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ABSTRACT During biofilm formation, the opportunistic pathogen Pseudomonas aeruginosa uses its type IV pili (TFP) to sense a surface, eliciting increased second-messenger production and regulating target pathways required to adapt to a surface lifestyle. The mechanisms whereby TFP detect surface contact are still poorly understood, although mechanosensing is often invoked, with few data supporting this claim. Using a combination of molecular genetics and single-cell analysis, with biophysical, biochemical, and genomics techniques, we show that force-induced changes mediated by the von Willebrand A (vWA) domain-containing, TFP tip-associated protein PilY1 are required for surface sensing. Atomic force microscopy shows that TFP/PilY1 can undergo force-induced, sustained conformational changes akin to those observed for mechanosensitive proteins like titin. We show that mutation of a single cysteine residue in the vWA domain of PilY1 results in modestly lower surface adhesion forces, reduced sustained conformational changes, and increased nanospring-like properties, as well as reduced c-di-GMP signaling and biofilm formation. Mutating this cysteine has allowed us to genetically separate a role for TFP in twitching motility from surface-sensing signaling. The conservation of this Cys residue in all P. aeruginosa PA14 strains and its absence in the ∼720 sequenced strains of P. aeruginosa PAO1 may contribute to explaining the observed differences in surface colonization strategies observed for PA14 versus PAO1. IMPORTANCE Most bacteria live on abiotic and biotic surfaces in surface-attached communities known as biofilms. Surface sensing and increased levels of the second-messenger molecule c-di-GMP are crucial to the transition from planktonic to biofilm growth. The mechanism(s) underlying TFP-mediated surface detection that triggers this c-di-GMP signaling cascade is unclear. Here, we provide key insight into this question; we show that the eukaryote-like vWA domain of the TFP tip-associated protein PilY1 responds to mechanical force, which in turn drives the production of a key second messenger needed to regulate surface behaviors. Our studies highlight a potential mechanism that may account for differing surface colonization strategies.

ABSTRACT The bacterial cell envelope is essential for viability, the environmental gatekeeper and first line of defense against external stresses. For most bacteria, the envelope biosynthesis is also the site of action of some of the most important groups of antibiotics. It is a complex, often multicomponent structure, able to withstand the internally generated turgor pressure. Thus, elucidating the architecture and dynamics of the cell envelope is important, to unravel not only the complexities of cell morphology and maintenance of integrity but also how interventions such as antibiotics lead to death. To address these questions requires the capacity to visualize the cell envelope in situ via high-spatial resolution approaches. In recent years, atomic force microscopy (AFM) has brought novel molecular insights into the assembly, dynamics, and functions of bacterial cell envelopes. The ultrafine resolution and physical sensitivity of the technique have revealed a wealth of ultrastructural features that are invisible to traditional optical microscopy techniques or imperceptible in their true physiological state by electron microscopy. Here, we discuss recent progress in our use of AFM imaging for understanding the architecture and dynamics of the bacterial envelope. We survey recent studies that demonstrate the power of the technique to observe isolated membranes and live cells at (sub)nanometer resolution and under physiological conditions and to track in vitro structural dynamics in response to growth or to drugs.