Downloaded from www.microbiologyresearch.org by IP: 54.160.90.203 On: Tue, 21 Jun 2016 03:14:43 A proteomic approach to the identification of the major virion structural proteins of the marine cyanomyovirus S-PM2 Martha R. J. Clokie, 1 3 Konstantinos Thalassinos, 2 3 Pascale Boulanger, 3 Susan E. Slade, 2 Svetla Stoilova-McPhie, 2 Matt Cane, 2 James H. Scrivens 2 and Nicholas H. Mann 2 Correspondence Martha R. J. Clokie mrjc1@le.ac.uk 1 Department of Infection, Immunity and Inflammation, Maurice Shock Medical Sciences Building, University of Leicester, Leicester LE1 9HN, UK 2 Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK 3 U IBBMC-CNRS UMR 8619, Ba ˆ t. 430 – Universite ´ de Paris-Sud, F-91405 Orsay, Paris, France Received 26 December 2007 Revised 5 March 2008 Accepted 12 March 2008 In this study, an MS-based proteomics approach to characterizing the virion structural proteins of the novel marine ‘photosynthetic’ phage S-PM2 is presented. The virus infects ecologically important cyanobacteria of the genus Synechococcus that make a substantial contribution to primary production in the oceans. The S-PM2 genome encodes 236 ORFs, some of which exhibit similarity to known phage virion structural proteins, but the majority (54 %) show no detectable homology to known proteins from other organisms. Using public and in-house bioinformatics tools the proteome of S-PM2 was predicted and a database compatible with MS-based search engines was constructed. S-PM2 virion proteins were resolved by SDS-PAGE, excised, tryptically digested and analysed by LC-ESI-MS/MS. The resulting MS data were searched against the database. A parallel control study was undertaken on the well-characterized coliphage T4 in order to assess the sensitivity and efficiency of this approach. In total, 11 of the 15 S-PM2 proteins, predicted to be virion proteins by bioinformatics approaches, were confirmed as such, together with the identification of a further 12 novel structural proteins. In the case of T4, 24 of the 39 known virion structural proteins were identified, including the major tail-fibre proteins. This approach has wide-ranging applicability and can be applied to any novel organism whose genome encodes ORFs with few detectable homologies in the public databases. INTRODUCTION Interest in the study of bacteriophages has increased dramatically in recent years, largely as a result of the growing recognition of their ecological importance (Fuhrman, 1999; Suttle, 2007). Consequently, viruses infecting bacteria and archaea are currently being isolated and characterized. Genomic and transcriptomic studies are revealing novel features of the interactions between phages and their hosts that significantly expand the paradigms derived from the study of the classical phage–host systems. One area where the study of these novel phages is not progressing so rapidly is their structural biology. Genomic data can often be used to identify structural components of the virion on the basis of homology, but this approach is usually restricted in its success. An example of this can be seen in genomes of phages that infect archaea where many phage genomes contain no ORFs with similarity to known proteins (Prangishvili & Garrett, 2004). Thus, while it is very useful to have whole-genome sequences for phages, additional characterization is often necessary. To establish which ORFs in novel phage genomes encode structural proteins, an MS-based proteomics and bioinformatics approach was undertaken. The phage selected for this study was S-PM2 which infects marine cyanobacteria of the genus Synechococcus. These organisms, together with their close relatives from the genus Prochlorococcus, make a substantial contribution to the primary productivity of the oceans (Field et al., 1998). This phage was originally isolated from water collected off the coast of Plymouth, UK (Wilson et al., 1993). It is a lytic myovirus with an icosahedral head and contractile tail and its genome has been sequenced (Mann et al., 2005). The archetypal myovirus T4 devotes more than 40 % of its genome to the synthesis and assembly of one of the most complex virus particles known (Miller et al., 2003a). S- PM2 is morphologically similar to T4 and therefore it is likely that a similar number of proteins are involved in 3These authors contributed equally to this work. Microbiology (2008), 154, 1775–1782 DOI 10.1099/mic.0.2007/016261-0 2007/016261 G 2008 SGM Printed in Great Britain 1775