Freshwater Cyanophage Research Articles

Cyanobacteria-cyanophage interactions between freshwater and marine ecosystems based on large-scale cyanophage genomic analysis

The disparities in harmful algal blooms dynamics are largely attributed to variations in cyanobacteria populations within aquatic ecosystems. However, cyanobacteria-cyanophage interactions and their role in shaping cyanobacterial populations has been previously underappreciated. To address this knowledge gap, we isolated and sequenced 42 cyanophages from diverse water sources in China, with the majority (n = 35) originating from freshwater sources. We designated these sequences as the “Novel Cyanophage Genome sequence Collection” (NCGC). NCGC displayed notable genetic variations, with 95 % (40/42) of the sequences representing previously unidentified taxonomic ranks. By integrating NCGC with public data of cyanophages and cyanobacteria, we found evidence for more frequent historical cyanobacteria-cyanophage interactions in freshwater ecosystems. This was evidenced by a higher prevalence of prophage integrase-related genes in freshwater cyanophages (37.97 %) than marine cyanophages (7.42 %). In addition, freshwater cyanophages could infect a broader range of cyanobacteria orders (n = 4) than marine ones (n = 0). Correspondingly, freshwater cyanobacteria harbored more defense systems per million base pairs in their genomes, indicating more frequent phage infections. Evolutionary and cyanophage epidemiological studies suggest that interactions between cyanobacteria and cyanophages in freshwater and marine ecosystems are interconnected, and that brackish water can act as a transitional zone for freshwater and marine cyanophages. In conclusion, our research significantly expands the genetic information database of cyanophage, offering a wider selection of cyanophages to control harmful cyanobacterial blooms. Additionally, we represent a pioneering large-scale and comprehensive analysis of cyanobacteria and cyanophage sequencing data, and it provides theoretical guidance for the application of cyanophages in different environments.

Corrected and Republished from: "Isolation and Characterization of the First Freshwater Cyanophage Infecting Pseudanabaena".

Cyanobacteria are the major primary producers in both freshwater and marine environments. However, the majority of freshwater cyanophages remain unknown due to the limited number of cyanophage isolates. In this study, we present a novel lytic freshwater cyanophage, PA-SR01, which was isolated from the Singapore Serangoon Reservoir. To our knowledge, this is the first isolate of a cyanophage that has been found to infect the cyanobacterium Pseudanabaena. PA-SR01 has a narrow host range, a short latent period, and is chloroform sensitive. PA-SR01 is a member of Siphoviridae with a long noncontractile tail. It is a double-stranded DNA virus with a 137,012-bp genome. Functional annotation for the predicted open reading frames (ORFs) of the PA-SR01 genome identified genes with putative functions related to DNA metabolism, structural proteins, lysis, host-derived metabolic genes, and DNA packaging. Out of 166 predicted ORFs, only 17 ORFs have homology with genes with known function. Phylogenetic analysis of the major capsid protein and terminase large subunit further suggests that phage PA-SR01 is evolutionary distinct from known cyanophages. Metagenomics sequence recruitment onto the PA-SR01 genome indicates that PA-SR01 represents a new evolutionary lineage of phage which shares considerable genetic similarities with phage sequences in aquatic environments and could play key ecological roles. IMPORTANCE This study presents the isolation of the very first freshwater cyanophage, PA-SR01, that infects Pseudanabaena, and fills an important knowledge gap on freshwater cyanophages as well as cyanophages infecting Pseudanabaena.

Correction for Zhang et al., "Isolation and Characterization of the First Freshwater Cyanophage Infecting Pseudanabaena".

Correction for Zhang et al., "Isolation and Characterization of the First Freshwater Cyanophage Infecting Pseudanabaena".

Genomic Analysis of a New Freshwater Cyanophage Lbo240-yong1 Suggests a New Taxonomic Family of Bacteriophages.

