Structural and Proteomic Analysis of ΦTE, a Phage Infecting Pectobacterium atrosepticum

Abstract

Bacteriophages are extremely diverse and are also far more numerous than any other organism on Earth. Despite this diversity, there still remain many unanswered questions regarding their structures, what (and how) forces direct morphological evolution, and structural characteristics such as the more specific interactions between the phage genome, proteome, and the final particle. One potential driving force in the evolution of phage morphology is CRISPR-Cas adaptive immunity found in bacteria. In order to contribute to knowledge of CRISPR-Cas as a potential driving force in phage morphological evolution, the aim was to solve the three dimensional structure of a relevant phage in high resolution and to obtain data on the phage proteome and tail for further analysis. To that end, phage isolation and purification techniques as well as cryo-electron microscopy techniques are employed so that a relevant phage could be obtained and viewed in its native, hydrated state. This then allows collection of several micrographs so that a three dimensional reconstruction of the native phage particle may be produced through the use of computer averaging tools. The bacteriophage reconstructed here, ΦTE, was found to have a T=13 capsid as well as a contracting tail structure indicating that it is a member of the Myoviridae family of Caudovirales phages. Proteomic and structural analysis of ΦTE, in conjunction with a previously sequenced genome, then revealed several key structural proteins of the bacteriophage, their relative abundance, as well as the peptides composing these proteins. Altogether, these results can be used in the future as a basis for experimentation toward understanding how CRISPR-Cas can potentially drive the evolution of phage morphology.