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 ...

bViruses play important roles in regulating the abundance, clonal diversity, and composition of their host populations. To assess their impact on the host populations, it is essential to understand the dynamics of virus infections in the natural environment. Cyanophages often carry host-like genes, including photosynthesis genes, which maintain host photosynthesis. This implies a diurnal pattern of cyanophage infection depending on photosynthesis. Here we investigated the infection pattern of Microcystis cyanophage by following the abundances of the Ma-LMM01-type phage tail sheath gene g91 and its transcript in a natural population. The relative g91 mRNA abundance within host cells showed a peak during the daylight hours and was lowest around midnight. The phage g91 DNA copy numbers in host cell fractions, which are predicted to indicate phage replication, increased in the afternoon, followed by an increase in the free-phage fractions. In all fractions, at least 1 of 71 g91 genotypes was observed (in tested host cell, free-phage, and RNA fractions), indicating that the replication cycle of the cyanophage (i.e., injection, transcription, replication, and release of progeny phages) was occurring. Thus, Microcystis cyanophage infection occurs in a diel cycle, which may depend on the light cycle. Additionally, our data show that the abundance of mature cyanophage produced within host cells was 1 to 2 orders of magnitude greater than that of released phages, suggesting that phage production may be higher than previously reported.

Aims:  We determined if the intra‐specific genetic diversity of Microcystis aeruginosa correlates with phenotypic characteristics. Methods and Results:  Microcystis aeruginosa isolates from various Japanese waters were characterized using genetic analyses based on the 16S–23S rDNA internal transcribed spacer (ITS) region and DNA‐independent RNA polymerase (rpoC1) gene sequences. In addition, morphological and biochemical properties, and the toxicity of M. aeruginosa strains were determined. We found a correlation in phylogenetic clusters of the ITS region and rpoC1 gene sequences. Using a polyphasic approach, genotypic and phenotypic variations in M. aeruginosa showed that the three genetic lineage groups are comprised of a particular phenotype or subgroup of closely related phenotypes. However, some strains had high phenotypic and genotypic diversity compared to the three lineage groups and did not show distinct lineages; therefore, these strains were designated as the ‘complex group’. Conclusions:  The ‘complex group’ consisted of genetically and phenotypically incoherent and high diverse populations in M. aeruginosa, although some genotypes or lineages displayed consistent phenotypes. Significance and Impact of the Study:  The polyphasic approach combining phenotypic and genetic characterization was effective for comprehending distinct lineages and discriminating the potential complexity of M. aeruginosa populations at the intra‐species level.

The cyanophage Ma-LMM01, specifically-infecting Microcystis aeruginosa, has an insertion sequence (IS) element that we named IS607-cp showing high nucleotide similarity to a counterpart in the genome of the cyanobacterium Cyanothece sp. We tested 21 strains of M. aeruginosa for the presence of IS607-cp using PCR and detected the element in strains NIES90, NIES112, NIES604, and RM6. Thermal asymmetric interlaced PCR (TAIL-PCR) revealed each of these strains has multiple copies of IS607-cp. Some of the ISs were classified into three types based on their inserted positions; IS607-cp-1 is common in strains NIES90, NIES112 and NIES604, whereas IS607-cp-2 and IS607-cp-3 are specific to strains NIES90 and RM6, respectively. This multiplicity may reflect the replicative transposition of IS607-cp. The sequence of IS607-cp in Ma-LMM01 showed robust affinity to those found in M. aeruginosa and Cyanothece spp. in a phylogenetic tree inferred from counterparts of various bacteria. This suggests the transfer of IS607-cp between the cyanobacterium and its cyanophage. We discuss the potential role of Ma-LMM01-related phages as donors of IS elements that may mediate the transfer of IS607-cp; and thereby partially contribute to the genome plasticity of M. aeruginosa.