Grant: Discovery Grants Program - Individual

Bacteria have evolved sophisticated adaptations to colonize specific niches. They can behave as multicellular masses that cooperate with each other to maximize their success. As biofilms they are remarkably resistant to eradication. Here we are studying large outer membrane proteins (adhesins) that function in both adhesion to an extracellular matrix and self-adhesion to form biofilms. One of several example we are studying is a 1.5-MDa adhesin from a marine bacterium (Marinomonas primoryensis) found under sea ice. At its C-terminal end it has a Type I secretion sequence common to RTX proteins and next to it an ice-binding domain. The other end attaches to the bacterial outer membrane. In between are ~120 identical repeats of an IgG-like domain that project the ice-binding domain away from the bacterial surface. Tandem arrays of the IgG-repeats bind to each other in an antiparallel fashion. Thus the adhesin not only binds the bacteria to ice but also links bacteria together in clusters to strengthen their adhesion to ice. Equally exciting is the role of a sugar-binding domain adjacent to the ice-binding domain that may serve to help bind the adhesin to its neighbours in the growing biofilm. These RTX adhesins are widely distributed in bacteria. One we will study is from the magnetotactic bacterium, Magnetospirillum magnetotacticum. These bacteria are well known for containing magnetic crystals that help the host to find their optimal niche of low (but essential) oxygen levels. We suggest that the magnetotactic bacteria self-adhere through their adhesins and cooperate in moving to the optimal niche. Another target bacterium is Marinobacter hydrocarbonoclasticus, which is able to form biofilms at the interface between oil and water to degrade the oil. Here we will use these bacteria as model organisms to develop strategies for disrupting self-association in the early phases of biofilm formation. We will follow bacterial association in custom designed microfluidics chambers monitored by microscopy. The microfluidics apparatus will allow us to change media, form gradients of attractants and repellents, and observe bacterial associations with substrate like ice, oil droplets, and cellulose. An integral part of this proposal will be the structural analysis of each domain and domain combinations used in adhesion. Recombinant proteins will be produced for structural and biophysical analysis. Functional analysis will involve mutagenesis, domain swap and deletion experiments, and transfer of adhesin genes to other hosts. These are all techniques and procedures that our lab excels in and will be the basis for the training of our three graduate students (T.Vance, C. Stevens and J. Arora) in our bacterial adhesin program. By the time they graduate they will have command of these biochemistry and structural biology methodologies and the ability to apply them to solve complex biological questions.