Profiling the interplay and coevolution of Microcystis aeruginosa and cyanosiphophage Mic1.

The cyanosiphophage Mic1 specifically infects the bloom-forming Microcystis aeruginosa FACHB 1339 from Lake Chaohu, China. Previous genomic analysis showed that its 92,627 bp double-stranded DNA genome consists of 98 putative open reading frames, 63% of which are of unknown function. Here, we investigated the transcriptome dynamics of Mic1 and its host using RNA sequencing. In the early, middle, and late phases of the 10 h lytic cycle, the Mic1 genes are sequentially expressed and could be further temporally grouped into two distinct clusters in each phase. Notably, six early genes, including gp49 that encodes a TnpB-like transposase, immediately reach the highest transcriptional level in half an hour, representing a pioneer cluster that rapidly regulates and redirects host metabolism toward the phage. An in-depth analysis of the host transcriptomic profile in response to Mic1 infection revealed significant upregulation of a polyketide synthase pathway and a type III-B CRISPR system, accompanied by moderate downregulation of the photosynthesis and key metabolism pathways. The constant increase of phage transcripts and relatively low replacement rate over the host transcripts indicated that Mic1 utilizes a unique strategy to gradually take over a small portion of host metabolism pathways after infection. In addition, genomic analysis of a less-infective Mic1 and a Mic1-resistant host strain further confirmed their dynamic interplay and coevolution via the frequent horizontal gene transfer. These findings provide insights into the mutual benefit and symbiosis of the highly polymorphic cyanobacteria M. aeruginosa and cyanophages. IMPORTANCE: The highly polymorphic Microcystis aeruginosa is one of the predominant bloom-forming cyanobacteria in eutrophic freshwater bodies and is infected by diverse and abundant cyanophages. The presence of a large number of defense systems in M. aeruginosa genome suggests a dynamic interplay and coevolution with the cyanophages. In this study, we investigated the temporal gene expression pattern of Mic1 after infection and the corresponding transcriptional responses of its host. Moreover, the identification of a less-infective Mic1 and a Mic1-resistant host strain provided the evolved genes in the phage-host coevolution during the multiple-generation cultivation in the laboratory. Our findings enrich the knowledge on the interplay and coevolution of M. aeruginosa and its cyanophages and lay the foundation for the future application of cyanophage as a potential eco-friendly and bio-safe agent in controlling the succession of harmful cyanobacterial blooms.

A Review of Cyanophage-Host Relationships: Highlighting Cyanophages as a Potential Cyanobacteria Control Strategy.

Harmful algal blooms (HABs) are naturally occurring phenomena, and cyanobacteria are the most commonly occurring HABs in freshwater systems. Cyanobacteria HABs (cyanoHABs) negatively affect ecosystems and drinking water resources through the production of potent toxins. Furthermore, the frequency, duration, and distribution of cyanoHABs are increasing, and conditions that favor cyanobacteria growth are predicted to increase in the coming years. Current methods for mitigating cyanoHABs are generally short-lived and resource-intensive, and have negative impacts on non-target species. Cyanophages (viruses that specifically target cyanobacteria) have the potential to provide a highly specific control strategy with minimal impacts on non-target species and propagation in the environment. A detailed review (primarily up to 2020) of cyanophage lifecycle, diversity, and factors influencing infectivity is provided in this paper, along with a discussion of cyanophage and host cyanobacteria relationships for seven prominent cyanoHAB-forming genera in North America, including: Synechococcus, Microcystis, Dolichospermum, Aphanizomenon, Cylindrospermopsis, Planktothrix, and Lyngbya. Lastly, factors affecting the potential application of cyanophages as a cyanoHAB control strategy are discussed, including efficacy considerations, optimization, and scalability for large-scale applications.

A Novel Freshwater Cyanophage, Mae-Yong924-1, Reveals a New Family.

