Cyanophage infection in the bloom-forming cyanobacteria Microcystis aeruginosa in surface freshwater.

Host-like genes are often found in viral genomes. To date, multiple host-like genes involved in photosynthesis and the pentose phosphate pathway have been found in phages of marine cyanobacteria Synechococcus and Prochlorococcus. These gene products are predicted to redirect host metabolism to deoxynucleotide biosynthesis for phage replication while maintaining photosynthesis. A cyanophage, Ma-LMM01, infecting the toxic cyanobacterium Microcystis aeruginosa, was isolated from a eutrophic freshwater lake and assigned as a member of a new lineage of the Myoviridae family. The genome encodes a host-like NblA. Cyanobacterial NblA is known to be involved in the degradation of the major light harvesting complex, the phycobilisomes. Ma-LMM01 nblA gene showed an early expression pattern and was highly transcribed during phage infection. We speculate that the co-option of nblA into Microcystis phages provides a significant fitness advantage to phages by preventing photoinhibition during infection and possibly represents an important part of the co-evolutionary interactions between cyanobacteria and their phages.

Real-time PCR detection of host-mediated cyanophage gene transcripts during infection of a natural Microcystis aeruginosa population.

The aim of this study was to develop a quantitative real-time reverse transcription-PCR (real-time RT-PCR) assay to detect and quantify mRNA of cyanophages within infected Microcystis aeruginosa cells in a freshwater pond. Laboratory-based data showed that the relative abundance of the cyanophage g91 mRNA within host cells increased before cyanophage numbers increased in culture. This transcriptional pattern indicated the kinetics of the viral infection suggesting the real-time RT-PCR method to be a potential tool for environmental monitoring of cyanophage infections. In this field survey, the numbers of infected M. aeruginosa cell populations estimated from cyanophage numbers were low at 0.01-2.9 cells mL(-1). The highest relative abundance of phage g91 RNA (10(-2) per rnpB transcript) was at about the same levels of expression as laboratory-based growth data for Ma-LMM01 (estimated density of infected host cells: 10(5) cells mL(-1)); and was observed when cyanophage numbers rapidly increased (as well as a decrease in host cell numbers). Quantification of cyanophage numbers is important to understand ecological relationships between the phage and its hosts. Our data suggest the quantification of phage gene transcripts within a natural host cell population to be a strong tool for investigating the quantitative effects of phage lysis during infection of the host population.

Ecological dynamics of the toxic bloom-forming cyanobacterium Microcystis aeruginosa and its cyanophages in freshwater.

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.

Ma-LMM01 infecting toxic Microcystis aeruginosa illuminates diverse cyanophage genome strategies.

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.

Dynamics of microcystin-producing and non-microcystin-producing Microcystis populations is correlated with nitrate concentration in a Japanese lake.

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.

Isolation and characterization of a cyanophage infecting the toxic cyanobacterium Microcystis aeruginosa.

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 anisometric 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.

B-lymphocyte-induced maturation protein-1 (Blimp-1) gene of torafugu (Takifugu rubripes).

Homologous gene of B-lymphocyte-induced maturation protein-1 (Blimp-1) of torafugu (Takifugu rubripes) was identified by tblast search analysis. RT-PCR and 5'RACE clearly defined the sequence of the UTR and coding region which has been ambiguously determined by tblast analysis. Fugu Blimp-1 was mainly expressed in the lymphoid organs. These finding imply that Blimp-1 would take a major role in the terminal differentiation of B-cells to plasma cells in fish.

Molecular cloning of the BCL-6 gene, a transcriptional repressor for B-cell differentiation, in torafugu (Takifugu rubripes).

B-cell lymphoma-6 (BCL-6) is a transcriptional repressor that prevents the terminal differentiation of mature B-cells to plasma cells, and is essential for germinal center formation in the primary lymphoid organs of mammals. In this study, we identified the BCL-6 gene in torafugu (Takifugu rubripes) using the torafugu genome database, and analyzed the expression of BCL-6 mRNA in various tissues of torafugu, using RT-PCR. The BCL-6 gene consisted of eight exons and seven introns spanning a genome of ca. 3.3 kb. BCL-6 mRNA contained a 2112 bp open reading frame encoding 703 amino acids, with a predicted protein size of 78.8 kDa. The predicted torafugu BCL-6 primary structure contains two conserved specific motifs, the BTB/POZ domain at the N-terminus and the sixC2H2-type zinc finger motifs at the C-terminal region. The homology of torafugu BCL-6 to those of zebrafish (Danio rerio), Xenopus laevis, mouse (Mus musculus) and human (Homo sapiens) is 76, 59, 60 and 60%, respectively. RT-PCR analysis revealed that BCL-6 mRNA is highly expressed in pronephros, thymus, intestine, ovary, brain, nasal cavity and muscle. These results imply that torafugu BCL-6 is involved in regulation of B-cell differentiation in torafugu.

