Reconstructing the Neisseria Type IV Pilus System in E.coli

Abstract

Type IV Pili, long, thread-like structures found on the surface of many species of bacteria, are important virulence factors involved in motility, DNA/phage uptake, biofilm formation, and adhesion. Energy for the system is supplied by a set of cytoplasmic, hexameric ATPases which interact with proteins within the bacterial membrane to traffic pilin monomers to and from the pilus. The identity of these proteins and how they cooperate to transmit the energy of ATP hydrolysis is yet unclear. Up to fifteen proteins are often considered necessary for pilus function; however, as few as six proteins may be sufficient for pilus assembly--the rest being involved in the disassembly process and/or regulation of the system. To better understand the assembly mechanism and to definitively identify the components of the assembly apparatus, a library of plasmids containing from one to nine component genes of the Type IV Pilus system from Neisseria gonorrhoeae has been constructed using a combinatorial assembly method. Expression of these sets of proteins in non-piliated E. coli is currently being tested to identify those sufficient for pilus assembly and to enable further mechanistic studies.