Cyanobacterial blooms are a worldwide ecological issue. Cyanophages are aquatic viruses specifically infecting cyanobacteria. Little is known about freshwater cyanophages. In this study, a freshwater cyanophage, Mae-Yong924-1, was isolated by the double-layer agar plate method using Microcystis aeruginosa FACHB-924 as an indicator host. Mae-Yong924-1 has several unusual characteristics: a unique shape, cross-taxonomic order infectivity and a very unique genome sequence. Mae-Yong924-1 contains a nearly spherical head of about 100 nm in diameter. The tail or tail-like structure (approximately 40 nm in length) is like the tassel of a round Chinese lantern. It could lyse six diverse cyanobacteria strains across three orders including Chroococcales, Nostocales and Oscillatoriales. The genome of the cyanophage is 40,325 bp in length, with a G + C content of 48.32%, and 59 predicted open reading frames (ORFs), only 12 (20%) of which were functionally annotated. Both BLASTn and BLASTx scanning resulted in "No significant similarity found", i.e., the Mae-Yong924-1 genome shared extremely low homology with sequences in NCBI databases. Mae-Yong924-1 formed a root node alone and monopolized a root branch in the proteomic tree based on genome-wide sequence similarities. The results suggest that Mae-Yong924-1 may reveal a new unknown family apparently distinct from other viruses.

A Novel Wide-Range Freshwater Cyanophage MinS1 Infecting the Harmful Cyanobacterium Microcystis aeruginosa.

Microcystis aeruginosa, as one of the major players in algal bloom, produces microcystins, which are strongly hepatotoxic, endangering human health and damaging the ecological environment. Biological control of the overgrowth of Microcystis with cyanophage has been proposed to be a promising solution for algal bloom. In this study, a novel strain of Microcystis cyanophage, MinS1, was isolated. MinS1 contains an icosahedral head approximately 54 nm in diameter and a 260 nm-long non-contractile tail. The phage genome consists of a linear, double-stranded 49,966 bp DNA molecule, which shares very low homology with known phages in the NCBI database (only 1% of the genome showed weak homology with known phages when analyzed by megablast). The phage contains 75 ORFs, of which 23 ORFs were predicted to code for proteins of known function, 39 ORFs were predicted to code for proteins of unknown function, and 13 ORFs showed no similarity to any protein sequences. Transmission electron microscopy and phylogenetic analysis showed that MinS1 belongs to the family Siphoviridae. Various experiments confirmed that the phage could infect several different orders of cyanobacteria, including Chroococcales, Nostocales, Oscillatoriales, Hormogonales, and Synechococcales, indicating that it has a very broad host range. In addition, MinS1 has no known antibiotic tolerance genes, virulence genes, and tRNAs, and it is tolerant to temperature, pH, UV, and salinity, suggesting that MinS1 has good potential for application as a biological control agent against cyanobacterial blooms. This study expands the diversity and knowledge of cyanophages, and it provides useful information for the development of novel prevention and control measures against cyanobacterial blooms.

Occurrence and diversity of viruses associated with cyanobacterial communities in a Brazilian freshwater reservoir.

As part of the phytoplankton of marine and freshwater environments around the world, cyanobacteria interact with viruses (cyanophages) that affect their abundance and diversity. Investigations focusing on cyanophages co-occurring with freshwater cyanobacteria are scarce, particularly in Brazil. The aim of this study was to assess the diversity of cyanophages associated with a Microcystis-dominated cyanobacterial bloom in a tropical reservoir. Samples were processed as viral fractions of water and cellular fractions, and temporal fluctuations in the abundance of Ma-LMM01-type cyanophages and their Microcystis hosts were determined by qPCR. We applied shotgun metagenomics to obtain a wider characterization of the cyanophage community. During the study period, Microcystis gene copies were quantified in all cellular fractions, and the copy number of the Ma-LMM01 phage gene tended to increase with host abundance. Metagenomic analysis demonstrated that Caudovirales was the major viral order associated with the cyanophage families Myoviridae (34-88%), Podoviridae (3-42%), and Siphoviridae (6-23%). The metagenomic analysis results confirmed the presence of Microcystis cyanophages in both viral and cellular fractions and demonstrated a high relative abundance of picocyanobacteria-related viruses and Prochlorococcus (36-52%) and Synechococcus (37-50%) phages. For other main cyanobacterial genera, no related cyanophages were identified, which was probably due to the scarce representation of cyanophage sequences in databanks. Thus, the studied reservoir hosted a diverse cyanophage community with a remarkable contribution of phages related to picoplanktonic cyanobacteria. These results provide insights that motivate future sequencing efforts to assess cyanophage diversity and recover complete genomes.

