Biological and structural diversity of bacterial type IV secretion systems

PROJECT SUMMARYThe transmission of macromolecules across biological membranes is a fundamental process in all cells. In theearliest studies of genetic exchange in bacteria dating back to the 1940's, the F plasmid (then termed `sexfactor') was shown to self-transfer and, through recombination, mediate the transfer of the entire E. colichromosome to recipient bacteria. In the ensuing ~75 years, studies established the broad medical importanceof F and other mobile genetic elements (MGEs) in the shaping of bacterial genomes and as vectors fordissemination of antibiotic resistance and other fitness traits among bacterial populations. MGEs also encodeconjugative pili or other cell surface adhesins, which promote intercellular contacts necessary for DNA transferand establishment robust, antibiotic-resistant biofilm communities. MGEs are transmitted intercellularly throughnanomachines termed type IV secretion systems (T4SSs). The T4SSs are present in most if not all bacterialspecies, where they have functionally diversified into two large subfamilies, the DNA transfer or conjugationsystems and the `effector translocators' that translocate effector proteins into eukaryotic host cells as a criticalfeature of infection processes. Over the past 27 years, my group has used molecular, genetic and biochemicalapproaches to identify many mechanistic and architectural features of T4SSs, including the first view of thetranslocation route for a DNA substrate through a T4SS. We have consistently implemented emergingtechnologies, and just within the past 1ｽ years we began to solve T4SS structures at unprecedentedresolution by in situ cryoelectron tomography (CryoET). These new structures are significantly advancing thefield, but also are raising important new questions relating to underlying mechanisms and signals governing i)assembly of envelope-spanning T4SS channels and conjugative pili, ii) early-stage substrate recruitment andprocessing reactions, and iii) establishment of direct contacts (mating junctions) with bacterial and eukaryoticcells. Moving forward, we will address these fundamental questions by (1) continuing to solve novel structuresencoded by the E. coli F T4SS using in situ CryoET, biochemical fractionation, super-resolution fluorescencemicroscopy, and single-particle CryoEM, (2) defining contributions of the newly visualized ATPase energycenter positioned at the channel entrance in binding and unfolding substrates and dissociating accessoryfactors using in vivo and in vitro biochemistry and ultrastructural approaches, (3) exploring the roles ofconjugative pili and cell surface adhesins in formation and disassembly of mating junctions using cytological,biochemical and biophysical approaches, and (4) exploiting our development of distinct model systems toidentify mechanistic themes and specialized mechanisms. We will continue to draw on the expertise of ourclose collaborations for a `team-science' and multidisciplinary focus. Our studies will generate important newinsights into the architecture, biogenesis, and mechanism of action of the T4SS superfamily. These findingswill lead to major paradigm shifts in this field, and set the stage for design of intervention therapies.