A worldwide ecological issue, cyanobacterial blooms in marine and freshwater have caused enormous losses in both the economy and the environment. Virulent cyanophages-specifically, infecting and lysing cyanobacteria-are key ecological factors involved in limiting the overall extent of the population development of cyanobacteria. Over the past three decades, reports have mainly focused on marine Prochlorococcus and Synechococcus cyanophages, while information on freshwater cyanophages remained largely unknown. In this study, a novel freshwater cyanophage, named Lbo240-yong1, was isolated via the double-layer agar plate method using Leptolyngbya boryana FACHB-240 as a host. Transmission electron microscopy observation illustrated the icosahedral head (50 ± 5 nm in diameter) and short tail (20 ± 5 nm in length) of Lbo240-yong1. Experimental infection against 37 cyanobacterial strains revealed that host-strain-specific Lbo240-yong1 could only lyse FACHB-240. The complete genome of Lbo240-yong1 is a double-stranded DNA of 39,740 bp with a G+C content of 51.99%, and it harbors 44 predicted open reading frames (ORFs). A Lbo240-yong1 ORF shared the highest identity with a gene of a filamentous cyanobacterium, hinting at a gene exchange between the cyanophage and cyanobacteria. A BLASTn search illustrated that Lbo240-yong1 had the highest sequence similarity with the Phormidium cyanophage Pf-WMP4 (89.67% identity, 84% query coverage). In the proteomic tree based on genome-wide sequence similarities, Lbo240-yong1, three Phormidium cyanophages (Pf-WMP4, Pf-WMP3, and PP), one Anabaena phage (A-4L), and one unclassified Arthronema cyanophage (Aa-TR020) formed a monophyletic group that was more deeply diverging than several other families. Pf-WMP4 is the only member of the independent genus Wumpquatrovirus that belongs to the Caudovircetes class. Pf-WMP3 and PP formed the independent genus Wumptrevirus. Anabaena phage A-4L is the only member of the independent Kozyakovvirus genus. The six cyanopodoviruses share similar gene arrangements. Eight core genes were found in them. We propose, here, to set up a new taxonomic family comprising the six freshwater cyanopodoviruses infecting filamentous cyanobacteria. This study enriched the field's knowledge of freshwater cyanophages.

Fine structure and assembly pattern of a minimal myophage Pam3

The myophage possesses a contractile tail that penetrates its host cell envelope. Except for investigations on the bacteriophage T4 with a rather complicated structure, the assembly pattern and tail contraction mechanism of myophage remain largely unknown. Here, we present the fine structure of a freshwater Myoviridae cyanophage Pam3, which has an icosahedral capsid of ~680 Å in diameter, connected via a three-section neck to an 840-Å-long contractile tail, ending with a three-module baseplate composed of only six protein components. This simplified baseplate consists of a central hub-spike surrounded by six wedge heterotriplexes, to which twelve tail fibers are covalently attached via disulfide bonds in alternating upward and downward configurations. Invitro reduction assays revealed a putative redox-dependent mechanism of baseplate assembly and tail sheath contraction. These findings establish a minimal myophage that might become a user-friendly chassis phage in synthetic biology.

Phylogenomics of five Pseudanabaena cyanophages and evolutionary traces of horizontal gene transfer

BackgroundAlong with the fast development and urbanization in developing countries, the waterbodies aside the growing cities become heavily polluted and highly eutrophic, thus leading to the seasonal outbreak of cyanobacterial bloom. Systematic isolation and characterization of freshwater cyanophages might provide a biological solution to control the awful blooms. However, genomic sequences and related investigations on the freshwater cyanophages remain very limited to date.ResultsFollowing our recently reported five cyanophages Pam1~Pam5 from Lake Chaohu in China, here we isolated another five cyanophages, termed Pan1~Pan5, which infect the cyanobacterium Pseudanabaena sp. Chao 1811. Whole-genome sequencing showed that they all contain a double-stranded DNA genome of 37.2 to 72.0 kb in length, with less than half of the putative open reading frames annotated with known functions. Remarkably, the siphophage Pan1 encodes an auxiliary metabolic gene phoH and constitutes, together with the host, a complete queuosine modification pathway. Proteomic analyses revealed that although Pan1~Pan5 are distinct from each other in evolution, Pan1 and Pan3 are somewhat similar to our previously identified cyanophages Pam3 and Pam1 at the genomic level, respectively. Moreover, phylogenetic analyses suggested that Pan1 resembles the α-proteobacterial phage vB_DshS-R5C, revealing direct evidence for phage-mediated horizontal gene transfer between cyanobacteria and α-proteobacteria.ConclusionIn addition to the previous reports of Pam1~Pam5, the present findings on Pan1~Pan5 largely enrich the library of reference freshwater cyanophages. The abundant genomic information provides a pool to identify novel genes and proteins of unknown function. Moreover, we found for the first time the evolutionary traces in the cyanophage that horizontal gene transfer might occur at the level of not only inter-species, but even inter-phylum. It indicates that the bacteriophage or cyanophage could be developed as a powerful tool for gene manipulation among various species or phyla.