Tracing the active genetic diversity of Microcystis and Microcystis phage through a temporal survey of Taihu.

Harmful algal blooms are commonly thought to be dominated by a single genus, but they are not homogenous communities. Current approaches, both molecular and culture-based, often overlook fine-scale variations in community composition that can influence bloom dynamics. We combined homology-based searches (BLASTX) and phylogenetics to distinguish and quantify Microcystis host and phage members across a summer season during a 2014 Microcystis- dominated bloom that occurred in Lake Tai (Taihu), China. We found 47 different genotypes of the Microcystis-specific DNA-dependent RNA polymerase (rpoB), which included several morphospecies. Microcystis flos-aquae and Microcystis wesenbergii accounted for ~86% of total Microcystis transcripts, while the more commonly studied Microcystis aeruginosa only accounted for ~7%. Microcystis genotypes were classified into three temporal groups according to their expression patterns across the course of the bloom: early, constant and late. All Microcystis morphospecies were present in each group, indicating that expression patterns were likely dictated by competition driven by environmental factors, not phylogeny. We identified three primary Microcystis-infecting phages based on the viral terminase, including a novel Siphoviridae phage that may be capable of lysogeny. Within our dataset, Myoviridae phages consistent with those infecting Microcystis in a lytic manner were positively correlated to the early host genotypes, while the Siphoviridae phages were positively correlated to the late host genotypes, when the Myoviridae phages express putative genetic markers for lysogeny. The expression of genes in the microcystin-encoding mcy cassette was estimated using mcyA, which revealed 24 Microcystis-specific genotypes that were negatively correlated to the early host genotypes. Of all environmental factors measured, pH best described the temporal shift in the Microcystis community genotypic composition, promoting hypotheses regarding carbon concentration mechanisms and oxidative stress. Our work expounds on the complexity of HAB events, using a well-studied dataset to highlight the need for increased resolution of community dynamics.

A novel freshwater cyanophage vB_MelS-Me-ZS1 infecting bloom-forming cyanobacterium Microcystis elabens.

Blooms of cyanobacteria cause enormous losses in both the economy and environment. Cyanophages are of great potential for fighting blooming cyanobacteria. Research report on cyanophage of bloom-forming cyanobacterium, Microcystis elabens is deficient. vB_MelS-Me-ZS1 (abbreviated as Me-ZS1) was isolated from fresh water by double-layer agar plate method using M. elabens. TEM exhibited that cyanosiphovirus Me-ZS1 has an icosahedral head about 60 nm in diameter, and a noncontractile tail approximately 260 nm. Experimental infection against 15 cyanobacterial strains showed that Me-ZS1 can infect 12 strains across taxonomic orders (Chroococcales, Nostocales and Oscillatoriales). High-throughput sequencing and bioinformatics analysis revealed that Me-ZS1 has a double-stranded DNA genome of 49,665 bp, with a G + C content of 58.22%, and 73 predicted open reading frames (ORFs). BLASTn and ORF comparisons showed that Me-ZS1 shares very low homology with the public sequences, and the phylogenetic tree based on TerL indicated that Me-ZS1 may delegate a novel and genetically distinct clade of Siphoviridae phages. In microcosm experiment, Me-ZS1 represented apparent effect on reducing relative abundance of cyanobacteria, increasing relative abundance of Saprospiraceae and protecting brocade carp (Carassius auratus) in cyanobacterial bloom water. This study isolated and characterized a novel broad-host-range Microcystis phage Me-ZS1 presenting a genetically distinct clade of freshwater cyanophage. The features of cyanophage Me-ZS1 provide a potential solution to the loss caused by cyanobacterial bloom.

Capsid Structure of a Freshwater Cyanophage Siphoviridae Mic1.

Cyanobacteria are the most abundant photosynthetic microorganisms, the global distribution of which is mainly regulated by the corresponding cyanophages. A systematic screening of water samples in the Lake Chaohu enabled us to isolate a freshwater siphocyanophage that infects Microcystis wesenbergii, thus termed Mic1. Using cryoelectron microscopy, we solved the 3.5-Å structure of Mic1 capsid. The major capsid protein gp40 of an HK97-like fold forms two types of capsomers, hexons and pentons. The capsomers interact with each other via the interweaved N-terminal arms of gp40 in addition to a tail-in-mouth joint along the three-fold symmetric axis, resulting in the assembly of capsid in a mortise-and-tenon pattern. The novel-fold cement protein gp47 sticks at the two-fold symmetric axis and further fixes the capsid. These findings provide structural insights into the assembly of cyanophages, and set up a platform to explore the mechanism of specific interactions and co-evolution with cyanobacteria.

