PROJECT SUMMARYBiofilms are surface-attached microbial communities that pose a significant clinical problem, in part becausebiofilm cells are highly antibiotic tolerant. Device-related biofilm infections incur costs of >1 billion dollarsannually. Moreover, lethal Pseudomonas aeruginosa biofilm infections are common in cystic fibrosis (CF) andother respiratory diseases. A better understanding of how microbial communities form is required to prevent orreverse biofilm formation. Our reported studies show that P. aeruginosa can detect surface contact via apathway requiring Type IV pili (TFP) and a membrane-bound signaling complex that generates the secondmessenger cAMP. Our recent findings using cell tracking of entire communities at single-cell resolution andcombined 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 theirintracellular cAMP. Rather, the surface induces phase-shifted temporal waves of intracellular cAMP levels andTFP activity that constitute a `memory' of the surface. This memory, which is multigenerational and surprisinglyrobust, allows planktonic descendants of surface-exposed cells to adapt to the surface and increase surfacecell populations orders of magnitude faster than their ancestors upon attachment. To understand this pivotalevent, we will use population-level and single-cell analyses, combined with molecular genetic approacheswithin a rigorous biophysical theoretical framework, to explore the mechanistic underpinnings of these earliestevents in biofilm formation. We will (1) test the hypothesis that surface contact induces phase-shifted waves ofcAMP levels and TFP activity that encode a memory of the surface, allowing for surface adaptation thatdrastically modifies behavior of cells on surfaces, (2) test the hypothesis that surface sensing is transmitted viaintegrating TFP mechanical retraction, inner membrane dynamics of PilA, and the formation of an innermembrane PilJ-PilA complex required for cAMP signaling. Upon completion of the proposed studies, we willhave established the mechanism by which P. aeruginosa senses and irreversibly attaches to a surface. Giventhat irreversible attachment is the first committed step in biofilm formation, our work uncovers a key aspect ofbacterial biology.