Structural and functional analyses of proteins involved in DNA uptake and competence-associated type IV pilus

Abstract

Natural competence plays an important role in horizontal gene transfer and therefore makes a significant contribution to bacterial adaptive evolution. Natural competence in many Gram-negative bacteria is associated with type IV pili and specialized competence pili have been shown to mediate DNA uptake. Previous work on the naturally competent Thermus thermophilus has identified a competence-associated locus containing five ORFs; mutational analyses revealed that pilA1-3 and comZ mutants are impaired in natural transformation, although piliation is maintained. This project aimed to study the structures and interactions of these specialized competence pilins. The competence-associated ComZ was shown to bind with high affinity in vitro to another competence-associated pilin, PilA2. Two other competence-associated pilins, PilA1 and PilA3 were also examined for binding to ComZ but no interactions could be established. ComZ was shown to bind dsDNA by EMSA and the gradual increase in an electrophoretic shift with concentrations of ComZ suggested multiple, relatively weak binding sites on the DNA. The crystal structures of ComZ and PilA2 were determined by using X-ray crystallography. ComZ consists of a type II secretion pseudopilin-like domain and an unusually large beta-solenoid domain. The segmental mobility between the two domains is constrained by extensive loop regions; this could play a part in the proposed role of ComZ, in complex with PilA2, as a tip DNA receptor of a competence-associated pilus for DNA binding in natural transformation. ComEC, a specialized channel protein for DNA translocation is required for transformation and is indispensable for the uptake of DNA across the cytoplasmic membrane. ComEC is highly conserved and its homolog can be found in various naturally transformable bacteria. Based on our conserved domain search, ComEC from T. thermophilus was found to be composed of three domains: an N-terminal DUF4131 domain, a competence domain, and a C-terminal domain belongs to the metallo-beta-lactamase superfamily. The C-terminal domain of ComEC was expressed, purified, and its interaction with DNA was studied by EMSA, in which it was shown to degrade dsDNA. From thermofluor assays, the metallo-beta-lactamase-like C-terminal domain of ComEC was shown to bind zinc ions. We propose that the domain binds two zinc ions per molecule as ligands, possibly acting as intermediates for the protein to bind and degrade a phosphate-containing molecule, such as DNA via a zinc-mediated catalysis mechanism, similar to a mechanism adopted by metallo-beta-lactamases.

Date of Award	21 Dec 2018
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jeremy Derrick (Main Supervisor)