Cooccurrence of Broad- and Narrow-Host-Range Viruses Infecting the Bloom-Forming Toxic Cyanobacterium Microcystis aeruginosa.

Viruses play important roles in regulating the abundance and composition of bacterial populations in aquatic ecosystems. The bloom-forming toxic cyanobacterium Microcystis aeruginosa is predicted to interact with diverse cyanoviruses, resulting in Microcystis population diversification. However, current knowledge of the genomes from these viruses and their infection programs is limited to those of Microcystis virus Ma-LMM01. Here, we performed a time series sampling at a small pond in Japan during a Microcystis bloom and then investigated the genomic information and transcriptional dynamics of Microcystis-interacting viruses using metagenomic and metatranscriptomic approaches. We identified 15 viral genomic fragments classified into three groups, groups I (including Ma-LMM01), II (high abundance and transcriptional activity), and III (new lineages). According to the phylogenetic distribution of Microcystis strains possessing spacers against each viral group, the group II-original viruses interacted with all three phylogenetically distinct Microcystis population types (phylotypes), whereas the groups I and III-original viruses interacted with only one or two phylotypes, indicating the cooccurrence of broad- (group II) and narrow (groups I and III)-host-range viruses in the bloom. These viral fragments showed the highest transcriptional levels during daytime regardless of their genomic differences. Interestingly, M. aeruginosa expressed antiviral defense genes in the environment, unlike what was seen with an Ma-LMM01 infection in a previous culture experiment. Given that broad-host-range viruses often induce antiviral responses within alternative hosts, our findings suggest that such antiviral responses might inhibit viral multiplication, mainly that of broad-host-range viruses like those in group II.IMPORTANCE The bloom-forming toxic cyanobacterium Microcystis aeruginosa is thought to have diversified its population through the interactions between host and viruses in antiviral defense systems. However, current knowledge of viral genomes and infection programs is limited to those of Microcystis virus Ma-LMM01, which was a narrow host range in which it can escape from the highly abundant host defense systems. Our metagenomic approaches unveiled the cooccurrence of narrow- and broad-host-range Microcystis viruses, which included fifteen viral genomic fragments from Microcystis blooms that were classified into three groups. Interestingly, Microcystis antiviral defense genes were expressed against viral infection in the environment, unlike what was seen in a culture experiment with Ma-LMM01. Given that viruses with a broad host range often induce antiviral responses within alternative hosts, our findings suggest that antiviral responses inhibit viral reproduction, especially that of broad-range viruses like those in group II. This paper augments our understanding of the interactions between M. aeruginosa and its viruses and fills an important knowledge gap.

Characterization of Cyanophages in Lake Erie: Interaction Mechanisms and Structural Damage of Toxic Cyanobacteria.

Cyanophages are abundant in aquatic environments and play a critical role in bloom dynamics, including regulation of cyanobacteria growth and photosynthesis. In this study, cyanophages from western Lake Erie water samples were screened for lytic activities against the host cell (Microcystis aeruginosa), which was also originated from Lake Erie and identified with real-time sequencing (Nanopore sequencing). M. aeruginosa was mixed with the cyanophages and their dynamic interactions were examined over two weeks using atomic force microscopy (AFM) as well as transmission electron microscopy (TEM), qPCR, phycocyanin and chlorophyll-a production, and optical absorbance measurements. The TEM images revealed a short-tailed virus (Podoviridae) in 300 nm size with unique capsid, knob-like proteins. The psbA gene and one knob-like protein gene, gp58, were identified by PCR. The AFM showed a reduction of mechanical stiffness in the host cell membranes over time after infection, before structural damage became visible. Significant inhibition of the host growth and photosynthesis was observed from the measurements of phycocyanin and chlorophyll-a concentrations. The results provide an insight into cyanobacteria-cyanophage interactions in bloom dynamics and a potential application of cyanophages for bloom control in specific situations.

