Genes and structural proteins of the phage SYN5 of the marine cyanobacteria, Synechococcus

• Main Author: Pope, Welkin Hazel
• Other Authors: Jonathan King.
• Format: Others
• Language: English
• Published: Massachusetts Institute of Technology 2008
• Subjects: Joint Program in Biological Oceanography.; Biology.; Woods Hole Oceanographic Institution.; Cyanobacteria Physiology
• Online Access: http://dspace.mit.edu/handle/1721.1/39190; http://hdl.handle.net/1721.1/39190

Thesis (Ph. D.)--Joint Program in Biological Oceanography (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), 2005. === Includes bibliographical references (p. 157-171). === Bacteriophage have been proposed to be the most abundant organisms on the planet, at an estimated 10³¹ particles globally (Hendrix et al., 1999). The majority of bacteriophage isolates (96%) are double-stranded DNA tailed phages (Caudovirales). These phages possess a distinctive icosehedral head, with a protein tail structure protruding from a single vertex. This organelle determines host specificity and provides the mechanism of passage of the phage genome into the host cell. Phages infecting differing microbial hosts may have access to a global pool of genes, albeit at different levels. Marine cyanobacteria of the genera Prochlorococcus and Synechococcus are numerically dominant photosynthetic cells in the large oligotrophic gyres of the open oceans, and contribute an estimated 30% to the oceanic photosynthetic budget. Cyanophages have been isolated which propagate on many strains of Synechococcus and Prochlorococcus. Cyanophages can effect community structure and succession through lytic infection of their hosts, and have implications in lateral gene transfer, mediated through lysogeny, mixed infections, pseudolysogeny, and transduction. === (cont.) The broad host ranges (between genera) observed in some phages indicates that lateral gene transfer is not confined to cells of the same strain. These phage/host interactions begin by host recognition by the tail of the infecting phage. Few studies have examined the structural proteins of cyanophage, partially due to the lack of a robust protocol for the growth and purification of phage particles. Cyanophage Syn5 is a short-tailed phage isolated from the Sargasso Sea by Waterbury and Valois (1993) which infects Synechococcus strain WH8109. Methods of growing the host cells and the phage, and concentrating the phage by PEG precipitation were developed. These methods led to highly concentrated purified phage stocks, to titers of 1012 particles/ml. Preliminary characterization of the growth of Syn5 gave a burst size of approximately 30 phage/cell and a lytic period of approximately 10 hours when inoculated into exponentially growing host cells acclimated to a temperature of 26⁰C and a light intensity of 50[mu]E m⁻² s⁻¹. Isolation of the phage nucleic acid yielded dsDNA molecules of approximately 40kb. The Syn5 particles were comprised of twelve structural proteins, as determined by SDS-PAGE. === (cont.) The most intense band on the gel was assigned to the capsid protein of Syn5 ([approx.] 35kDa). However, it was not possible to distinguish putative tail proteins via this method. Purified Syn5 particles were sent to the Pittsburgh Bacteriophage Institute for genome sequencing. The completed Syn5 genome was 46,214 bp long with a 237bp terminal repeat. Annotation of the completed Syn5 genome identified 61 putative ORFs, and revealed that Syn5 appeared closely related to the enteric phage T7 and cyanophages P-SSP7 and P60, as determined by gene similarity and synteny, although the genome was [approx.] 10kb longer than T7. Syn5 appeared to possess a more extensive DNA replisome that T7, containing copies of genes that encoded proteins of known T7 host co-factors, such as thioredoxin, utilized by the T7 DNAP. Several large ORFs were identified between the gene encoding the putative tail fiber and the gene encoding the putative terminase. These ORFs encoded proteins similar to some fibrous sequences within the NCBI non- redundant (nr) gene sequence database as of March, 2005; but had unknown functions within the phage. Unlike other recently sequenced cyanophages, SynS did not contain any photosynthetic genes. === (cont.) The structural proteins of SynS, as visualized by SDS-PAGE, were characterized by mass-spectroscopy and N-terminal sequencing. This allowed the assignment of sequences to putative ORFs within the Syn5 genome. The Syn5 particle was comprised of eleven discreet protein chains of molecular weight 152kDa, 139kDa, 99kDa, 90kDa, 66kDa, 60kDa, 47kDa, 35kDa, 22kDa, 21kDa, and 16kDa. The identified proteins included the portal, capsid, two tail tube proteins, and three internal virion proteins. Each of the genes encoding these proteins were found in the same gene order in the Syn5 genome as the corresponding genes were ordered in the T7 genome. There were three unidentifiable proteins within the particle (66kDa, 47kDa, and 16kDa). These mapped to the area of the SynS genome between the gene encoding the putative tail fiber and the gene encoding the putative terminase. No minor capsid or decorative capsid proteins were detected. The copy numbers of the corresponding protein chains were similar to those known for T7, with the exception of the tail fiber, which was present at a number of three chains per particle in comparison to T7's eighteen per particle. === (cont.) Polyclonal antibodies were raised against Syn5 particles. A Western blot with these antibodies showed that the tail fiber and the two unknown fibrous sequences were highly antigenic. This evidence implies that the unknown structures may act as host recognition proteins in addition to the tail fiber. Characterization of these novel proteins may provide insight to the host recognition abilities of cyanophages. An additional study was also carried out, investigating the high temperature limit of the growth of phage P22. The results revealed that the production of infectious particles was limited by the temperature sensitivity of the folding and assembly of the P22 tailspike protein. This work has been published and is included in the Appendix. === by Welkin Hazel Pope. === Ph.D.