Cyanobacteria and their phages are significant microbial components of the freshwater and marine environments. We identified a lytic phage, Ma-LMM01, infecting Microcystis aeruginosa, a cyanobacterium that forms toxic blooms on the surfaces of freshwater lakes. Here, we describe the first sequenced freshwater cyanomyovirus genome of Ma-LMM01. The linear, circularly permuted, and terminally redundant genome has 162,109 bp and contains 184 predicted protein-coding genes and two tRNA genes. The genome exhibits no colinearity with previously sequenced genomes of cyanomyoviruses or other Myoviridae. The majority of the predicted genes have no detectable homologues in the databases. These findings indicate that Ma-LMM01 is a member of a new lineage of the Myoviridae family. The genome lacks homologues for the photosynthetic genes that are prevalent in marine cyanophages. However, it has a homologue of nblA, which is essential for the degradation of the major cyanobacteria light-harvesting complex, the phycobilisomes. The genome codes for a site-specific recombinase and two prophage antirepressors, suggesting that it has the capacity to integrate into the host genome. Ma-LMM01 possesses six genes, including three coding for transposases, that are highly similar to homologues found in cyanobacteria, suggesting that recent gene transfers have occurred between Ma-LMM01 and its host. We propose that the Ma-LMM01 NblA homologue possibly reduces the absorption of excess light energy and confers benefits to the phage living in surface waters. This phage genome study suggests that light is central in the phage-cyanobacterium relationships where the viruses use diverse genetic strategies to control their host's photosynthesis.The cyanobacterium Microcystis aeruginosa is a toxic, bloomforming bacteria found in eutrophic freshwaters throughout the world (12). The bacterium produces potent hepatotoxins, cyclic peptides called "microcystins," which inhibit eukaryotic protein phosphatase types 1 and 2A and can cause hepatocellular carcinoma (42,53,82). Blooms of M. aeruginosa can lead to the deaths of livestock and humans and pose serious problems for water management (12, 13).The mechanisms controlling bloom initiation and termination remain unclear; however, there have been many studies concerning the effects of environmental factors on M. aeruginosa growth (57). Recently, viral mortality of algae was recognized as one of the factors involved in the termination of algal blooms, including M. aeruginosa blooms (10,52,71,74,75). We previously reported culturing the lytic cyanophage Ma-LMM01 infecting the toxic M. aeruginosa strain NIES298 (81). Currently, M. aeruginosa NIES298 and Ma-LMM01 are the sole culture host/virus system to study interactions between a toxic cyanobacterium and its phage.Ma-LMM01 is a member of the Myoviridae family with a contractile tail (81), the distinctive morphological feature of this viral family. Myoviridae has at least six subgroups: T4-, P1-, P2-, Mu-, SPO1-, and H-like phages (23). These subgroups sh...

We isolated a cyanophage (Ma-LMM01) that specifically infects a toxic strain of the bloom-forming cyanobacterium Microcystis aeruginosa. Transmission electron microscopy showed that the virion is composed of an isometric head and a tail complex consisting of a central tube and a contractile sheath with helical symmetry. The morphological features and the host specificity suggest that Ma-LMM01 is a member of the cyanomyovirus group. Using semi-one-step growth experiments, the latent period and burst size were estimated to be 6 to 12 h and 50 to 120 infectious units per cell, respectively. The size of the phage genome was estimated to be ca. 160 kbp using pulse-field gel electrophoresis; the nucleic acid was sensitive to DNase I, Bal31, and all 14 restriction enzymes tested, suggesting that it is a linear double-stranded DNA having a low level of methylation. Phylogenetic analyses based on the deduced amino acid sequences of two open reading frames coding for ribonucleotide reductase alpha-and beta-subunits showed that Ma-LMM01 forms a sister group with marine and freshwater cyanobacteria and is apparently distinct from T4-like phages. Phylogenetic analysis of the deduced amino acid sequence of the putative sheath protein showed that Ma-LMM01 does not form a monophyletic group with either the T4-like phages or prophages, suggesting that Ma-LMM01 is distinct from other T4-like phages that have been described despite morphological similarity. The host-phage system which we studied is expected to contribute to our understanding of the ecology of Microcystis blooms and the genetics of cyanophages, and our results suggest the phages could be used to control toxic cyanobacterial blooms.Microcystis aeruginosa is one of the highly noxious cyanobacteria that frequently form dense blooms in eutrophic freshwaters throughout the world (9). M. aeruginosa produces potent hepatotoxins (microcystins) that specifically inhibit eukaryotic protein phosphatase types 1 and 2A and cause hepatocellular carcinoma (21,26,48). Hence, M. aeruginosa blooms are often responsible for the death of livestock and wildlife and cause serious problems in water management (9).Despite studies of the effects of various environmental factors on the growth of Microcystis species, the mechanisms that determine bloom dynamics and termination have not been studied sufficiently (27). Recent observations have shown that in addition to physical factors such as temperature and irradiation, chemical factors such as nutrients, and biological factors (predators), mortality induced by virus may be one of the important factors that control these algal blooms (8,23,44).There are a great number of viruses in natural waters, in both marine and freshwater environments (5), and it is suspected that a large proportion of these viruses are infectious for bacteria or cyanobacteria (30). The first isolation of freshwater cyanophages was reported about 40 years ago, and during the following two decades numerous cyanophage strains were isolated (2,3,14,(31)(32)(33)(34). In the...