A Novel Freshwater Cyanophage Mae-Yong1326-1 Infecting Bloom-Forming Cyanobacterium Microcystis aeruginosa.

Microcystis aeruginosa is a major harmful cyanobacterium causing water bloom worldwide. Cyanophage has been proposed as a promising tool for cyanobacterial bloom. In this study, M. aeruginosa FACHB-1326 was used as an indicator host to isolate cyanophage from Lake Taihu. The isolated Microcystis cyanophage Mae-Yong1326-1 has an elliptical head of about 47 nm in diameter and a slender flexible tail of about 340 nm in length. Mae-Yong1326-1 could lyse cyanobacterial strains across three orders (Chroococcales, Nostocales, and Oscillatoriales) in the host range experiments. Mae-Yong1326-1 was stable in stability tests, maintaining high titers at 0–40 °C and at a wide pH range of 3–12. Mae-Yong 1326-1 has a burst size of 329 PFU/cell, which is much larger than the reported Microcystis cyanophages so far. The complete genome of Mae-Yong1326-1 is a double-stranded DNA of 48, 822 bp, with a G + C content of 71.80% and long direct terminal repeats (DTR) of 366 bp, containing 57 predicted ORFs. No Mae-Yong1326-1 ORF was found to be associated with virulence factor or antibiotic resistance. PASC scanning illustrated that the highest nucleotide sequence similarity between Mae-Yong1326-1 and all known phages in databases was only 17.75%, less than 70% (the threshold to define a genus), which indicates that Mae-Yong1326-1 belongs to an unknown new genus. In the proteomic tree based on genome-wide sequence similarities, Mae-Yong1326-1 distantly clusters with three unclassified Microcystis cyanophages (MinS1, Mwe-Yong1112-1, and Mwes-Yong2). These four Microcystis cyanophages form a monophyletic clade, which separates at a node from the other clade formed by two independent families (Zierdtviridae and Orlajensenviridae) of Caudoviricetes class. We propose to establish a new family to harbor the Microcystis cyanophages Mae-Yong1326-1, MinS1, Mwe-Yong1112-1, and Mwes-Yong2. This study enriched the understanding of freshwater cyanophages.

Comparative genomic analysis of five freshwater cyanophages and reference-guided metagenomic data mining

BackgroundAs important producers using photosynthesis on Earth, cyanobacteria contribute to the oxygenation of atmosphere and the primary production of biosphere. However, due to the eutrophication of urban waterbodies and global warming, uncontrollable growth of cyanobacteria usually leads to the seasonal outbreak of cyanobacterial blooms. Cyanophages, a group of viruses that specifically infect and lyse cyanobacteria, are considered as potential environment-friendly agents to control the harmful blooms. Compared to the marine counterparts, only a few freshwater cyanophages have been isolated and genome sequenced to date, largely limiting their characterizations and applications.ResultsHere, we isolated five freshwater cyanophages varying in tail morphology, termed Pam1~Pam5, all of which infect the cyanobacterium Pseudanabaena mucicola Chao 1806 that was isolated from the bloom-suffering Lake Chaohu in Anhui, China. The whole-genome sequencing showed that cyanophages Pam1~Pam5 all contain a dsDNA genome, varying in size from 36 to 142 Kb. Phylogenetic analyses suggested that Pam1~Pam5 possess different DNA packaging mechanisms and are evolutionarily distinct from each other. Notably, Pam1 and Pam5 have lysogeny-associated gene clusters, whereas Pam2 possesses 9 punctuated DNA segments identical to the CRISPR spacers in the host genome. Metagenomic data-based calculation of the relative abundance of Pam1~Pam5 at the Nanfei estuary towards the Lake Chaohu revealed that the short-tailed Pam1 and Pam5 account for the majority of the five cyanophages. Moreover, comparative analyses of the reference genomes of Pam1~Pam5 and previously reported cyanophages enabled us to identify three circular and seven linear contigs of virtual freshwater cyanophages from the metagenomic data of the Lake Chaohu.ConclusionsWe propose a high-throughput strategy to systematically identify cyanophages based on the currently available metagenomic data and the very limited reference genomes of experimentally isolated cyanophages. This strategy could be applied to mine the complete or partial genomes of unculturable bacteriophages and viruses. Transformation of the synthesized whole genomes of these virtual phages/viruses to proper hosts will enable the rescue of bona fide viral particles and eventually enrich the library of microorganisms that exist on Earth.4WYBiJEHr_CXoYqKn7ZoJMVideo abstract

