Molecular Analysis of the Assembly of Bacterial Type IV Pili

Virginia Polytechnic Institute And State University, Blacksburg VA

Investigators

Abstract

Intellectual merit. Type IV pili (TFP) are bacterial surface fibers that have a range of functions including twitching and gliding motility, adhesion to cells and surfaces, and formation of microbial biofilms. In some metal-reducing bacteria, they function as nanowires capable of carrying an electric current. TFP are assembled by a protein complex spanning the inner membrane and the peptidoglycan of Gram(+) bacteria as well as the outer membrane of Gram(-) bacteria. The core assembly complex includes the pilin subunits themselves, an assembly ATPase, and an inner membrane protein; for Gram(-) bacteria, there is also an outer membrane secretin (channel) through which the assembled pilus passes. Many TFP also utilize a retraction ATPase to disassemble pili, which facilitates twitching motility and tight adherence to surfaces. The force generated by the retraction ATPase has been measured at >100 piconewtons, making it the strongest molecular motor known in biology. However, the mechanism by which the pilin monomers are polymerized into a growing pilus is still not well understood. This project will a comprehensive atomic-level model that can account for pilin polymerization in an energetically favorable fashion. The approach is to reconstitute a functional TFP assembly apparatus in lipid vesicles using purified proteins, derived from the Gram(+) bacterium Clostridium perfringens. Experiments will also measure the rate of pilin polymerization using fluorescently tagged proteins. The direct output from this project will be (1) identification of the minimal components that comprise a functional TFP assembly apparatus, (2) direct measurements of TFP assembly rate kinetics, and (3) measurement of the energy input (ATP) required to translocate pilin monomers from a hydrophobic membrane environment to another hydrophobic environment found in the core of the pilus filament. These results will have wide applications in microbial physiology and ecology, as well as nanosystems biology. Broader impacts. Graduate students will be heavily involved in this research project. In addition, minorities and other underrepresented individuals will be encouraged to participate as either graduate students or undergraduate research interns. The principal investigator will continue to participate in an institutional PREP program, which is also dedicated to recruiting and retaining members of underrepresented groups into graduate school and scientific careers. The investigator will continue training female graduate students and undergraduate students, which in the past have included one student with a disability. The investigator will incorporate findings from this research into course lectures and presentations local grammar schools. The intent is to get young people excited about biology and microbiology and to encourage them to pursue careers in science and math. Since bacterial pili play an essential role in many bacterial environments, the results will likely benefit society in such areas as agricultural safety practices, microbial ecology, geobiology, as well as nanotechnology applications for industry.