Molecular prediction of lytic vs lysogenic states for Microcystis phage: Metatranscriptomic evidence of lysogeny during large bloom events.

Microcystis aeruginosa is a freshwater bloom-forming cyanobacterium capable of producing the potent hepatotoxin, microcystin. Despite increased interest in this organism, little is known about the viruses that infect it and drive nutrient mobilization and transfer of genetic material between organisms. The genomic complement of sequenced phage suggests these viruses are capable of integrating into the host genome, though this activity has not been observed in the laboratory. While analyzing RNA-sequence data obtained from Microcystis blooms in Lake Tai (Taihu, China), we observed that a series of lysogeny-associated genes were highly expressed when genes involved in lytic infection were down-regulated. This pattern was consistent, though not always statistically significant, across multiple spatial and temporally distinct samples. For example, samples from Lake Tai (2014) showed a predominance of lytic virus activity from late July through October, while genes associated with lysogeny were strongly expressed in the early months (June-July) and toward the end of bloom season (October). Analyses of whole phage genome expression shows that transcription patterns are shared across sampling locations and that genes consistently clustered by co-expression into lytic and lysogenic groups. Expression of lytic-cycle associated genes was positively correlated to total dissolved nitrogen, ammonium concentration, and salinity. Lysogeny-associated gene expression was positively correlated with pH and total dissolved phosphorous. Our results suggest that lysogeny may be prevalent in Microcystis blooms and support the hypothesis that environmental conditions drive switching between temperate and lytic life cycles during bloom proliferation.

The molecular ecology of Microcystis sp. blooms in the San Francisco Estuary.

Harmful blooms of the cyanobacterium Microcystis sp. have become increasingly pervasive in the San Francisco Estuary Delta (USA) since the early 2000s and their rise has coincided with substantial decreases in several important fish species. Direct and indirect effects Microcystis blooms may have on the Delta food web were investigated. The Microcystis population was tracked for 2 years at six sites throughout the Delta using quantitative PCR. High-throughput amplicon sequencing and colony PCR sequencing revealed the presence of 10 different strains of Microcystis, including 6 different microcystin-producing strains. Shotgun metagenomic analysis identified a variety of Microcystis secondary metabolite pathways, including those for the biosynthesis of: aeruginosin, cyanopeptolin, microginin, microviridin and piricyclamide. A sizable reduction was observed in microbial community diversity during a large Microcystis bloom (H' = 0.61) relative to periods preceding (H' = 2.32) or following (H' = 3.71) the bloom. Physicochemical conditions of the water column were stable throughout the bloom period. The elevated abundance of a cyanomyophage with high similarity to previously sequenced isolates known to infect Microcystis sp. was implicated in the bloom's collapse. Network analysis was employed to elucidate synergistic and antagonistic relationships between Microcystis and other bacteria and indicated that only very few taxa were positively correlated with Microcystis.

Cyanophages Infection of Microcystis Bloom in Lowland Dam Reservoir of Sulejów, Poland.

An increased incidence of cyanobacterial blooms, which are largely composed of toxigenic cyanobacteria from the Microcystis genus, leads to a disruption of aquatic ecosystems worldwide. Therefore, a better understanding of the impact of environmental parameters on the development and collapse of blooms is important. The objectives of the present study were as follows: (1) to investigate the presence and identity of Microcystis-specific cyanophages capable of cyanobacterial cell lysis in a lowland dam reservoir in Central Europe; (2) to investigate Microcystis sensitivity to phage infections with regard to toxic genotypes; and (3) to identify key abiotic parameters influencing phage infections during the summer seasons between 2009 and 2013. Sequencing analysis of selected g91 gene amplification products confirmed that the identified cyanophages belonged to the family Myoviridae (95 % homology). Cyanophages and Microcystis hosts, including toxic genotypes, were positively correlated in 4 of the 5 years analyzed (r = 0.67-0.82). The average percentage of infected Microcystis cells varied between 0.1 and 32 %, and no particular sensitivity of the phages to toxigenic genotypes was recorded. The highest number of cyanophages (>10(4) gene copy number per microliter) was observed in the period preceded by the following: an increase of the water retention time, growth of the water temperature, optimum nutrient concentrations, and the predomination of Microcystis bloom.

