This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Although double-stranded DNA-containing bacteriophages and eukaryotic viruses all have the same core genes for morphogenesis, the length of bacteriophage genomes varies from 20 Kb to 670 Kb;1,200 Kb eukaryotic virus genomes have been found. The long-genome bacteriophages are of interest for understanding how and why complexity evolves in biological systems. The data indicate a systematic under-sampling of long-genome bacteriophages during isolation from the environment. Serwer recently has found procedures to more comprehensively isolate long-genome bacteriophages. A result was the isolation of bacteriophage Phi8-36, a virulent double-stranded DNA bacteriophage that, however, clears bacterial cultures only when grown in dilute agarose gels. This bacteriophage has atypical tail fibers that appear to be corkscrew-shaped when observed by electron microscopy. Fluorescence microscopy analysis of this phage revealed that it also (1) partitions to one of two liquid phases that form in part because of the presence of the bacteriophage particles and (2) forms aggregates that range in size from 3-20 particles to hundreds of bacteriophage particles. Thus, the data indicate that it is programmed for complex interactions in the extracellular environment. Our sequencing of the 218.948 Kb genome (6.479 Kb terminal repeat) revealed that, while it had the classical core genes for virus morphogenesis, it also had many others;55 total proteins were found in its capsid by SDSPAGE/mass spectrometry. All genes were highly diverged from known genes. This is twice the number for bacteriophage T4, the most complex bacteriophage previously studied Thomas et al., 2007). The long-range objective of the proposal is to determine the reason(s) that double- stranded DNA bacteriophages in the Phi8-36 class have numerous bacteriophage particle- associated proteins that are not classical bacteriophage/virus proteins. The working hypothesis is that these proteins were selected to optimize complex interactions of bacteriophage particles in the environment. Testing this hypothesis will provide a leading edge prototype of the evolution of biological complexity. Understanding both environmental interactions and the genomes of these bacteriophages are also important for (1) constructing models for the evolution of the bacterial hosts, (2) optimizing bacteriophage therapy of infectious disease, (3) analyzing vertical gene descent with minimal interference from horizontal gene transfer, (4) developing improved, large capacity cloning and display vectors, including those useful for both DNA and protein vaccines and (5) setting standards for conducting controlled evolution. We propose to use cryo-electron microscopy to provide a basis in detailed structure for determining the function of the 'extra'proteins. To understand the function of the 'extra'capsid proteins, we will perform either mutagenesis or controlled evolution and then compare genotype to phenotype for the modified bacteriophages. We propose to make cryo-electron microscopy-derived structure a major aspect of the phenotype. We will also compare the structure of Phi8-36 to the structure of other known corkscrew fibered bacteriophages.
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