Grant: Discovery Grants Program - Individual

This research program will explore the hypotheses that adhesion proteins (adhesins) are the first points of contact between bacteria and substrates they colonize and on which they may form biofilms; that the ligand-binding domains of the adhesins determine the niches inhabited by the bacteria; and that adhesins have a role in both adhesion to a surface and cohesion to each other or to the resulting biofilm. Having determined the 3-D structures of the ligand-binding domains and identified their ligands, we will explore ways to interrupt or modify these interactions, and to engineer changes to adhesins to alter their binding locations and partners. For this study we will focus on the "Repeats-in-Toxin" (RTX) adhesins that are widespread in Gram-negative bacteria, including many environmentally interesting and important species. The bacterial adhesion systems we are currently investigating include Pseudomonas fluorescens, a beneficial colonizer of plant roots; Marinomonas primoryensis that binds to ice and diatoms; the oil degrader, Marinobacter hydrocarbonoclasticus, which forms transient biofilms at the interface between water and oil as it metabolizes the latter; and Aeromonas hydrophila that is a fish pathogen. Their adhesins are gigantic single proteins, often coded by the largest open reading frame in the bacteria. Work funded during the last 5+ years on my NSERC DG revealed that two variable regions lying between the adhesin termini are crucial for their function. One is the extender region that determines the reach of the adhesin. The other is the ligand-binding region near the far end of the adhesin that can house one or more ligand-binding domains responsible for surface attachment. We estimate there are well over a dozen different types of these domains used in various combinations in different bacteria, only some of which have homologues in the present databases. We will continue to discover and characterize new adhesin ligand-binding domains, solve their crystal structures, identify their ligands and research how they cooperate to define the specific niche inhibited by the host bacterium. To address our hypotheses using adhesion engineering, we have two systems in play where we can swap ligand-binding domains and alter the surfaces bound by bacteria. One is a scaled down version of the ice-binding adhesin of M. primoryensis expressed from a plasmid in E. coli. The other is the 18-kDa N-terminal domain of Pseudomonas borealis ice nucleation protein that has been used extensively in E. coli as a surface display system to place enzymes and antigens on the bacterial surface. Twenty-one HQPs (6 MSc/PhD + 15 undergraduates) will be trained in this 5-yr program for work in the Canadian economy. The knowledge we discover on how to promote colonization by beneficial bacteria and block binding by harmful bacteria will benefit agriculture, aquaculture, and environmental remediation, and inform microbiological research.