Genome analysis and gene nblA identification of Microcystis aeruginosa myovirus (MaMV-DC) reveal the evidence for horizontal gene transfer events between cyanomyovirus and host.

The genome sequence, genetic characterization and nblA gene function of Microcystis aeruginosa myovirus isolated from Lake Dianchi in China (MaMV-DC) have been analysed. The genome DNA is 169 223 bp long, with 170 predicted protein-coding genes (001L­170L) and a tRNA gene. About one-sixth of these genes have homologues in the host cyanobacteria M. aeruginosa. The genome carries a gene homologous to host nblA, which encodes a protein involved in the degradation of cyanobacterial phycobilisome. Its expression during MaMV-DC infection was confirmed by reverse transcriptase PCR and Western blot detection and abundant expression was companied by the significant decline of phycocyanin content and massive release of progeny MaMV-DC. In addition, expressing MaMV-DC nblA reduced the phycocyanin peak and the phycocyanin to chlorophyll ratio in model cyanobacteria. These results confirm that horizontal gene transfer events have occurred between cyanobacterial host and cyanomyovirus and suggest that MaMV-DC carrying host-derived genes (such as 005L, that codes for NblA) is responsible for more efficient expression of cyanophage genes and release of progeny cyanophage. This study provides novel insight into the horizontal gene transfer in cyanophage and the interactions between cyanophage and their host.

Development of a real-time PCR assay for the quantification of Ma-LMM01-type Microcystis cyanophages in a natural pond.

UNLABELLED: Microcystis aeruginosa forms toxic cyanobacterial blooms throughout the world where its infectious phages are thought to influence host population dynamics. To assess the cyanophage impact on the host dynamics, we previously monitored Ma-LMM01-type phage abundance using a real-time PCR with a primer set designed based on the sequence of Microcystis phage Ma-LMM01; and we estimated the phage-infected host cell abundance. However, a recent study shows the Ma-LMM01 g91 gene sequence belongs to the smallest group, group III, of the three genotype groups, suggesting Ma-LMM01-type phage abundance was underestimated. Therefore, to re-evaluate the effect of Ma-LMM01-type phages on their hosts, we monitored the abundance of Ma-LMM01-type phages using real-time PCR with a new primer set designed based on the sequences of genotype groups I-III. We found phage abundance between 10(3) and 10(4) ml(-1) using the new primer set in samples where previously these phages were not detected using the old primer set. The frequency of Ma-LMM01-type phage-infected cells to Ma-LMM01-type phage-susceptible host cells may be as high as 30%, suggesting the phages may occasionally affect not only shifts in the genetic composition but also the dynamics of Ma-LMM01-type phage-susceptible host populations. SIGNIFICANCE AND IMPACT OF THE STUDY: Phages are one of the factors that may control the ecology of their host blooms. Therefore, it is essential to estimate phage abundance to understand phage impact on host populations. A real-time PCR assay was improved to detect a larger range of Microcystis cyanophages in natural surroundings where no phages were detected using a previous method by re-designing a new primer set based on sequences from three Ma-LMM01-type phage genetic groups. The new method allows us to determine the distribution, dynamics and infection cycle of the phage to help understand the interaction between the phages and the hosts.

Genetic diversity of Microcystis cyanophages in two different freshwater environments.

Bacteriophages rapidly diversify their genes through co-evolution with their hosts. We hypothesize that gene diversification of phages leads to locality in phages genome. To test this hypothesis, we investigated the genetic diversity and composition of Microcystis cyanophages using 104 sequences of Ma-LMM01-type cyanophages from two geographically distant sampling sites. The intergenetic region between the ribonucleotide reductase genes nrdA and nrdB was used as the genetic marker. This region contains the host-derived auxiliary metabolic genes nblA, an unknown function gene g04, and RNA ligase gene g03. The sequences obtained were conserved in the Ma-LMM01 gene order and contents. Although the genetic diversity of the sequences was high, it varied by gene. The genetic diversity of nblA was the lowest, suggesting that nblA is a highly significant gene that does not allow mutation. In contrast, g03 sequences had many point mutations. RNA ligase is involved in the counter-host's phage defense mechanism, suggesting that phage defense also plays an important role for rapid gene diversification. The maximum parsimony network and phylogenic analysis showed the sequences from the two sampling sites were distinct. These findings suggest Ma-LMM01-type phages rapidly diversify their genomes through co-evolution with hosts in each location and eventually provided locality of their genomes.

