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 ...

Using resting cells and extracts of Streptomyces clavuligerus NP1, we have been able to convert penicillin G (benzylpenicillin) to deacetoxycephalosporin G. Conversion was achieved by increasing by 45؋ the concentration of FeSO 4 (1.8 mM) and doubling the concentration of ␣-ketoglutarate (1.28 mM) as compared with standard conditions used for the normal cell-free conversion of penicillin N to deacetoxycephalosporin C. ATP, MgSO 4 , KCl, and DTT, important in cellfree expansion of penicillin N, did not play a significant role in the ring expansion of penicillin G by resting cells or cell-free extracts. When these conditions were used with 14 other penicillins, ring expansion was achieved in all cases. Biosynthesis of cephalosporins in Cephalosporium acremoniumand Streptomyces clavuligerus proceeds through a biosynthetic pathway that includes expansion of the five-membered thiazolidine ring of the intermediate penicillin N into the sixmembered dihydrothiazine ring of deacetoxycephalosporin C (DAOC) (for reviews, see refs. 1 and 2). This step is catalyzed by the ␣-ketoglutarate-dependent dioxygenase, DAOC synthase (''expandase'') (3). In the fungus C. acremonium, the activity of expandase resides in a bifunctional enzyme (4) that catalyzes not only ring expansion but also the hydroxylation of the methyl group of DAOC to deacetylcephalosporin C whereas in the bacterium S. clavuligerus the two activities are associated with separate proteins (5, 6).We and others (3-10) have found that the enzyme, in cell-free extracts as well as after purification, has a very narrow substrate specificity and no detectable activity on readily available and inexpensive penicillins (such as penicillin G and V produced by Penicillium chrysogenum). Indeed, chemical ring expansion plus an enzymatic removal of the phenylacetyl side chain is used in industry to convert penicillin G into 7-aminodeacetoxycephalosporanic acid, an important intermediate for the manufacture of semisynthetic cephalosporins. However, this process requires several steps and is expensive and polluting. A simple biological route might replace the chemical process, requiring only two steps, i.e., ring expansion and enzymatic deacylation, thereby reducing costs and environmental problems. We now have identified the conditions that allow conversion of penicillin G to deacetoxycephalosporin G (DAOG; phenylacetyl-7-aminodeacetoxycephalosporanic acid). In this report, we describe the conversion of a number of penicillins, including penicillin G, into cephalosporins by using resting cells and cell-free extracts of the cephalosporin-deficient mutant S. clavuligerus NP1 (11). MATERIALS AND METHODSMicroorganism. S. clavuligerus NP1, a mutant producing only trace levels of cephalosporins (11), was used in this work. The absence of significant levels of cephalosporins in this strain facilitated detection of cephalosporins produced from added penicillins by ring expansion.Media and Culture Conditions. Mycelia were obtained by using 250-ml baffled flasks containing 40 ml of...