Capsid Structure of Anabaena Cyanophage A-1(L).

A-1(L) is a freshwater cyanophage with a contractile tail that specifically infects Anabaena sp. PCC 7120, one of the model strains for molecular studies of cyanobacteria. Although isolated for half a century, its structure remains unknown, which limits our understanding on the interplay between A-1(L) and its host. Here we report the 3.35 Å cryo-EM structure of A-1(L) capsid, representing the first near-atomic resolution structure of a phage capsid with a T number of 9. The major capsid gp4 proteins assemble into 91 capsomers, including 80 hexons: 20 at the center of the facet and 60 at the facet edge, in addition to 11 identical pentons. These capsomers further assemble into the icosahedral capsid, via gradually increasing curvatures. Different from the previously reported capsids of known-structure, A-1(L) adopts a noncovalent chainmail structure of capsid stabilized by two kinds of mortise-and-tenon inter-capsomer interactions: a three-layered interface at the pseudo 3-fold axis combined with the complementarity in shape and electrostatic potential around the 2-fold axis. This unique capsomer construction enables A-1(L) to possess a rigid capsid, which is solely composed of the major capsid proteins with an HK97 fold. IMPORTANCE Cyanobacteria are the most abundant photosynthetic bacteria, contributing significantly to the biomass production, O2 generation, and CO2 consumption on our planet. Their community structure and homeostasis in natural aquatic ecosystems are largely regulated by the corresponding cyanophages. In this study, we solved the structure of cyanophage A-1(L) capsid at near-atomic resolution and revealed a unique capsid construction. This capsid structure provides the molecular details for better understanding the assembly of A-1(L), and a structural platform for future investigation and application of A-1(L) in combination with its host Anabaena sp. PCC 7120. As the first isolated freshwater cyanophage that infects the genetically tractable model cyanobacterium, A-1(L) should become an ideal template for the genetic engineering and synthetic biology studies.

Cyanophage A-1(L) Adsorbs to Lipopolysaccharides of Anabaena sp. Strain PCC 7120 via the Tail Protein Lipopolysaccharide-Interacting Protein (ORF36).

Ecological functions of cyanophages in aquatic environments depend on their interactions with cyanobacterial hosts. The first step of phage-host interaction involves adsorption to the cell surface. We report that adsorption of a cyanophage, A-1(L), to the outer membrane of Anabaena sp. strain PCC 7120 is based on the binding of a tail protein, ORF36, to the O antigen of lipopolysaccharides (LPS). Removal of O antigen by gene inactivation abolished infection by A-1(L); consistently, preincubation of the cyanophage with extracted Anabaena LPS partially blocked infection. In contrast, inactivation of major outer membrane protein genes in Anabaena or addition of Synechocystis LPS showed no effect on infection. ORF35 and ORF36 are two predicted tail proteins of A-1(L). Antibodies against either ORF35 or ORF36 strongly inhibited infection. Enzyme-linked immunosorbent assay showed a specific interaction between ORF36 and the LPS of Anabaena sp. strain PCC 7120. These findings indicate that ORF35 and ORF36 are probably both required for adsorption of A-1(L) to the cell surface, but ORF36 specifically binds to the O antigen of LPS.IMPORTANCE Cyanophages play an important role in regulating the dynamics of cyanobacterial communities in aquatic environments. Hitherto, the mechanisms for cyanophage infection have been barely investigated. In this study, the first cyanophage tail protein that binds to the receptor (LPS) on cell surface was identified and shown to be essential for the A-1(L) infection of Anabaena sp. strain PCC 7120. The protein-LPS interaction may represent an important route for adsorption of cyanophages to their hosts.

[Isolation and phylogenetic analysis of related genes of cultivable cyanophages in Daqing Wetland].

Objective: To provide scientific data for studying the ecology of cyanophages isolated from Daqing wetland by analyzing their genetic diversity and phylogenetic positions. Methods: Liquid enrichment culture and double-layer plate methods were used to isolate cultivable cyanophages by using host cyanobacteria Anabaena PCC7120, and the DNA of cultivable cyanophages was extracted. The biomarker genes of g20 encoding capsid assembly protein and pol encoding DNA polymerase in podoviruses were PCR amplified. The PCR products were cloned and sequenced. The sequences were constructed with references sequences into the phylogenetic trees to clarify the phylogenetic positions of cultivable cyanophage. Results: One g20 sequence and four pol sequences were obtained. Phylogenetic analysis showed that the g20 sequence belongs to the cultivable cyanophage group (Cluster δ). Three pol sequences were closely related to cyanophage groups PG-Pol-I and PG-Pol-II that were observed in an alkaline paddy floodwater in Da'an, Jilin province, China; another pol sequence formed a unique clade. Conclusion: The g20 gene from cultivable cyanophages infecting Anabaena PCC7120 belongs to the Cluster δ, and the pol genes are closely related to those of paddy floodwater in Da'an, China.

