Visualizing virus assembly intermediates inside marine cyanobacteria.

Cyanobacteria are photosynthetic organisms responsible for ∼25% of organic carbon fixation on the Earth. These bacteria began to convert solar energy and carbon dioxide into bioenergy and oxygen more than two billion years ago. Cyanophages, which infect these bacteria, have 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 electron cryo-tomography (cryoET). This imaging modality yields dramatic 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 identify distinct Syn5 assembly intermediates. Our results indicate that the procapsid releases scaffolding proteins and expands its volume at an early stage of genome packaging. Later in the assembly process, we detected full particles with a tail either with or without an additional horn. The morphogenetic pathway we describe here is highly conserved and was probably established long before that of double-stranded DNA viruses infecting more complex organisms.

Two novel proteins of cyanophage Syn5 compose its unusual horn structure.

The marine cyanophage Syn5 can be propagated to a high titer in the laboratory on marine photosynthetic Synechococcus sp. strain WH8109. The purified particles carry a novel slender horn structure projecting from the vertex opposite the tail vertex. The genome of Syn5 includes a number of genes coding for novel proteins. Using immune-electron microscopy with gold-labeled antibodies, we show that two of these novel proteins, products of genes 53 and 54, are part of the horn structure. A third novel protein, the product of gene 58, is assembled onto the icosahedral capsid lattice. Characterization of radioactively labeled precursor procapsids by sucrose gradient centrifugation shows that there appear to be three classes of particles-procapsids, scaffold-deficient procapsids, and expanded capsids. These lack fully assembled horn appendages. The horn presumably assembles onto the virion just before or after DNA packaging. Antibodies raised to the recombinant novel Syn5 proteins did not interfere with phage infectivity, suggesting that the functions of these proteins are not directly involved in phage attachment or infection of the host WH8109. The horn structure may represent some adaption to the marine environment, whose function will require additional investigation.

Intracellular assembly of cyanophage Syn5 proceeds through a scaffold-containing procapsid.

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 ∼45 min, a latent phase of ∼60 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.

Cloning, characterization and heterologous expression of the aspartokinase and aspartate semialdehyde dehydrogenase genes of cephamycin C-producer Streptomyces clavuligerus.

Carbon flow through the lysine branch of the aspartate biosynthetic pathway is a rate-limiting step in the formation of cephamycin C, a broad spectrum beta-lactam antibiotic produced by Streptomyces clavuligerus. In this study, genes which encode the enzymes catalyzing the first two steps of the aspartate pathway, ask (aspartokinase) and asd (aspartate semialdehyde dehydrogenase), in S. clavuligerus NRRL 3585 were cloned and sequenced. Nucleotide sequencing and codon preference analysis revealed three complete open reading frames (ORFs). ORF2 starts within ORF1 and terminates by utilizing the same stop codon as ORF1, an arrangement typical of many ask genes. ORF3 is located 2 nucleotides downstream of ORF1,2. Database comparisons with these proteins identified ORF1 as the large (alpha) subunit of aspartokinase, ORF2 as the small (beta) subunit of aspartokinase and ORF3 as the aspartate semialdehyde dehydrogenase. The cloned genes were functionally expressed in auxotrophic Escherichia coli strains, CGSC5074 (ask(-)) and E. coli CGSC5080 (asd(-)), the two enzymes were partially purified from E. coli cell extracts and their kinetic parameters were determined. The effects of end product amino acids and diaminopimelic acid on the activity of Ask and Asd enzymes were also described.

Protruding knob-like proteins violate local symmetries in an icosahedral marine virus.

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.

Homologous expression of aspartokinase (ask) gene in Streptomyces clavuligerus and its hom-deleted mutant: effects on cephamycin C production.

In this study, the effect of homologous multiple copies of the ask gene, which encodes aspartokinase catalyzing the first step of the aspartate pathway, on cephamycin C biosynthesis in S. clavuligerus NRRL 3585 and its hom mutant was investigated. The intracellular pool levels of aspartate pathway amino acids accorded well with the Ask activity levels in TB3585 and AK39. When compared with the control strain carrying vector alone without any gene insert, amplification of the ask gene in the wild strain resulted in a maximum of 3.1- and 3.3-fold increase in specific, 1.7- and 1.9-fold increase in volumetric cephamycin C production when grown in trypticase soy broth (TSB) and a modified chemically defined medium (mCDM), respectively. However, expression of multicopy ask gene in a hom-deleted background significantly decreased cephamycin C yields when the cells were grown in either TSB or mCDM, most probably due to physiological disturbance resulting from enzyme overexpression and high copy number plasmid burden in an auxotrophic host, respectively.