SummaryCyanobacteria are photosynthetic organisms responsible for ~25% of organic carbon fixation on earth. These bacteria began to convert solar energy and carbon dioxide into bioenergy and oxygen billions of years ago. Cyanophages, which infect these bacteria, play an important role in regulating the marine ecosystem by controlling cyanobacteria community organization and mediating lateral gene transfer. Here we visualize the maturation process of cyanophage Syn5 inside its host cell, Synechococcus, using Zernike Phase Contrast (ZPC) electron cryo-tomography (cryoET) 1,2 . This imaging modality yields significant enhancement of image contrast over conventional cryoET and thus facilitates the direct identification of subcellular components, including thylakoid membranes, carboxysomes and polyribosomes, as well as phages, inside the congested cytosol of the infected cell. By correlating the structural features and relative abundance of viral progeny within cells at different stages of infection, we identified distinct Syn5 assembly Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms The averaged density maps of the procapsid, expanded capsid and the DNA-containing capsid have been deposited in the EBI under accession codes EMD-5742, EMD-5743, EMD-5744, EMD-5745, and EMD-5746, respectively. The authors declare no competing financial interests.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Fig. 1a-b) are roughly concentric, the thylakoid membrane does not fully enclose the inner compartment of the cell, nor does it seem to directly interact with the cell membrane. This differs from the organization seen in other cyanobacteria 9,10 . Cyanobacteria also contain carboxysomes, polyhedral compartments encapsulating enzymes for carbon fixation 11,12 . Each WH8109 cell has, on average, four or five carboxysomes, with diameters ranging from 920 to 1160Å (Fig. 1c). Ribosomes are abundant and widespread, forming numerous intracellular patches that contain polyribosomes (Fig. 1d). HHS Public AccessCyanophage Syn5 that infects WH8109 cells is a short-tailed icosahedral phage with a unique horn appendage at the vertex opposite to the tail 13 (Extended Data Fig. 2). Initial segmentation of our tomograms of infected cells identified Syn5 particles on the cell surface, floating in the extracellular medium, and Syn5 progeny inside the cell. Multiple full and empty phage particles are seen attached to the cell surface. Injection of viral DNA occurs at multiple sites on the bacterial envelope and does not appear to be a coordinated process. Fig. 1e shows a tubular density extending from the phage tail through the periplasm to the cytoplasm (Supplementary video 4), similar to observations in other phage-infected bacteria 14,15 . As infection progresses, increasing numbers of Syn5 phage progeny ...

Syn5 is a marine cyanophage that is propagated on the marine photosynthetic cyanobacterial strain Synechococcus sp. WH8109 under laboratory conditions. Cryoelectron images of this double-stranded DNA (dsDNA) phage reveal an icosahedral capsid with short tail appendages and a single novel hornlike structure at the vertex opposite the tail. Despite the major impact of cyanophages on life in the oceans, there is limited information on cyanophage intracellular assembly processes within their photosynthetic hosts. The one-step growth curve of Syn5 demonstrated a short cycle with an eclipse period of ϳ45 min, a latent phase of ϳ60 min, and a burst size of 20 to 30 particles per cell at 28°C. SDS-PAGE and Western blot analysis of cell lysates at different times after infection showed the synthesis of major virion proteins and their increase as the infection progressed. The scaffolding protein of Syn5, absent from virions, was identified in the lysates and expressed from the cloned gene. It migrated anomalously on SDS-PAGE, similar to the phage T7 scaffolding protein.Particles lacking DNA but containing the coat and scaffolding proteins were purified from Syn5-infected cells using CsCl centrifugation followed by sucrose gradient centrifugation. Electron microscopic images of the purified particles showed shells lacking condensed DNA but filled with protein density, presumably scaffolding protein. These findings suggest that the cyanophages form infectious virions through the initial assembly of scaffolding-containing procapsids, similar to the assembly pathways for the enteric dsDNA bacteriophages. Since cyanobacteria predate the enteric bacteria, this procapsid-mediated assembly pathway may have originated with the cyanophages.

Background: Cyanophages are dominant viruses in the ocean while little has been known on their nucleic acid metabolism. Results: The RNA polymerase of cyanophage Syn5 has been purified and characterized and the Syn5 promoters identified. Conclusion: The Syn5 RNA polymerase and promoters have unique and ocean-adapted features. Significance: The first characterized single-subunit RNA polymerase from marine organisms.

Marine viruses play crucial roles in shaping the dynamics of oceanic microbial communities and in the carbon cycle on Earth. Here we report a 4.7-Å structure of a cyanobacterial virus, Syn5, by electron cryo-microscopy and modelling. A Cα backbone trace of the major capsid protein (gp39) reveals a classic phage protein fold. In addition, two knob-like proteins protruding from the capsid surface are also observed. Using bioinformatics and structure analysis tools, these proteins are identified to correspond to gp55 and gp58 (each with two copies per asymmetric unit). The non 1:1 stoichiometric distribution of gp55/58 to gp39 breaks all expected local symmetries and leads to non-quasi-equivalence of the capsid subunits, suggesting a role in capsid stabilization. Such a structural arrangement has not yet been observed in any known virus structures.