Stimulation of the <i>Caulobacter crescentus</i> surface sensing pathway by deletion of a specialized minor pilin-like gene

ABSTRACT Bacteria colonize surfaces through complex mechanisms of surface sensing. Pili are dynamic bacterial appendages that play an important role in this process. In Caulobacter crescentus , the tension on retracting, surface-bound pili triggers the rapid synthesis of holdfast, an adhesive that permanently attaches cells to surfaces. However, the mechanosensory elements in pilus-mediated surface sensing remain unknown. In this study, we used a genetic screen to isolate mutants with altered pilus dynamics, to identify genes potentially involved in pilus-mediated surface sensing. This screen identified cpaL , whose deletion led to reduced piliation levels, but surprisingly also to increased holdfast production and attachment—an unexpected finding given the importance of pili in these processes. Interestingly, this phenotype was absent under conditions that restrict the surface-sensing pathway of holdfast production. Furthermore, when pilus retraction was blocked to artificially simulate surface contact, ∆ cpaL cells did not show increased holdfast production as seen in wild-type cells. Together, these observations suggest that loss of cpaL stimulates surface sensing even in the absence of mechanical cues, consistent with a negative regulatory role for CpaL in the surface-sensing pathway of holdfast production in C. crescentus . Structural predictions suggest that CpaL is a minor pilin fused to a mechanosensitive von Willebrand factor type A (vWA) domain, which can be accommodated at the pilus tip alongside other minor pilins. These results collectively position CpaL as a strong candidate for the mechanosensory element in pilus-mediated surface sensing in C. crescentus . IMPORTANCE Surface sensing allows bacteria to colonize surfaces and form biofilms, with wide-ranging implications for bacterial survival, ecology, and human health. In Caulobacter crescentus , tight adherence (Tad) pili play an important role in surface sensing and attachment, however the molecular mechanisms of pilus-mediated mechanosensing remain unknown. Here, we demonstrate that CpaL, a potential pilus tip mechanosensory protein, could be a major regulatory element controlling Tad pilus-mediated surface attachment and colonization in C. crescentus . Specifically, CpaL plays a regulatory role in holdfast synthesis upon surface contact. By identifying CpaL as a key player in surface recognition, our work offers valuable insights into the mechanisms of bacterial adhesion.