Project Summary The transmission of macromolecules across biological membranes is a fundamental process in all cells. In the earliest studies of genetic exchange in bacteria dating back to the 1940’s, the F plasmid (then termed ‘sex factor’) was shown to self-transfer and, through recombination, can mediate the transfer of the entire E. coli chromosome to recipient bacteria. In the ensuing ~80 years, studies have established the broad medical importance of F and other mobile genetic elements (MGEs) in shaping of bacterial genomes and as vectors for dissemination of antibiotic resistance and other fitness traits among bacterial populations. MGEs also encode conjugative pili or other cell surface adhesins, which promote intercellular contacts necessary for DNA transfer and establishment of robust, antibiotic-resistant biofilm communities. MGEs are transmitted intercellularly through nanomachines termed type IV secretion systems (T4SSs). The T4SSs are present in most bacterial species, where they have functionally diversified into two large subfamilies, the DNA transfer or conjugation systems and the ‘effector translocators’ that deliver effector proteins into eukaryotic host cells during infection. In our 33 years of investigations, my group has identified many mechanistic and architectural features of these nanomachines, including the first view of the translocation route for a DNA substrate through a T4SS. We have consistently implemented emerging technologies and established collaborations to diversify our approaches, which is especially evident from this cycle of MIRA-supported research. Collaboratively, we solved structures of 4 model T4SSs in their native contexts of the bacterial cell envelope by in situ cryoelectron tomography (CryoET), and we solved a structure of a large subassembly of the F-encoded T4SS at near-atomic resolution by single-particle cryoelectron microscopy (spCryoEM). Independently, we pioneered studies of F pilus dynamics by labeling Cys-derivatized pilins with fluorescent maleimide conjugates, systematically deciphered contributions of F-encoded machine subunits to assembly of the translocation channel and F pilus, and probed functions of substrate processing factors and surface adhesins in early and late stages of substrate transfer. Moving forward, we will address three major gaps in our knowledge of these dynamic nanomachines: 1) How are substrates recruited to and delivered into and through the T4SS channel? 2) How do T4SSs elaborate conjugative pili and for what biological ends? 3) How do T4SSs promote formation and disassembly of ‘mating junctions’ and how are these junctions architecturally configured? We will continue to use powerful in vivo biochemical approaches, such as site-directed photocrosslinking, in combination with state-of-the-art super- resolution fluorescence microscopy, in situ CryoET, and spCryoEM approaches to further probe T4SS structure-function relationships. We anticipate our multidisciplinary...