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, ?29, 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.
Bacteriophages have profound impacts on microbial communities, ecosystems, and human health, while phage infection initiation is the least understood aspect in phage biology. We combine traditional genetic and molecular biology with cutting-edge imaging techniques to provide novel mechanistic insights into phage adsorption and genome transfer into the host cytoplasm. The proposed in vivo study also has implications on nanotechnology, antibacterial therapeutics, and development of novel DNA translocation systems.
| Qin, Zhuan; Hu, Bo; Liu, Jun (2018) Visualizing Chemoreceptor Arrays in Bacterial Minicells by Cryo-Electron Tomography and Subtomogram Analysis. Methods Mol Biol 1729:187-199 |
| Farley, Madeline M; Tu, Jiagang; Kearns, Daniel B et al. (2017) Ultrastructural analysis of bacteriophage ?29 during infection of Bacillus subtilis. J Struct Biol 197:163-171 |
| Tu, Jiagang; Park, Taehyun; Morado, Dustin R et al. (2017) Dual host specificity of phage SP6 is facilitated by tailspike rotation. Virology 507:206-215 |
| Farley, Madeline M; Hu, Bo; Margolin, William et al. (2016) Minicells, Back in Fashion. J Bacteriol 198:1186-95 |
| Morado, Dustin R; Hu, Bo; Liu, Jun (2016) Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography. J Vis Exp :e53608 |
| Hu, Bo; Margolin, William; Molineux, Ian J et al. (2015) Structural remodeling of bacteriophage T4 and host membranes during infection initiation. Proc Natl Acad Sci U S A 112:E4919-28 |
| Hu, Bo; Morado, Dustin R; Margolin, William et al. (2015) Visualization of the type III secretion sorting platform of Shigella flexneri. Proc Natl Acad Sci U S A 112:1047-52 |
| Hu, Bo; Margolin, William; Molineux, Ian J et al. (2013) The bacteriophage t7 virion undergoes extensive structural remodeling during infection. Science 339:576-9 |
