool for studies of the ecological impact of cyanophages on the Microcystis bloom. However, throughout these experiments, we did not detect any phages lytic to M. aeruginosa strain NIES298. This suggests three hypotheses: 1) diversity of host specificity in phages, 2) dominance of defective cyanophages in nature, and 3) lysogeny in the examined host strain NIES298. Key words: Microcystis aeruginosa, cyanobacteria, cyanophage, real-time PCR, quantitative detection
The cyanobacterium Microcystis aeruginosa forms noxious blooms in freshwater throughout the world2). Some strains of M. aeruginosa produce heptapeptides called microcystins that have hepatotoxicity and specifically inhibit eukaryotic protein phosphatase types 1 and 2A11,33). Thus, blooms of M. aeruginosa cause the deaths of livestock and wildlife5) and create serious problems for water management. The effects of environmental and chemical factors such as temperature26,28), irradiation26,28), and nutrients27,28) on the growth of M. aeruginosa are well studied; however, the mechanisms involved in bloom dynamics * Corresponding author. E-mail address:
[email protected]; Tel.: +81–770–52–6300; Fax: +81–770–52–6003.
and their termination are unknown16). Viruses and phages are significant factors in the mortality of algal blooms21). In the marine environment, cyanophages are abundant29) and are considered to play an important role in controlling the structure of cyanobacterial communities23). With Microcystis blooms, reports suggest that phages play an important role in regulating bloom dynamics. Manage et al.12) enumerated plaques on M. aeruginosa lawns using an enrichment method; and showed that increases in the number of plaques correlated with decreases in the number of M. aeruginosa. Tucker and Pollard25) observed two types of Podovirus-like particles that inhibited the growth of M. aeruginosa in lakewater samples during a bloom. These results suggest a host-phage
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relationship that affects bloom dynamics; however, cyanophages infectious to M. aeruginosa have never been isolated. We previously isolated a myovirus, Ma-LMM01, that specifically infects a toxic strain of M. aeruginosa32). A phylogenetic analysis of the deduced amino acid sequence of a putative sheath protein showed that Ma-LMM01 is distinctive from other T4-like phages despite its morphological similarity32). Recently, monitoring methods using a realtime PCR have been applied to detect and quantify microorganisms, such as harmful algae8), cyanobacteria31) and virus3) in environments. In the present report, we describe the development and application of a real-time PCR method for the quantification of cyanophages infecting M. aeruginosa in environmental samples.
Materials and Methods Cyanobacterial culture and growth conditions Microcystis aeruginosa strain NIES298 was obtained from the National Institute for Environmental Studies (NIES), Environmental Agency, Tsukuba, Japan. It was grown at 30°C in CB medium9) at a light intensity of ca. 40 µmol photons m−2 s−1 (white light fluorescent lamps) under a 12-h light 12-h dark cycle.
Cyanophage isolation and characterization Surface water samples were collected from Lake Mikata in Fukui Prefecture, Japan (35°33'N, 135°33'E) on 25 August 2003 and Hirosawanoike Pond in Kyoto Prefecture, Japan (35°02'N, 135°41'E) on 24 August 2003. Cyanophages were isolated as described by Yoshida et al.32). Briefly, 100-ml aliquots of the water samples were filtered through 0.2-µm polycarbonate membranes (Advantec Toyo, Tokyo, Japan); and 100 µl of the filtrate was inoculated into 900 µl of exponentially growing culture of M. aeruginosa strain NIES298 and incubated under the conditions described above for one week. After host cell lysis was observed, clonal isolates of cyanophages were obtained using three
cycles of the extinction dilution procedure13). Briefly, each lysate was diluted with CB medium in a series of 10-fold dilution steps. Aliquots (100 µl) of each dilution were added to 900 µl of exponentially growing culture of M. aeruginosa strain NIES298, and incubated as described above. The lysate in the most diluted wells of the second assay was serially diluted and transferred into a fresh host culture. This procedure was repeated three times, and the resultant lysate was regarded as the clonal cyanophage suspension. The clonal isolates were observed by transmission electron microscope as described previously32). The genome size for each clonal cyanophage was estimated by pulsed-field gel electrophoresis as described previously32).
Sequencing of three ORFs in the cyanophage isolates Lysates were subjected to two cycles of 2 min at 95°C and then 2 min at 4°C. Using oligonucleotide primer sets (Table 2), PCR was performed for 35 cycles: 30 sec at 94°C, 30 sec at 56°C, and 1 min at 72°C; concluding with a final extension step of 5 min at 72°C. The nucleotide sequences of the PCR products were determined using each primer shown in Table 2 (except for SheathRTF and SheathRTR), as described previously30).
