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 80infectious 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.

Freshwater Cyanophages

Cyanophages are double-stranded DNA viruses that infect cyanobacteria, and they can be found in both freshwater and marine environments. They have a complex pattem of host ranges and play important roles in controlling cyanobacteria population. Unlike marine cyanophages, for which there have been a number of recent investigations, very little attention has been paid to freshwater cyanophages. This review summarizes the taxonomy and morphology, host range, distribution, seasonal dynamics, and complete genomes of freshwater cyanophages, as well as diagnostic markers that can be used to identify them.

Characterization and Genomic Analysis of a Plaque Purified Strain of Cyanophage PP

Cyanophages are ubiquitous and essential components of the aquatic environment and play an important role in the termination of algal blooms.As such,they have attracted widespread interest.PP was the first isolated cyanophage in China,which infects Plectonema boryanum and Phormidium foveolarum.In this study,this cyanophage was purified three times by a double-agar overlay plaque assay and characterized.Its genome was extracted,totally sequenced and analyzed.Electron microscopy revealed a particle with an icosahedral head connected to a short stubby tail.Bioassays showed that PP was quite virulent.The genome of PP is a 42,480 base pair(bp),linear,double-stranded DNA molecule with 222 bp terminal repeats.It has high similarity with the known Pf-WMP3 sequence.It contains 41 open reading frames(ORFs),17 of which were annotated.Intriguingly,the genome can be divided into two completely different parts,which differ both in orientation and function.

The First Isolation of a Cyanophage-Synechococcus System from the East China Sea

A cyanophage strain and its host Synechococcus were isolated from the East China Sea. The host Synechococcus sp. S J01 was characterized by its 16S rRNA, ITS, andpsbA gene sequences as well as by its morphological appearance and pigmentation. The cyanophage, strain S-SJ2, was able to cause a lytic infection of the coastal Synechococcus. TEM of negative-stained specimens showed that the phage isolate has an isometric head with a diameter of 68 nm and a long tail with a length of 280 nm. The cyanophage-Synechococcus system from the East China Sea shares many properties with other marine cyanophage-Synechocoecus systems worldwide.

Synthesis and assembly of full-length cyanophage A-4L genome

Artificial cyanophages are considered to be an effective biological method to control harmful cyanobacterial bloom.However,no synthetic cyanophage genome has been constructed and where its obstacles are unclear.Here,we survey a stretch of 16 kb length sequence of cyanophage A-4L that is unclonable in Escherichia coli.We test 12 predicted promoters of cyanophage A-4L which were verified all active in E.coli.Next,we screen for eight ORFs that hindered the assembly of intermediate DNA fragments in E.coli and describe that seven ORFs in the 16 kb sequence could not be separately cloned in E.coli.All of unclonable ORFs in high-copy-number plasmid were successfully cloned using low-copy-number vector,suggesting that these ORFs were copy-number-dependent.We propose a clone strategy abandoned the promotor and the start codon that could be applied for unclonable ORFs.Last,we de novo synthesized and assembled the full-length genome of cyanophage A-4L.This work deepens the understanding of synthetic cyanophages studies.

Viral Metagenomics Analysis of Planktonic Viruses in East Lake,Wuhan,China

East Lake(Lake Donghu),located in Wuhan,China,is a typical city freshwater lake that has been experiencing eutrophic conditions and algal blooming during recent years.Marine and fresh water are considered to contain a large number of viruses.However,little is known about their genetic diversity because of the limited techniques for culturing viruses.In this study,we conducted a viral metagenomic analysis using a high-throughput sequencing technique with samples collected from East Lake in Spring,Summer,Autumn,and Winter.The libraries from four samples each generated 234,669,71,837,12,820,and 34,236 contigs(>90 bp each),respectively.The genetic structure of the viral community revealed a high genetic diversity covering 23 viral families,with the majority of contigs homologous to DNA viruses,including members of Myoviridae,Podoviridae,Siphoviridae,Phycodnaviridae,and Microviridae,which infect bacteria or algae,and members of Circoviridae,which infect invertebrates and vertebrates.The highest viral genetic diversity occurred in samples collected in August,then December and June,and the least diversity in March.Most contigs have low-sequence identities with known viruses.PCR detection targeting the conserved sequences of genes(g20,psbA,psbD,and DNApol)of cyanophages further confirmed that there are novel cyanophages in the East Lake.Our viral metagenomic data provide the first preliminary understanding of the virome in one freshwater lake in China and would be helpful for novel virus discovery and the control of algal blooming in the future.

Genetic diversity and temporal dynamics of phytoplankton viruses in East Lake, China

Phytoplankton viruses are important components of aquatic ecosystems. However, their prevalence and genetic diversity in marine and freshwater systems are largely under estimated owing to the immense size of water bodies and limitations in virus discovery techniques. In this study, we conducted a 1-year survey of phytoplankton virus communities by collecting surface water monthly from an inland lake(East Lake) in China between May 2012 and April 2013. We examined four phytoplankton viruses, i.e., myoviruses, podoviruses, siphoviruses, and phycodnaviruses, and seven sets of primers were used to target conserved genes within these four species. In this year-long investigation, a total of 358 different virus-related sequences from four virus families were obtained. All virus families were detected in all months, except for cyanopodoviruses, which were only identified during eight of the 12 months surveyed. Moreover, virus abundance and diversity changed dynamically over time. Phylogenetic analysis revealed that the majority of viral sequences from East Lake, China displayed distinct clustering patterns compared with published sequences. These results supported the existence of a highly diverse and unique phytoplankton virus community in East Lake, China.