LapA of Pf0-1 belongs to a diverse family of cell surface associated bacterial adhesins that are secreted via the type-1 secretion system (T1SS). We previously reported that the periplasmic protease LapG cleaves the N-terminus of LapA at a canonical dialanine motif to release the adhesin from the cell surface under conditions unfavorable to biofilm formation, thus decreasing biofilm formation. Here, we characterize LapA as the first type 1 secreted substrate that does not follow the "one-step" rule of T1SS. Rather, a novel N-terminal element, called the retention module (RM), localizes LapA at the cell surface as a secretion intermediate. Our genetic, biochemical, and molecular modeling analysis support a model wherein LapA is tethered to the cell surface through its T1SS outer membrane TolC-like pore, LapE, until LapG cleaves LapA in the periplasm. We further demonstrate this unusual retention strategy is likely conserved among LapA-like proteins, and reveals a new subclass of T1SS ABC transporters involved in transporting this group of surface-associated, LapA-like adhesins. These studies demonstrate a novel cell surface retention strategy used throughout the Proteobacteria and highlight a previously unappreciated flexibility of function for T1SS. Bacteria have evolved multiple secretion strategies to interact with their environment. For many bacteria, the secretion of cell surface associated adhesins is key for initiating contact with a preferred substratum to facilitate biofilm formation. Our work demonstrates that uses a previously unrecognized secretion strategy to retain the giant adhesin LapA at its cell surface. Further, we identify likely LapA-like adhesins in various pathogenic and commensal Proteobacteria and provide phylogenetic evidence that these adhesins are secreted by a new subclass of T1SS ABC transporters.
Over the past decades, it has become clear that bacteria often prefer a sessile over a free‐swimming lifestyle. The transition from a mobile to an immobile, social lifestyle, called a biofilm, is induced in response to a variety of environmental stimuli, such as carbon or phosphate sources, linked to signaling via the second messenger cyclic‐di‐GMP (c‐di‐GMP). In gammaproteobacteria, c‐di‐GMP regulates cell adhesion to biotic and abiotic surfaces through the transmembrane Lap system that controls the surface presentation of large adhesin proteins. Signaling specificity is achieved through direct physical interaction between a c‐di‐GMP‐producing diguanylate cyclases (DGCs) and LapD, the c‐di‐GMP receptor central to the Lap system. To date, ten DGCs have been identified as regulators of LapD, affecting biofilm formation. However, how they differ functionally and the stimuli to which they respond are often not well understood. To shed light on the environmental sensing mechanism for DGC activation, we have determined the structure of the periplasmic domain of two DGCs in P. fluorescens, a model organism for biofilm research. These domains, belonging to the CACHE family of protein domains, protrude into the periplasm where they encounter their respective ligands, which correlates with their activation. By comparing the structures of CACHE domains and their ligand binding profiles, we identify molecular signatures that contribute to c‐di‐GMP signaling specificity, environmental sensing, and biofilm formation.
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