Bacteriophages are the most abundant biological entity in the biosphere and are responsible for much of bacterial
evolution. Most phages utilize elaborate tail machines to translocate their viral DNA and proteins into a host cell.
During the last grant period, we made considerable progress in documenting infection initiation from several
classic phages (T7, T4, P22, SP6, F29, and ¿). Our studies provided new molecular insights into the mechanisms
by which these tailed phages overcome the multiple barriers of the bacterial cell envelope and to deliver their
genetic material into the host cell cytoplasm. Here we focus entirely on T7, because a complete mechanistic
description of how T7 infection is initiated and how DNA translocation is effected remains elusive. The signal
triggering protein ejection is not known. How do the core proteins penetrate the outer membrane, and how is the
cytoplasmic membrane breached? How does the extended tail nanomachine function to deliver its cargo – the
phage genome – into a cell? Equally importantly, how are the conformational changes in the tail and fibers
coordinated so that the initial interaction of a phage with a susceptible cell almost inexorably leads to infection?
A completely unanticipated observation for any phage system is that T7 recruits the host F1FO ATP synthase at
the initiation of infection. Our central hypothesis is that T7 undergoes massive conformational changes to
facilitate adsorption, channel formation, and DNA translocation. Our collaborative experimental approach where
structural biology in situ is intimately coupled to genetics and physiology will address these fundamental
questions. Three specific aims are: (1) Dissect the structure and function of the T7 genome ejection machine;
(2) determine how the extended T7 tail spans the cell envelope; (3) illuminate mechanisms of adsorption and
recruitment of the F1FO ATP synthase.