Heterocapsa circularisquama RNA virus (HcRNAV) has at least two ecotypes (types UA and CY) that have intraspecies host specificities which are complementary to each other. We determined the complete genomic RNA sequence of two typical HcRNAV strains, HcRNAV34 and HcRNAV109, one of each ecotype. The nucleotide sequences of the viruses were 97.0% similar, and each had two open reading frames (ORFs), ORF-1 coding for a putative polyprotein having protease and RNA-dependent RNA polymerase (RdRp) domains and ORF-2 encoding a single major capsid protein. Phylogenetic analysis of the RdRp amino acid sequence suggested that HcRNAV belongs to a new previously unrecognized virus group. Four regions in ORF-2 had amino acid substitutions when HcRNAV34 was compared to HcRNAV109. We used a reverse transcriptionnested PCR system to amplify the corresponding regions and also examined RNAs purified from six other HcRNAV strains with known host ranges. We also looked at natural marine sediment samples. Phylogenetic dendrograms for the amplicons correlated with the intraspecies host specificities of the test virus strains. The cloned sequences found in sediment also exhibited considerable similarities to either the UA-type or CY-type sequence. The tertiary structure of the capsid proteins predicted using computer modeling indicated that many of the amino acid substitutions were located in regions on the outside of the viral capsid proteins. This strongly suggests that the intraspecies host specificity of HcRNAV is determined by nanostructures on the virus surface that may affect binding to suitable host cells. Our study shows that capsid alterations can change the phytoplankton-virus (host-parasite) interactions in marine systems.Only five RNA viruses are known to infect marine eukaryotic microorganisms. The following four viruses are singlestranded RNA (ssRNA) viruses: Heterosigma akashiwo RNA virus (HaRNAV) infects the noxious bloom-forming raphidophyte Heterosigma akashiwo (Raphidophyceae) (26); Rhizosolenia setigera RNA virus infects the bloom-forming diatom Rhizosolenia setigera (20); Heterocapsa circularisquama RNA virus (HcRNAV) infects the bivalve-killing bloom-forming dinoflagellate Heterocapsa circularisquama (30); and Schizochytrium sp. single-stranded RNA virus (SssRNAV) infects the marine fungoid protist Schizochytrium sp. (Labyrinthulae, Thraustochytriaceae) (27). One of the five viruses is a doublestranded RNA (dsRNA) virus (Micromonas pusilla RNA virus) that infects the cosmopolitan phytoplankton Micromonas pusilla (1). Detailed genomic analysis has been performed for two of these viruses, HaRNAV (18) and SssRNAV (Takao et al., unpublished data). Hence, genomic studies of the three other marine RNA viruses are required. In this paper we describe the genome of HcRNAV, which infects H. circularisquama.HcRNAV infection is strain specific rather than species specific because about 6,000 combinations of cross-infection tests between H. circularisquama strains and HcRNAV strains showed that HcRNAV strains are divided rou...

