Probing Large Protein Adhesin Molecules on <I>Pseudomonas fluorescens</I> with Atomic Force Microscopy

Protein adhesins are an important type of surface structure, and are components of some Gram-negative bacterial outer membranes, such as Pseudomonas fluorescens. The LapA adhesin is a major surface protein on this bacterium, which is required for irreversible adhesion and the initiation of biofilm formation. Atomic force microscopy (AFM) was used to characterize surface structures of P. fluorescens Pf0-1. The wild-type and three genetically modified strains were studied, namely a strain consisting of a single cross-over knockout mutation disrupting the lapA gene, a strain consisting of a single cross-over knockout mutation disrupting the lapB gene, (LapA is maintained in the cytoplasm and not transported to the cell surface), and lapG (LapA is unable to be cleaved from the cell surface). AFM data was modeled using the Alexander de Gennes (A-dG) relation for steric repulsion. We calculated the equilibrium layer thickness of the surface structures as well as spacing between adhesins. The wild-type strain and the lapA and lapB mutants all showed similar spacing for surface proteins. The strain lacking LapG had the smallest spacing between molecules. This suggests that the absence of the LapG protease allowed the LapA protein to accumulate, thus decreasing the overall molecular spacing of the protein on the bacterial surface compared to the wild-type strain. We found that the lapG mutant strain of P. fluorescens behaved like a classical polymer brush, in which the spacing between molecules was very small (3.3 nm), which would allow intermolecular interactions between protein units. Recent work has shown that the lapG mutant has greater adhesion and biofilm formation than the wild-type, lapA, and lapB strains, and exhibits stiffer conformation of LapA due to higher protein density and aggregation. Taken together, our results and these recent studies support the finding that LapA adhesin conformation is related to irreversible bacterial