Unraveling the genome structure of cyanobacterial podovirus A-4L with long direct terminal repeats.

The freshwater cyanobacterial virus (cyanophage) A-4L, a podovirus, can infect the model cyanobacterium Anabaena sp. strain PCC 7120 resulting in a high burst size and forming concentric plaques on its lawns. The complete genome sequence of A-4L was determined by the combination of high-throughput sequencing, terminal transferase-mediated polymerase chain reaction and restriction mapping. It contains 41,750 bp with 810 bp direct terminal repeats and 38 potential open reading frames. As compared with other cyanobacterial podoviruses in diverse ecosystems, the A-4L has the longest terminal repeat and shares similar genome organizations with freshwater members. Furthermore, phylogenetic analysis based on concatenated sequences of eight core proteins indicated that freshwater cyanobacterial podoviruses were clustered together and distinct from marine counterparts, suggesting a clear divergence in the cyanobacterial podovirus lineage between freshwater and marine ecosystems. Our findings uncover the unique genome structure of A-4L which contains long direct terminal repeats, and create the first model system to address knowledge gaps in understanding cyanobacterial virus-host interactions at the molecular level.

Genome-derived insights into the biology of the hepatotoxic bloom-forming cyanobacterium Anabaena sp. strain 90.

BACKGROUND: Cyanobacteria can form massive toxic blooms in fresh and brackish bodies of water and are frequently responsible for the poisoning of animals and pose a health risk for humans. Anabaena is a genus of filamentous diazotrophic cyanobacteria commonly implicated as a toxin producer in blooms in aquatic ecosystems throughout the world. The biology of bloom-forming cyanobacteria is poorly understood at the genome level. RESULTS: Here, we report the complete sequence and comprehensive annotation of the bloom-forming Anabaena sp. strain 90 genome. It comprises two circular chromosomes and three plasmids with a total size of 5.3 Mb, encoding a total of 4,738 genes. The genome is replete with mobile genetic elements. Detailed manual annotation demonstrated that almost 5% of the gene repertoire consists of pseudogenes. A further 5% of the genome is dedicated to the synthesis of small peptides that are the products of both ribosomal and nonribosomal biosynthetic pathways. Inactivation of the hassallidin (an antifungal cyclic peptide) biosynthetic gene cluster through a deletion event and a natural mutation of the buoyancy-permitting gvpG gas vesicle gene were documented. The genome contains a large number of genes encoding restriction-modification systems. Two novel excision elements were found in the nifH gene that is required for nitrogen fixation. CONCLUSIONS: Genome analysis demonstrated that this strain invests heavily in the production of bioactive compounds and restriction-modification systems. This well-annotated genome provides a platform for future studies on the ecology and biology of these important bloom-forming cyanobacteria.

Identification of a diagnostic marker to detect freshwater cyanophages of filamentous cyanobacteria.

Cyanophages are viruses that infect the cyanobacteria, globally important photosynthetic microorganisms. Cyanophages are considered significant components of microbial communities, playing major roles in influencing host community diversity and primary productivity, terminating cyanobacterial water blooms, and influencing biogeochemical cycles. Cyanophages are ubiquitous in both marine and freshwater systems; however, the majority of molecular research has been biased toward the study of marine cyanophages. In this study, a diagnostic probe was developed to detect freshwater cyanophages in natural waters. Oligonucleotide PCR-based primers were designed to specifically amplify the major capsid protein gene from previously characterized freshwater cyanomyoviruses that are infectious to the filamentous, nitrogen-fixing cyanobacterial genera Anabaena and Nostoc. The primers were also successful in yielding PCR products from mixed virus communities concentrated from water samples collected from freshwater lakes in the United Kingdom. The probes are thought to provide a useful tool for the investigation of cyanophage diversity in freshwater environments.