Diatoms are important components of the biological community and food web in the aquatic environment. Here, we report the characteristics of a single-stranded RNA (ssRNA) virus (CtenRNAV01) that infects the marine diatom Chaetoceros tenuissimus Meunier (Bacillariophyceae). The ca. 31-nm virus particle is icosahedral and lacks a tail. CtenRNAV01 forms crystalline arrays occupying most of the infected host's cytoplasm. By growth experiments, the lytic cycle and the burst size were estimated to be <24 h and ϳ1 ؋ 10 4 infectious units per host cell, respectively. Stationary-phase C. tenuissimus cultures were shown to be more sensitive to CtenRNAV01 than logarithmic-phase cultures. The most noticeable feature of this virus is its exceptionally high yields of ϳ10 10 infectious units ml ؊1 ; this is much higher than those of any other algal viruses previously characterized. CtenRNAV01 has two molecules of ssRNA of approximately 8.9 and 4.3 kb and three major proteins (33.5, 31.5, and 30.0 kDa). Sequencing of the total viral genome has produced only one large contig [9,431 bases excluding the poly(A) tail], suggesting considerable overlapping between the two RNA molecules. The monophyly of CtenRNAV01 compared to another diatom-infecting virus, Rhizosolenia setigera RNA virus, was strongly supported in a maximum likelihood phylogenetic tree constructed based on the concatenated amino acid sequences of the RNA-dependent RNA polymerase domains. Although further analysis is required to determine the detailed classification and nomenclature of this virus, these data strongly suggest the existence of a diatom-infecting ssRNA virus group in natural waters.

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Diatoms are a major phytoplankton group that play important roles in maintaining oxygen levels in the atmosphere and sustaining the primary nutritional production of the aquatic environment. Among diatoms, the genus Chaetoceros is one of the most abundant and widespread. Temperature, climate, salinity, nutrients, and predators were regarded as important factors controlling the abundance and population dynamics of diatoms. Here we show that a viral infection can occur in the genus Chaetoceros and should therefore be considered as a potential mortality source. Chaetoceros salsugineum nuclear inclusion virus (CsNIV) is a 38-nm icosahedral virus that replicates within the nucleus of C. salsugineum. The latent period was estimated to be between 12 and 24 h, with a burst size of 325 infectious units per host cell. CsNIV has a genome structure unlike that of other viruses that have been described. It consists of a single molecule of covalently closed circular single-stranded DNA (ssDNA; 6,005 nucleotides), as well as a segment of linear ssDNA (997 nucleotides). The linear segment is complementary to a portion of the closed circle creating a partially double-stranded genome. Sequence analysis reveals a low but significant similarity to the replicase of circoviruses that have a covalently closed circular ssDNA genome. This new host-virus system will be useful for investigating the ecological relationships between bloom-forming diatoms and other viruses in the marine system. Our study supports the view that, given the diversity and abundance of plankton, the ocean is a treasury of undiscovered viruses.

Diatoms are one of the most significant primary producers in the ocean, and the importance of viruses as a potential source of mortality for diatoms has recently been recognized. Thus far, eight different diatom viruses infecting the genera Rhizosolenia and Chaetoceros have been isolated and characterized to different extents. We report the isolation of a novel diatom virus (ClorDNAV), which causes the lysis of the bloomforming species Chaetoceros lorenzianus, and show its physiological, morphological, and genomic characteristics. The free virion was estimated to be ϳ34 nm in diameter. The arrangement of virus particles appearing in cross-section was basically a random aggregation in the nucleus. Occasionally, distinctive formations such as a ring-like array composed of 9 or 10 spherical virions or a centipede-like array composed of rod-shaped particles were also observed. The latent period and the burst size were estimated to be <48 h and 2.

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 Ma-LMM01 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 Ma-LMM01-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 detectionThe cyanobacterium Microcystis aeruginosa forms noxious blooms in freshwater throughout the world 2) . Some strains of M. aeruginosa produce heptapeptides called microcystins that have hepatotoxicity and specifically inhibit eukaryotic protein phosphatase types 1 and 2A 11,33) . Thus, blooms of M. aeruginosa cause the deaths of livestock and wildlife 5) and create serious problems for water management. The effects of environmental and chemical factors such as temperature 26,28) , irradiation 26,28) , and nutrients 27,28) on the growth of M. aeruginosa are well studied; however, the mechanisms involved in bloom dynamics and their termination are unknown 16) . Viruses and phages are significant factors in the mortality of algal blooms 21) . In the marine environment, cyanophages are abundant 29) and are considered to play an important role in controlling the structure of cyanobacterial communities 23) . 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 Pollard 25) 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