title: Single-cell and single-molecule analysis deciphers the localization, adhesion, and mechanics of the biofilm adhesin LapA.
abstract: The large adhesin protein LapA mediates adhesion and biofilm formation by Pseudomonas fluorescens. Although adhesion is thought to involve the long multiple repeats of LapA, very little is known about the molecular mechanism by which this protein mediates attachment. Here we use atomic force microscopy to unravel the biophysical properties driving LapA-mediated adhesion. Single-cell force spectroscopy shows that expression of LapA on the cell surface via biofilm-inducing conditions (i.e., phosphate-rich medium) or deletion of the gene encoding the LapG protease (LapA+ mutant) increases the adhesion strength of P. fluorescens toward hydrophobic and hydrophilic substrates, consistent with the adherent phenotypes observed in these conditions. Substrate chemistry plays an unexpected role in modulating the mechanical response of LapA, with sequential unfolding of the multiple repeats occurring only on hydrophilic substrates. Biofilm induction also leads to shortening of the protein extensions, reflecting stiffening of their conformational properties. Using single-molecule force spectroscopy, we next demonstrate that the adhesin is randomly distributed on the surface of wild-type cells and can be released into the solution. For LapA+ mutant cells, we found that the adhesin massively accumulates on the cell surface without being released and that individual LapA repeats unfold when subjected to force. The remarkable adhesive and mechanical properties of LapA provide a molecular basis for the "multi-purpose" adhesion function of LapA, thereby making P. fluorescens capable of colonizing diverse environments.
authors.affiliation: Institute of Life Sciences, Université catholique de Louvain , Croix du Sud, 1, bte L7.04.01, B-1348 Louvain-la-Neuve, Belgium.
authors.forename: Sofiane
authors.initials: S
authors.lastname: El-Kirat-Chatel
authors.forename: Audrey
authors.initials: A
authors.lastname: Beaussart
authors.forename: Chelsea D
authors.initials: CD
authors.lastname: Boyd
authors.forename: George A
authors.initials: GA
authors.lastname: O'Toole
authors.forename: Yves F
authors.initials: YF
authors.lastname: Dufrêne
mesh_terms: D018829:Adhesins, Bacterial / Q000032:analysis* / Q000378:metabolism*; D001422:Bacterial Adhesion; D018441:Biofilms / Q000254:growth & development*; D001696:Biomechanical Phenomena; D057927:Hydrophobic and Hydrophilic Interactions; D018625:Microscopy, Atomic Force; D011551:Pseudomonas fluorescens / Q000166:cytology / Q000502:physiology*
chemical_list: D018829:Adhesins, Bacterial