PROJECT SUMMARY Biofilms are surface-attached microbial communities that pose a significant clinical problem, in part because biofilm cells are highly antibiotic tolerant. Device-related biofilm infections incur costs of >1 billion dollars annually. Moreover, lethal Pseudomonas aeruginosa biofilm infections are common in cystic fibrosis (CF) and other respiratory diseases. A better understanding of how microbial communities form is required to prevent or reverse biofilm formation. Our reported studies show that P. aeruginosa can detect surface contact via a pathway requiring Type IV pili (TFP) and a membrane-bound signaling complex that generates the second messenger cAMP. Our recent findings using cell tracking of entire communities at single-cell resolution and combined with a cAMP reporter lead to our Central Hypothesis: Surface sensing is predicated on cAMP-TFP- based memory, and does not occur by gradually increasing surface residence times of attached cells and their intracellular cAMP. Rather, the surface induces phase-shifted temporal waves of intracellular cAMP levels and TFP activity that constitute a `memory' of the surface. This memory, which is multigenerational and surprisingly robust, allows planktonic descendants of surface-exposed cells to adapt to the surface and increase surface cell populations orders of magnitude faster than their ancestors upon attachment. To understand this pivotal event, we will use population-level and single-cell analyses, combined with molecular genetic approaches within a rigorous biophysical theoretical framework, to explore the mechanistic underpinnings of these earliest events in biofilm formation. We will (1) test the hypothesis that surface contact induces phase-shifted waves of cAMP levels and TFP activity that encode a memory of the surface, allowing for surface adaptation that drastically modifies behavior of cells on surfaces, (2) test the hypothesis that surface sensing is transmitted via integrating TFP mechanical retraction, inner membrane dynamics of PilA, and the formation of an inner membrane PilJ-PilA complex required for cAMP signaling. Upon completion of the proposed studies, we will have established the mechanism by which P. aeruginosa senses and irreversibly attaches to a surface. Given that irreversible attachment is the first committed step in biofilm formation, our work uncovers a key aspect of bacterial biology.

PROJECT NARRATIVE Biofilms are surface-attached microbial communities that pose a significant clinical problem, and biofilm infections lead to hundreds of millions of dollars in healthcare costs annually. We aim to understand how these microbial communities form so that we can prevent or reverse biofilm formation, and focus specifically on Pseudomonas aeruginosa, which contributes to lethal infections in cystic fibrosis, ventilator-associated pneumonia and burn wounds.