Characterisation of host growth after infection with a broad-range freshwater cyanopodophage.

Freshwater cyanophages are poorly characterised in comparison to their marine counterparts, however, the level of genetic diversity that exists in freshwater cyanophage communities is likely to exceed that found in marine environments, due to the habitat heterogeneity within freshwater systems. Many cyanophages are specialists, infecting a single host species or strain; however, some are less fastidious and infect a number of different host genotypes within the same species or even hosts from different genera. Few instances of host growth characterisation after infection by broad host-range phages have been described. Here we provide an initial characterisation of interactions between a cyanophage isolated from a freshwater fishing lake in the south of England and its hosts. Designated ΦMHI42, the phage is able to infect isolates from two genera of freshwater cyanobacteria, Planktothrix and Microcystis. Transmission Electron Microscopy and Atomic Force Microscopy indicate that ΦMHI42 is a member of the Podoviridae, albeit with a larger than expected capsid. The kinetics of host growth after infection with ΦMHI42 differed across host genera, species and strains in a way that was not related to the growth rate of the uninfected host. To our knowledge, this is the first characterisation of the growth of cyanobacteria in the presence of a broad host-range freshwater cyanophage.

Cultivation and characterization of the MaMV-DC cyanophage that infects bloom-forming cyanobacterium Microcystis aeruginosa.

The MaMV-DC cyanophage, which infects the bloom-forming cyanobacterium Microcystis aeruginosa, was isolated from Lake Dianchi, Kunming, China. Twenty-one cyanobacterial strains were used to detect the host range of MaMV-DC. Microcystic aeruginosa FACHB-524 and plaque purification were used to isolate individual cyanophages, and culturing MaMV-DC with cyanobacteria allowed us to prepare purified cyanophages for further analysis. Electron microscopy demonstrated that the negatively stained viral particles are tadpole-shaped with an icosahedral head approximately 70 nm in diameter and a contractile tail approximately 160 nm in length. Using one-step growth experiments, the latent period and burst size of MaMV-DC were estimated to be 24-48 hours and approximately 80 infectious units per cell, respectively. Restriction endonuclease digestion and agarose gel electrophoresis were performed using purified MaMV-DC genomic DNA, and the genome size was estimated to be approximately 160 kb. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed four major structural proteins. These results support the growing interest in using freshwater cyanophages to control bloom-forming cyanobacterium.

Rapid microcystis cyanophage gene diversification revealed by long- and short-term genetic analyses of the tail sheath gene in a natural pond.

Viruses influence the abundance of host populations through virus-mediated host cell lysis. Viruses contribute to the generation and maintenance of host diversity, which also results in viral diversity throughout their coevolution. Here, to determine the phage gene diversification throughout the coevolution of host and phage in a natural environment, we investigated the genetic diversity and temporal changes in Microcystis cyanophage populations using a total of 810 sequences of the Ma-LMM01-type cyanophage tail sheath gene (g91) from 2006 to 2011 in a natural pond. The sequences obtained were highly diverse and assigned to 419 different genotypes (GT1 to GT419) clustered at 100% nucleotide sequence similarity. A maximum-parsimony network showed that the genotypes were largely divided into three sequence groups, which were dominated by major genotypes (more than 24 sequences: GT2, GT53, and GT163 in group I; GT25 in group II; and GT1 in group III). These major genotypes coexisted and oscillated throughout the sampling periods, suggesting that the Microcystis-cyanophage coevolution was partly driven by a negative frequency-dependent selection. Meanwhile, the high viral genetic diversity observed was derived from a large number of the variants of each major and moderately frequent genotype (including 7 to 18 sequences: GT7, GT26, GT56, GT149, and GT182 in group I; GT152 in group II) (1 or 2 nucleotide substitutions). The variants almost always co-occurred with their origin genotypes. This manner of variant emergence suggests that increased contact frequency within a host-phage population promotes rapid coevolution in a form of "arms race."