Advisor(s)

Jacqueline M. Piret

Contributor(s)

Veronica S. Godoy-Carter, Kostia Bergman, Slava S. Epstein, Jonathan King

Date of Award

2012

Date Accepted

4-2012

Degree Grantor

Northeastern University

Degree Level

Ph.D.

Degree Name

Doctor of Philosophy

Department or Academic Unit

College of Science. Department of Biology.

Keywords

biology, microbiology, virology, bacteriophage, cyanobacteria, cyanophage, procapsids, Syn5

Subject Categories

Cyanobacteria - Genetics, Bacterial genetics

Disciplines

Biology | Microbiology

Abstract

Syn5 is a marine cyanophage, which is propagated under laboratory conditions on the marine cyanobacterial strain Synechococcus sp. WH8109. Its genome has high homology with enteric bacteriophage T7 but it also contains several genes encoding protein sequences with no matches to phage proteins in the NCBI database. Cryo-EM images of Syn5 revealed a novel structure, a so-called horn, on the vertex opposite the tail.

Synchronized, exponentially growing host cultures were cultivated and the one-step growth curve of Syn5 was performed. Syn5 exhibited a short life cycle with an eclipse period of about 43 min and a latent phase of about 60 min with a burst size of 20-30 particles per cell at 28ºC. SDS-PAGE and Western analysis of cell lysates at different times after infection showed the synthesis of the major virion proteins and their increase as the infection progressed.

Most of the genes of the novel Syn5 proteins as well as the coat, scaffolding and portal proteins were cloned, expressed in E. coli and purified, with the exception of the coat. The coat protein was insoluble when expressed on its own and its co-expression with the putative Syn5 scaffolding protein did not improve its solubility. The recombinant portal, coat and scaffolding proteins exhibit properties similar to those of homologs in other bacteriophages, especially to their homologs in T7. The characteristics of the recombinant novel proteins gp53 and gp54 suggest that they may be capable of polymerization.

Immunogold labeling of infectious Syn5 particles with polyclonal antibodies raised against novel proteins strongly suggests that gp58 is positioned on the capsid head, possibly building the decoration knobs, while gp53 and gp54 contribute to the horn structure.

Treatment of Syn5 infectious particles with polyclonal antibodies raised against novel proteins gp58, gp53, and gp54 did not affect the ability of Syn5 to infect host cells while anti-Syn5 antibodies raised against whole virus particles inactivated the phage.

Western blots of Syn5-infected cell lysates showed that the putative scaffolding protein (gp38) is expressed during infection. Particles lacking DNA, but containing the coat and scaffolding proteins, were purified from Syn5-infected cells, about 50 min after infection, using CsCl centrifugation followed by sucrose gradients. Electron microscopic images of the purified particles showed shells lacking condensed DNA, but filled with protein density, presumably scaffolding protein.

The findings described here strongly suggest that cyanophages such as Syn5 form infectious virions through 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 was probably inherited by enteric phages from more ancient bacteriophages including cyanophages.

Document Type

Dissertation

Rights Holder

Desislava Andreeva Raytcheva


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