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Journal of Bacteriology, March 2005, p. 2127-2137, Vol. 187, No. 6
0021-9193/05/$08.00+0     doi:10.1128/JB.187.6.2127-2137.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Brian H. Lower,1,{dagger} Ruchirej Yongsunthon,2 F. Paul Vellano III,2 and Steven K. Lower2,{dagger}*

Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington,1 Ohio State University, Columbus, Ohio2

Received 25 August 2004/ Accepted 22 November 2004

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.

* Corresponding author. Mailing address: Ohio State University, 275 Mendenhall Laboratory, 125 South Oval Mall, Columbus, OH 43210. Phone: (614) 292-1571. Fax: (614) 292-7688. E-mail: lower.9{at}osu.edu.

{dagger} B.H.L. and S.K.L. contributed equally to the manuscript.

Journal of Bacteriology, March 2005, p. 2127-2137, Vol. 187, No. 6
0021-9193/05/$08.00+0     doi:10.1128/JB.187.6.2127-2137.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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