Cyanophage dynamics in a tropical urban freshwater lake

Considerable studies have been conducted to quantify and examine free cyanophage particles across a wide range of aquatic systems, but few studies have analyzed the dynamics of host cell fraction cyanophages, and even less is known about tropical freshwater cyanophages. Here, we employed qPCR to examine the abundances of four ecologically relevant novel cyanophages (PA-SR01, S-SRP01, S-SRP02 and S-SRM01) in a tropical urban lake in Singapore from July to November 2019. Besides quantifying the extracellular free cyanophage particles, host cell fraction cyanophage were also examined to represent the abundance of intracellular and attached phages. Our study revealed differing fate and dynamics of each cyanophage target. Host cell fraction phage abundance was found to be more abundant than free phage abundance, indicating high level of infection and rapid dispersal of phage particles upon lysis. By further investigating the correlations between free phage particle abundance and numerous biotic (total cyanobacteria, Microcystis abundances, etc) as well as abiotic (nutrients and physiochemical water quality) variables, multiple correlations across different sampling sites were obtained, suggesting pH and rainfall to be potential key factors driving shifts in both cyanophage and cyanobacteria population. This study demonstrated the ecological importance and key roles of the cyanophage PA-SR01, S-SRP01, S-SRP02 and S-SRM01 in tropical freshwater lakes not only as regulators of the cyanobacterial community, but also as promoters of certain cyanotoxin production.

Genome sequence of the novel freshwater Microcystis cyanophage Mwe-Yong1112-1.

The freshwater cyanophage Mwe-Yong1112-1 was isolated using Microcystis wesenbergii as a host and found to have an icosahedral head, about 45 nm in diameter, and a flexible tail, approximately 133 nm in length and 4.5 nm in width. The complete genome of the cyanophage is 39,679 bp in length with a G+C content of 66.6%. Mwe-Yong1112-1 shared the highest pairwise average nucleotide identity (ANI) value of 67.7% (below the ≥95% boundary to define a species) and the highest nucleotide sequence similarity of 17.48% (below the >70% boundary to define a genus) with the most closely related phage. In a proteomic tree, Mwe-Yong1112-1 and three unclassified phages formed a monophyletic clade between the families Saparoviridae and Pyrstoviridae, but Mwe-Yong1112-1 occupied a separate branch from the other three phages, suggesting that it represents a new evolutionary lineage. This study enriches the available information about freshwater cyanophages.

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.

Genomic Characterization of a Novel Freshwater Cyanophage Reveals a New Lineage of Cyanopodovirus.

Cyanobacteria are one of the dominant autotrophs in tropical freshwater communities, yet phages infecting them remain poorly characterized. Here we present the characterization of cyanophage S-SRP02, isolated from a tropical freshwater lake in Singapore, which infects Synechococcus sp. Strain SR-C1 isolated from the same lake. S-SRP02 represents a new evolutionary lineage of cyanophage. Out of 47 open reading frames (ORFs), only 20 ORFs share homology with genes encoding proteins of known function. There is lack of auxiliary metabolic genes which was commonly found as core genes in marine cyanopodoviruses. S-SRP02 also harbors unique structural genes highly divergent from other cultured phages. Phylogenetic analysis and viral proteomic tree further demonstrate the divergence of S-SRP02 from other sequenced phage isolates. Nonetheless, S-SRP02 shares synteny with phage genes of uncultured phages obtained from the Mediterranean Sea deep chlorophyll maximum fosmids, indicating the ecological importance of S-SRP02 and its related viruses. This is further supported by metagenomic mapping of environmental viral metagenomic reads onto the S-SRP02 genome.

Structure and assembly pattern of a freshwater short-tailed cyanophage Pam1

Despite previous structural analyses of bacteriophages, quite little is known about the structures and assembly patterns of cyanophages. Using cryo-EM combined with crystallography, we solve the near-atomic-resolution structure of a freshwater short-tailed cyanophage, Pam1, which comprises a 400-Å-long tail and an icosahedral capsid of 650Å in diameter. The outer capsid surface is reinforced by trimeric cement proteins with a β-sandwich fold, which structurally resemble the distal motif of Pam1's tailspike, suggesting its potential role in host recognition. At the portal vertex, the dodecameric portal and connected adaptor, followed by a hexameric needle head, form a DNA ejection channel, which is sealed by a trimeric needle. Moreover, we identify a right-handed rifling pattern that might help DNA to revolve along the wall of the ejection channel. Our study reveals the precise assembly pattern of a cyanophage and lays the foundation to support its practical biotechnological and environmental applications.