Concentration of phage particles In order to develop an efficient phage concentration method, four different methods were compared using MaLMM01 particles. (i) BSA-GF/F method: Ma-LMM01 were trapped on a GF/F filter (Whatman International Ltd, Maidstone, UK) pre-coated with 3 ml of 1% bovine serum albumin (BSA)7). (ii) Ultracentrifugation: a 10-ml Ma-LMM01 suspension was centrifuged using a SW40Ti rotor (Beckman Coulter, Inc., Fullerton, CA, USA) at 111,000×g for 1.5 h at 4°C. The pellets were re-suspended in sterile distilled water. (iii) PEG precipitation: Ma-LMM01 was precipitated using 10% (w/v) polyethylene glycol 6000 (Nacalai Tesque, Inc., Kyoto, Japan)17). (iv) Ultrafiltration: Ma-LMM01 was concentrated using an Amicon filter with a 100-kDa molecular weight cutoff (Millipore corp., Bed-
Table 1. Characteristics of the four clonal cyanophage isolates used in this study Strain
a b
Station
Date
Head (nm)a
Tailb (nm)a
Genome size (kbp)
References Yoshida et al.32) this study this study this study
Ma-LMM01
Lake Mikata
08/25/03
86±12
90±13~227±15
165
Ma-LMM02 Ma-LMM03 Ma-HPM05
Lake Mikata Lake Mikata Hirosawanoike Pond
08/25/03 08/25/03 08/24/03
96±14 97±14 96±9
90±13~223±20 88±8~227±28 90±12~220±27
168 165 171
Values are means±standard deviations. length of contracted sheath and elongated sheath.
Quantitative Detection of Ma-LMM01-Type Phages
209
ford, MA, USA). The Ma-LMM01 phage particles were counted prior to and after each concentration method to determine the rate of phage recovery. One milliliter of each concentrated sample was stained with 25 µl of 100×SYBR Gold (Invitrogen Corp., Carlsbad, CA, USA) for 15 min and filtered onto a 0.02-µm (25 mm diameter) Anodisc filter (Whatman International Ltd, Maidstone, UK) and counted using blue excitation with a fluorescent microscope at a magnification of 1,000. At least 400 viral particles per sample were counted in 10 to 20 randomly chosen fields.
DNA extraction procedure The efficiency with which DNA was extracted from phage particles by two different methods, proteinase K treatment32) and the xanthogenate-SDS procedure24), was compared. For environmental samples, 10-ml aliquots of water samples collected from Lake Mikata and Hirosawanoike Pond on the date shown in Table 3 were filtered through 0.2-µm polycarbonate filters (Advantec Toyo, Tokyo, Japan) and concentrated to 500 µl as above. To avoid contamination with dissolved DNA, the filtrate was treated with DNase I
(1 U µl−1; Takara Bio Inc., Otsu, Japan) at 37°C for 1 h; then, the phage DNA was extracted as described above.
Real-time PCR Real-time PCR primers (SheathRTF and SheathRTR) were designed to amplify a 132-bp region of the sheath protein coding gene (Table 2). They were then used to query the NCBI GenBank DNA database sequences using BLAST1) to ensure there were no non-specific matches with other viruses. A standard curve was used for quantification and was plotted using threshold cycle values (Ct values) against 5.4×102 to 5.4×105 phage particles ml−1. Each realtime PCR mixture contained 25 µl of 2×SYBR Green Realtime PCR Master Mix (TOYOBO, Co., Ltd, Osaka Japan), 200 nM of each primer, 1 µl of DNA, and sterile ultra-pure water. The PCR cycles consist of an initial preheating step for 1 min at 95°C, followed by 35 cycles: 95°C for 15 s, 58°C for 15 s, and 82°C for 30 s. The assays were performed using an iCycler real-time PCR machine (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The iCycler software analysis program (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to calculate the Ct values to determine the sample concentrations based on the standard
Table 2. Primers for sequencing of four genes in cyanophage isolates and detection of Ma-LMM01 particles Target gene Ribonucleotide reductase alpha subunit
Ribonucleotide reductase beta subunit
Sheath protein
Primer
Sequence (5' to 3')
reducAF1 reducAF2 reducAF3 reducAF4 reducAF5 reducAR1 reducAR2 reducAR3 reducBF1 reducBF2 reducBR1 reducBR2 sheathF1 sheathF2 sheathF3 sheathR1 sheathR2 sheathR3 sheathR4 sheathR5 SheathRTF SheathRTR
CAA TCT CTA TTT GAA GGA CGA C CAG CGA TGA GTG CTA ATG G GAA ACT ATT CGA AGA CAT CTG C CGG TGG AGT GAA TCT ATG CGT TG GCT TTG ATA TAA GCG GCG CTG CTT CTT CTT ACG AAT GCG AC GCC CTT ACT CCA CTC ACT AC GCC GAT AGC GAC ATC AAG TAG TAC TGT GCT CTA CTG CCA ATA C ATT GGA CTT GGT GTG AAT GG CTC GAA TAC AGT AGG TAA GCC T CAA CGG CAT TAG TGA ATA TCT C GGC GGG ATA GAT TAA GAC AAC CC GTA GTC GGT GCG GCC C GAC TAT GTT AGC ATC ATC CGT ACT CTA G GGT TAG GTA GGT CGC CG GGC CAT CAC TAT CAC TAG CAC GTT GGC AAC CTG TCC GCT AC GAA ATG AAT AGC GGG ATC CGG CGC TGC AGG ACT GAC AAA GAA G ACA TCA GCG TTC GTT TCG G CAA TCT GGT TAG GTA GGT CG
210
TAKASHIMA et al. Table 3. Comparison of Ma-LMM01 quantification using real-time PCR and plaque assay of environmental samples Station
Date
Ma-LMM01 particles (particles ml−1)
Infectious phages (titer ml−1)
Total phages (particles ml−1)
Ma-LMM01 proportion (%)a
Microcystis (cells ml−1)
Hirosawanoike Pond
10/19/05
9.7×105
0.483
6.2×105
11/15/05
NDb ND
2.0×108
3
8
2.0×10
0.001
ND
9.4×107
—
1.5×105 ND
ND
7
Lake Mikata
04/12/05 05/23/05
a b
2.2×10 ND ND
4
08/23/05
2.3×10
09/13/05
1.8×105
—
ND
ND
8
1.8×10
0.013
3.4×101
ND
8.8×107
0.205
1.5×102
7.9×10
Ma-LMM01 proportion (%)=Ma-LMM01 particles/Total phages×100. ND=not detected.
