Single-Cell and Single-Molecule Analysis Deciphers
the Localization, Adhesion, and Mechanics of the Biofilm Adhesin LapA
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Abstract
The large adhesin protein LapA mediates
adhesion and biofilm formation
by <i>Pseudomonas fluorescens</i>. 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 <i>via</i> biofilm-inducing conditions (<i>i.e.</i>, phosphate-rich
medium) or deletion of the gene encoding the LapG protease (LapA+
mutant) increases the adhesion strength of <i>P. fluorescens</i> 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 <i>P. fluorescens</i> capable of colonizing diverse environments