Novel Freshwater Cyanophages Provide New Insights into Evolutionary Relationships between Freshwater and Marine Cyanophages.

ABSTRACTCyanobacteria and cyanophages are present widely in both freshwater and marine environments. However, freshwater cyanophages remain unknown largely due to the small numbers of cyanophage isolates despite their ecological and environmental significance. In this study, we present the characterization of two novel lytic freshwater cyanophages isolated from a tropical inland lake in Singapore, namely, cyanopodovirus S-SRP01 and cyanomyovirus S-SRM01, infecting two different strains of Synechococcus spp. Functional annotation of S-SRP01 and S-SRM01 genomes revealed a high degree of homology with marine cyanophages. Phylogenetic trees of concatenated genes and whole-genome alignment provided further evidence that S-SRP01 is close evolutionarily to marine cyanopodoviruses, while S-SRM01 is evolutionarily close to marine cyanomyoviruses. Few genetic similarities between freshwater and marine cyanophages have been identified in previous studies. The isolation of S-SRP01 and S-SRM01 expand current knowledge on freshwater cyanophages infecting Synechococcus spp. Their high degree of gene sharing provides new insights into the evolutionary relationships between freshwater and marine cyanophages. This relatedness is further supported by the discovery of similar phenomenon from other freshwater viral metagenomes.IMPORTANCE This study expands the current knowledge on freshwater cyanophage isolates and cyanophage genetic diversity, indicating that freshwater and marine cyanophages infecting Synechococcus spp. may share close genetic similarity and evolutionary relationships.

Cyanophage Distribution Across European Lakes of the Temperate-Humid Continental Climate Zone Assessed Using PCR-Based Genetic Markers

Studies of the diversity and distribution of freshwater cyanophages are generally limited to the small geographical areas, in many cases including only one or few lakes. Data from dozens of various lakes distributed at a larger distance are necessary to understand their spatial distribution and sensitivity to biotic and abiotic factors. Thus, the objective of this study was to analyze the diversity and distribution of cyanophages within the infected cells using marker genes (psbA, nblA, and g91) in 21 Polish and Lithuanian lakes. Physicochemical factors that might be related to them were also analyzed. The results demonstrated that genetic markers representing cyanophages were observed in most lakes studied. The frequently detected gene was psbA with 88% of cyanophage-positive samples, while nblA and g91 were found in approximately 50% of lakes. The DNA sequence analyses for each gene demonstrated low variability between them, although the psbA sequences branched within the larger cluster of marine Synechoccocuss counterparts. The principal component analysis allowed to identify significant variation between the lakes that presented high and low cyanobacterial biomass. The lakes with high cyanobacterial biomass were further separated by country and the different diversity of cyanobacteria species, particularly Planktothrix agardhii, was dominant in the Polish lakes and Planktolyngbya limnetica in the Lithuanian lakes. The total phosphorous and the presence of cyanophage genes psbA and nblA were the most important factors that allowed differentiation for the Polish lakes, while the pH and the genes g91 and nblA for the Lithuanian lakes.

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 60nm in diameter, and a noncontractile tail approximately 260nm. 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 49,665bp, 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.

Isolation and Characterization of the First Freshwater Cyanophage Infecting Pseudanabaena.

Cyanobacteria are the major primary producers in both freshwater and marine environments. However, the majority of freshwater cyanophages remain unknown due to the limited number of cyanophage isolates. In this study, we present a novel lytic freshwater cyanophage, PA-SR01, which was isolated from the Singapore Serangoon Reservoir. To our knowledge, this is the first isolate of a cyanophage that has been found to infect the cyanobacterium Pseudanabaena PA-SR01 has a narrow host range, a short latent period, and is chloroform sensitive. Distinct from the majority of cyanophage isolates, PA-SR01 has a tailless morphology. It is a double-stranded DNA virus with a 137,012-bp genome. Functional annotation for the predicted open reading frames (ORFs) of the PA-SR01 genome identified genes with putative functions related to DNA metabolism, structural proteins, lysis, host-derived metabolic genes, and DNA packaging. Out of 166 predicted ORFs, only 17 ORFs have homology with genes with known function. Phylogenetic analysis of the major capsid protein and terminase large subunit further suggests that phage PA-SR01 is evolutionary distinct from known cyanophages. Metagenomics sequence recruitment onto the PA-SR01 genome indicates that PA-SR01 represents a new evolutionary lineage of phage which shares considerable genetic similarities with phage sequences in aquatic environments and could play key ecological roles.IMPORTANCE This study presents the isolation of the very first freshwater cyanophage, PA-SR01, that infects Pseudanabaena, and fills an important knowledge gap on freshwater cyanophages as well as cyanophages infecting Pseudanabaena.