[Phagoresistance of filamentous cyanobacteria clones]. / Priroda fagorezistentnosti klonov nitchatykh tsianobacterii.

The paper deals with formation regularities of phagoresistant clones of cyanobacteria in two productive virus-cell systems: heterocyst cyanobacterium Nostoc linckia--cyanophage N-2, and mutant in heterocysts strain of Anabaena variabilis--cyanophage A-1. Frequency of spontaneous formation of phagoresistant clones of cyanobacterium N. linckia varies within 1.0-8.0 x 10(-6) per a cell, A. variabilis--5.0 x 10(-6)-7.0 x 10(-7) per cell. All the studied phagoresistant clones of N. linckia have identical morpho-cultural properties and do not differ from those of the initial culture. Phagoresistant clones of A. variabilis are presented by two groups. One of them, as to its properties, does not practically differ from the wild type culture. The second group differs considerably from the initial culture A. variabilis as to a number of characteristics--time of colonies appearance, their amount, length of trichomas, specific rate of growth and biomass accumulation. Spontaneous transfer of cyanophages to the culture liquid of clones resistant forms of cyanobacteria has not been revealed. Lysis of cells of the studied clones also was not induced under the effect of mytomycin C, thermal treatment and UV-irradiation. Cyanophage N-2 is not adsorbed by the cells of resistant cloned forms of cyanobacteria N. linckia. Only nonspecific adsorption takes place on the cells of phage-resistant clones of A. variabilis of both groups: about 20% of virions introduced in the adsorption mixture. Basing on the data obtained, it is supposed that phage-resistance of stable clones of filamentous cyanobacteria under the conditions of the given experiment is determined by the structure modification of cells receptors.

[Key enzymes of biosynthesis of amino acids of the glutamic series in the virus-cell system Anabaena variabilis + A-1]. / Kliuchevye fermenty biosinteza aminokislot glutaminovogo riada v virus-kletochnoi sisteme Anabaena variabilis +A-1.

The influence of cyanophage A-1 reproduction on glutamate dehydrogenase (GDG) and glutamine synthetase (GS) in A. variabilis cells was studied. It was determined that the both enzymes are intensified by 70% and 30%, accordingly. Isoenzymes of GDG and GS were isolated from native and infected cells of cyanobacteria, they had various physicochemical properties. It is concluded that cyanophage development causes the specific modification of cell enzymes.

Lipopolysaccharide dependence of cyanophage sensitivity and aerobic nitrogen fixation in Anabaena sp. strain PCC 7120.

Fox- mutants of Anabaena sp. strain PCC 7120 are unable to fix dinitrogen in the presence of oxygen. A fragment of the DNA of Anabaena sp. was cloned by complementation of a spontaneous Fox-, cyanophage-resistant mutant, R56, and characterized. Random insertion of transposon Tn5 delimited the complementing DNA to a 0.6-kb portion of the cloned fragment. Sequencing of this region and flanking DNA showed one complete open reading frame (ORF) similar to the gene rfbP (undecaprenyl-phosphate galactosephosphotransferase) and two partial ORFs similar to genes rfbD (GDP-D-mannose dehydratase) and rfbZ (first mannosyl transferase), all of which are active in the synthesis of the O antigen unit of the lipopolysaccharide (LPS) component of the outer membrane of gram-negative bacteria. In a transposon (Tn5-1087b)-induced, Fox-, cyanophage-resistant mutant, B14, the transposon was found within the same rfbP-like ORF. The three ORFs were insertionally inactivated with the omega cassette (P. Prentki and H. M. Krisch, Gene 29:303-313, 1984) or with Tn5::omega. Only the insertions in the rfbZ- and rfbP-like ORFs led to resistance to cyanophages A-1(L) and A-4(L) and to a Fox- phenotype. Electrophoretic analysis showed that interruption of the rfbZ- and rfbP-like ORFs resulted in a change in or loss of the characteristic pattern of the lengths of the LPS, whereas interruption of the rfbD-like ORF merely changed the distribution of the lengths of the LPS to one with a greater prevalence of low molecular weights. According to electron microscopy, interruption of the rfbP-like ORF may have led to aberrant deposition of the layers of the heterocyst envelope, resulting in increased leakage of oxygen into the heterocyst. The results suggest that modified LPS may prevent cyanophage infection of Anabaena sp. vegetative cells and the formation of a functional heterocyst envelope.