Identification of genes encoding critical factors regulating B-cell terminal differentiation in torafugu (Takifugu rubripes).

Many transcription factors, and associated co-factors, are involved in the regulation of B-cell terminal differentiation in mammals. In the teleost and cartilaginous fish, although evidence has strongly suggested the existence of B-cell like lymphocytes, the mechanism of terminal differentiation of B-cells remains to be elucidated. In the present study, we searched for the nucleotide and amino acid sequences similar to the critical regulatory factors facilitating the terminal differentiation of B-cells using the fugu BLAST server. We cloned the following cDNAs from Takifugu rubripes: (1) B-lymphocyte-induced maturation protein-1 (Blimp-1), which plays a major role in promoting plasma cell differentiation by repressing the transcription of many genes that participate in maintaining the differentiation of mature B-cells; (2) Bcl-6, which facilitates germinal center formation and represses Blimp-1 expression; (3) X-box binding protein-1 (XBP-1), which operates Ig secretion by activating transcription of the ER-stress responsible genes; (4) Pax-5, which suppresses XBP-1 and enhances the expression of activation-induced cytidine deaminase (AID), an inducer of somatic hypermutation and class-switch recombination of the immunoglobulin gene; and (5) TLE-3, one of the Groucho family proteins, a co-factor for Blimp-1. We also identified other co-factors and many target genes of Blimp-1 by in silico and/or cDNA cloning. These finding indicates that the basal process of B-cell terminal differentiation in fish is controlled by factors identical to those in mammals.

Coordination of DNA replication and cell division in cyanobacteria Microcystis aeruginosa.

Little is known about the cyanobacterial cell cycle. When either nalidixic acid or hydroxyurea was added to a synchronized culture of Microcystis aeruginosa to block DNA replication, cell division did not occur. Furthermore, transcription of the essential cell division gene, ftsZ was repressed. After DNA replication, ftsZ transcription, as well as cell division, was not affected by hydroxyl urea, suggesting that the DNA replication and cell division of M. aeruginosa are coordinated and that this coordination is partly controlled by ftsZ transcription depending on DNA replication.

Genetic diversity of the toxic cyanobacterium Microcystis in Lake Mikata.

The aim of the present study was to clarify the bloom dynamics and community composition of hepatotoxin microcystin-producing and non-microcystin-producing Microcystis genotypes in the environment. In Lake Mikata (Fukui, Japan) from April 2003 to January 2004, seasonal variation in the number of cells with microcystin (mcy) genotypes and the genetic diversity of the total population were investigated using quantitative competitive PCR and a 16S rDNA clone library, respectively. Using competitive PCR, cells with mcyA genotypes were quantified in August and October, and the ratio of the number of these mcyA genotypes to colony-forming Microcystis cells was 0.37 and 2.37, respectively. The 16S rDNA clones obtained could be divided into 12 ribotypes: a-l. Sixty-one Microcystis strains isolated from Lake Mikata during the sampling period were subjected to toxicity tests using HPLC and ELISA, PCR-based detection of the mcyA gene, and sequence analysis of the 16S rDNA. All isolates could be differentiated into 11 ribotypes (a, b, d, f, h, i, and m-q). Ribotypes b, f, i, m, n, and p had at least one strain that was a microcystin producer. In natural communities ribotypes b and f accounted for 85% of the 16S rDNA clones in August, and ribotypes b and i accounted for 24% of the clones in October. Thus, in some bloom stages the presence of microcystin genotypes identified using the 16S rDNA clone library correlated with that of mcy genotypes determined using competitive PCR.

Generation of a strong promoter for Escherichia coli from eukaryotic genome DNA.

Improvement of a gene product by introducing mutations into the gene is usually applied for improving structural genes. In this study the procedure was applied for generation and improvement of a genetic signal to drive gene expression. By adding various concentrations of Mn2+ to the PCR reaction mixture, mutations were introduced into a DNA fragment at various ratios. An appropriate condition was employed to introduce mutations into a DNA fragment with no promoter activity. The mutated fragment was introduced at an upstream site of the lacZ gene in a plasmid vector to see if the fragment carries promoter activity. Lysate of an Escherichia coli transformant with the vector was assayed for beta-galactosidase expression as an indicator of the promoter activity. Mutated DNA fragments were generated by error prone PCR with a condition which leads to introduction of 1.5% of mutation into a DNA fragment during the process. The strongest promoter was chosen by beta-galactosidase assay after error prone PCR and subjected to another step of the PCR. These processes were repeated four times to improve its activity to 1.94-fold to that by the tac promoter. When the luciferase gene was expressed by the strongest promoters, a similar expression level was noted. These results indicate that by randomly introducing mutations into a DNA fragment, it is relatively easy to generate and improve a prokaryotic promoter.