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...

Temporal changes in hepatotoxin microcystin-producing and non-microcystin-producing Microcystis aeruginosa populations were examined in Lake Mikata, Japan. To monitor the densities of the total M. aeruginosa population and the potential microcystin-producing subpopulation, we used a quantitative real-time PCR assay targeting the phycocyanin intergenic spacer and the microcystin synthetase gene (mcyA), respectively. During the sampling period, the ratio of the mcyA subpopulation to the total M. aeruginosa varied considerably, from 0.5% to 35%. When surface nitrate concentrations increased, there was a rise in the relative abundance of the mcyA subpopulation. This was a positive correlation with the nitrate concentrations (r=0.53, P<0.05, n=14); whereas temperature and ortho-phosphate had no significant correlation with the presence of mcyA. Our data suggest that high nitrate loading may be a significant factor promoting the growth of the microcystin subpopulations within M. aeruginosa communities in Lake Mikata.

The abundance of potentially Microcystis aeruginosa-infectious cyanophages in freshwater was studied using g91 real-time PCR. A clear increase in cyanophage abundance was observed when M. aeruginosa numbers declined, showing that these factors were significantly negatively correlated. Furthermore, our data suggested that cyanophage dynamics may also affect shifts in microcystin-producing and non-microcystin-producing populations.The major bloom-forming cyanobacterial species Microcystis aeruginosa forms noxious blooms in many eutrophic freshwater lakes, ponds, and reservoirs. A limited population (limited number of strains) of M. aeruginosa in the environment produces potent hepatotoxins called microcystins (7). These potent toxins in the M. aeruginosa blooms have caused many cases of animal and human poisoning (3,8,16).Previously, most studies have focused on relationships among the cyanobacterial bloom dynamics and the changes in physicochemical factors (e.g., nutrient supply, light, and temperature) that influence cyanobacterial growth in the aquatic environment (28). Since the discovery that viruses are widespread in marine ecosystems (4), cyanophages that can infect cyanobacteria have been thought to be an alternative factor that may control the succession of cyanobacterial blooms (12,14,15,18,19). In addition, cyanophages can also influence the clonal composition of the host Synechococcus communities (14, 27) and could account for some of the cyanobacterial diversity observed in natural communities (22,25,30). Nevertheless, little is known about how freshwater cyanophages can affect the abundance and clonal composition of cyanobacterial blooms in lakes over time.Our aim is to determine if the cyanophages have potential quantitative and qualitative effects on the M. aeruginosa communities in Lake Mikata in Japan. We performed two independent real-time PCR assays to monitor the dynamics of M. aeruginosa and its cyanophage communities. To quantify M. aeruginosa, we used the phycocyanin intergenic spacer (PC-IGS) that was previously used to examine total M. aeruginosa numbers (9, 32). A second real-time PCR assay was used to quantitatively detect potentially M. aeruginosa-infectious cyanophages using the primers targeting the viral sheath proteinencoding gene (g91) previously identified by Takashima et al.(21). To determine the effect of the cyanophages on the internal dynamics of the total M. aeruginosa communities, we examined the fluctuation in the abundance of potentially microcystin-producing M. aeruginosa populations using the real-time PCR and microcystin synthetase gene (mcyA)-specific primers (32) and monitored the relative size of the microcystin-producing subpopulation compared to the total population in relation to the cyanophage numbers using a field survey of M. aeruginosa blooms in a Japanese lake.Water samples were collected from the surface layer (0.5 m) once per month from April to October in 2006 at a fixed point (35°33ЈN, 135°53ЈE) in Lake Mikata (Fig. 1). The cyanobacterial cells used for ...

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