Simultaneous Force and Fluorescence Measurements of a Protein That Forms a Bond between a Living Bacterium and a Solid Surface

Abstract

All microbial biofilms are initiated through direct physical contact between a bacterium and a solid surface, a step that is controlled by inter-and intramolecular forces. Atomic force microscopy and confocal laser scanning microscopy were used simultaneously to observe the formation of a bond between a fluorescent chimeric protein on the surface of a living Escherichia coli bacterium and a solid substrate in situ. The chimera was composed of a portion of outer membrane protein A (OmpA) fused to the cyan-fluorescent protein AmCyan. Sucrose gradient centrifugation and fluorescent confocal slices through bacteria demonstrated that the chimeric protein was targeted and anchored to the external cell surface. The wormlike chain theory predicted that this protein should exhibit a nonlinear force-extension "signature" consistent with the sequential unraveling of the AmCyan and OmpA domains. Experimentally measured force-extension curves revealed a unique pair of "sawtooth" features that were present when a bond formed between a silicon nitride surface (atomic force microscopy tip) and E. coli cells expressing the OmpA-AmCyan protein. The observed sawtooth pair closely matched the wormlike chain model prediction for the mechanical unfolding of the AmCyan and OmpA substructures in series. These sawteeth disappeared from the measured force-extension curves when cells were treated with proteinase K. Furthermore, these unique sawteeth were absent for a mutant stain of E. coli incapable of expressing the AmCyan protein on its outer surface. Together, these data show that specific proteins exhibit unique force signatures characteristic of the bond that is formed between a living bacterium and another surface.A nearly universal trait of bacteria is their ability to attach to solid surfaces. Attached cells often form a biofilm, which is defined as a structured community of bacteria enclosed in a self-produced polymeric matrix adherent to an inert or living surface (6). Abundant evidence suggests that proteins on the outer wall of a bacterium play a critical role in initiating contact with another surface. Surface-induced expression of genes coding for various adhesins (e.g., flagella, pili, and fimbriae) have been characterized in the literature (28,39,45,48,51,54). Many of these studies rely on the mutation of genes of interest followed by a comparative analysis of the number of cells (wild type versus mutant) that are attached to a solid substrate. While these types of studies provide detailed characterizations of the proteins involved in mediating contact with another surface, the inherent surface-sensing mechanism remains hidden. The actual mechanism that allows a bacterium to sense or perceive another surface is the physical force(s) that exists in the nanometer-scale interface between a cell and that other surface. This type of elementary force, which includes van der Waals, electrostatic, solvation, and steric interactions (21,22,26), controls the way in which a protein on a bacterium's cell wall physically touches anoth...

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