count. The melting temperature for the real-time PCR products was determined using the iCycler software. All tests were performed in triplicate. The PCR products were cloned into pCRTMII vector (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer’s instructions. Positive clones were randomly selected, and plasmid DNA was amplified by PCR using M13 forward and reverse primers, and then sequenced30).
Plaque assay and host counts To enumerate infectious cyanophages, the plaque assay was performed using a solid medium with low melting agarose according to Shirai et al.18). One milliliter of each filtrate described above was serially diluted 10-fold, and 1 ml of each dilution was added to 1 ml of exponentially growing M. aeruginosa strain NIES298. After incubation at 30°C for 30 min, 2.5 ml of 0.72% (w/v) agarose LM (Nacalai Tesque, Inc., Kyoto, Japan) was added, mixed gently, and poured evenly onto a CB agarose plate18). The plates were incubated under the same conditions as described above for 10–14 days. Microcystis cell numbers were counted under a microscope using the morphological criteria proposed by Komárek et al.10).
Nucleotide sequence accession numbers
and one isolate from Hirosawanoike Pond. They had a polyhedral head of 96 nm and a complex contractile tail (20–28 nm in width, length: 220–227 nm elongated and 90 nm contracted) showing the typical morphological features of myoviruses (Table 1). The clonal isolates were subsequently termed Ma-LMM02, Ma-LMM03, and Ma-HPM05 according to Suttle21). As determined using pulsed-field gel electrophoresis, the genome size for each of the isolates was ca. 165 to 171 kb (Table 1). Sequences of three genes, those for the alpha- and beta-subunits of ribonucleotide reductase and for the sheath protein, including the upstream and downstream regions of Ma-LMM02, Ma-LMM03, and MaHPM05 were identical to those of Ma-LMM01. Representative data are shown in Fig. 1. Thus, these phages were closely related to Ma-LMM01 and were designated as MaLMM01-type phages. To detect and quantify Ma-LMM01type phages, we designed a real-time PCR primer set (sheathRTF/sheathRTR) to amplify the gene encoding the sheath protein, which is conserved among these phages. A BLAST search with the primer sequences showed no genomic cross-reactivity with other viruses.
Real-time PCR for Ma-LMM01-type phages Ultracentrifugation was the most efficient method to
The nucleotide sequences were deposited in the DDBJ/ EMBL/GenBank databases under accession numbers AB258332 to AB258340 for sequences from the cyanophage isolates and AB258341 to AB258356 for the sequences of clones isolated from the lake samples.
Results Designation of a real-time PCR primer to quantify Ma-LMM01-type phages We obtained two cyanophage isolates from Lake Mikata
Fig. 1. Structural organization of g91 (sheath protein) and flanking regions. The arrows indicate the positions and directions of PCR and sequencing primers. Lines indicate the expected PCR product length.
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Fig. 3. Alignment of deduced amino acid sequences of the gene encoding the sheath protein. PCR products from the Hirosawanoike Pond sample taken on 19 October 2005 were sequenced after the construction of a clone library. Among 16 sequences obtained, 12 clones belong to type 1, one clone to type 2, one clone to type 3, and two clones to type 4.
Fig. 2. Recovery rates and DNA extraction efficiency of Ma-LMM01. (A) Ma-LMM01 concentration recovery rates. (a) BSA-GF/F method; (b) Ultracentrifugation; (c) PEG precipitation; (d) Ultrafiltration. All tests were duplicated. (B) DNA extraction efficiency of Ma-LMM01; (a) proteinase K treatment method; (b) the xanthogenate-SDS method. The phage DNA extracted from 106, 105, and 104 particles ml−1 of Ma-LMM01 using each method was amplified by PCR with the primer sets, reducBF2/reducBR2.
recover Ma-LMM01 particles (Fig. 2A). When the extracted DNA was subjected to PCR amplification with reducBF2/ reducBR2, the largest amount of product was obtained using the xanthogenate-SDS method (Fig. 2B). We found a positive log linear correlation (R2=0.963, n=4, P