Crystal structure of a novel fold protein Gp72 from the freshwater cyanophage Mic1.

Cyanophages, widespread in aquatic systems, are a class of viruses that specifically infect cyanobacteria. Though they play important roles in modulating the homeostasis of cyanobacterial populations, little is known about the freshwater cyanophages, especially those hypothetical proteins of unknown function. Mic1 is a freshwater siphocyanophage isolated from the Lake Chaohu. It encodes three hypothetical proteins Gp65, Gp66, and Gp72, which share an identity of 61.6% to 83%. However, we find these three homologous proteins differ from each other in oligomeric state. Moreover, we solve the crystal structure of Gp72 at 2.3 Å, which represents a novel fold in the α + β class. Structural analyses combined with redox assays enable us to propose a model of disulfide bond mediated oligomerization for Gp72. Altogether, these findings provide structural and biochemical basis for further investigations on the freshwater cyanophage Mic1.

An uncultured marine cyanophage encodes an active phycobilisome proteolysis adaptor protein NblA.

Phycobilisomes (PBS) are large water-soluble membrane-associated complexes in cyanobacteria and some chloroplasts that serve as light-harvesting antennae for the photosynthetic apparatus. When deplete of nitrogen or sulphur, cyanobacteria readily degrade their phycobilisomes allowing the cell to replenish these vanishing nutrients. The key regulator in the degradation process is NblA, a small protein (∼6 kDa), which recruits proteases to the PBS. It was discovered previously that not only do cyanobacteria possess nblA genes but also that they are encoded by genomes of some freshwater cyanophages. A recent study, using assemblies from oceanic metagenomes, revealed genomes of a novel uncultured marine cyanophage lineage, representatives of which contain genes coding for the PBS degradation protein. Here, we examined the functionality of nblA-like genes from these marine cyanophages by testing them in a freshwater model cyanobacterial nblA knockout. One of the viral NblA variants could complement the non-bleaching phenotype and restore PBS degradation. Our findings reveal a functional NblA from a novel marine cyanophage lineage. Furthermore, we shed new light on the distribution of nblA genes in cyanobacteria and cyanophages.

A New Freshwater Cyanosiphovirus Harboring Integrase

Pelagic cyanobacteria are key players in the functioning of aquatic ecosystems, and their viruses (cyanophages) potentially affect the abundance and composition of cyanobacterial communities. Yet, there are few well-described freshwater cyanophages relative to their marine counterparts, and in general, few cyanosiphoviruses (family Siphoviridae) have been characterized, limiting our understanding of the biology and the ecology of this prominent group of viruses. Here, we characterize S-LBS1, a freshwater siphovirus lytic to a phycoerythrin-rich Synechococcus isolate (Strain TCC793). S-LBS1 has a narrow host range, a burst size of ∼400 and a relatively long infecting step before cell lysis occurs. It has a dsDNA 34,641 bp genome with putative genes for structure, DNA packing, lysis, replication, host interactions, DNA repair and metabolism. S-LBS1 is similar in genome size, genome architecture, and gene content, to previously described marine siphoviruses also infecting PE-rich Synechococcus, e.g., S-CBS1 and S-CBS3. However, unlike other Synechococcus phages, S-LBS1 encodes an integrase, suggesting its ability to establish lysogenic relationships with its host. Sequence recruitment from viral metagenomic data showed that S-LBS1-like viruses are diversely present in a wide range of aquatic environments, emphasizing their potential importance in controlling and structuring Synechococcus populations. A comparative analysis with 16 available sequenced cyanosiphoviruses reveals the absence of core genes within the genomes, suggesting high degree of genetic variability in siphoviruses infecting cyanobacteria. It is likely that cyanosiphoviruses have evolved as distinct evolutionary lineages and that adaptive co-evolution occurred between these viruses and their hosts (i.e., Synechococcus, Prochlorococcus, Nodularia, and Acaryochloris), constituting an important driving force for such phage diversification.