title: 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.
authors.affiliation: Pacific Northwest National Laboratory, Richland, Washington, USA.
authors.forename: Brian H
authors.initials: BH
authors.lastname: Lower
authors.forename: Ruchirej
authors.initials: R
authors.lastname: Yongsunthon
authors.forename: F Paul
authors.initials: FP
authors.lastname: Vellano
authors.forename: Steven K
authors.initials: SK
authors.lastname: Lower
mesh_terms: D001422:Bacterial Adhesion / Q000502:physiology*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D018441:Biofilms; D002499:Centrifugation, Density Gradient; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D018625:Microscopy, Atomic Force; D018613:Microscopy, Confocal; D008954:Models, Biological; D010957:Plasmids; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D013314:Stress, Mechanical; D013395:Sucrose
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C078167:OMPA outer membrane proteins; D013395:Sucrose
references.citation: Appl Microbiol Biotechnol. 1996 Mar;45(1-2):112-9
references.pmid: 8920186
references.citation: Nature. 1997 May 15;387(6630):308-12
references.pmid: 9153398
references.citation: J Physiol. 1999 Oct 1;520 Pt 1:5-14
references.pmid: 10517795
references.citation: J Bacteriol. 1996 Oct;178(19):5776-80
references.pmid: 8824625
references.citation: Biochem Biophys Res Commun. 2001 Jul 6;285(1):9-14
references.pmid: 11437364
references.citation: J Bacteriol. 1998 May;180(9):2442-9
references.pmid: 9573197
references.citation: Science. 1996 Feb 9;271(5250):795-9
references.pmid: 8628994
references.citation: Science. 1997 Sep 5;277(5331):1453-62
references.pmid: 9278503
references.citation: Science. 1999 May 21;284(5418):1318-22
references.pmid: 10334980
references.citation: Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):462-7
references.pmid: 11209050
references.citation: FEMS Microbiol Rev. 2003 Oct;27(4):505-23
references.pmid: 14550943
references.citation: Proc Natl Acad Sci U S A. 2000 Jan 4;97(1):139-44
references.pmid: 10618384
references.citation: Biotechnology (N Y). 1993 Apr;11(4):491-5
references.pmid: 7763519
references.citation: J Bacteriol. 1984 Jul;159(1):191-8
references.pmid: 6735979
references.citation: Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12283-8
references.pmid: 9770478
references.citation: Nat Struct Biol. 2000 Dec;7(12):1133-8
references.pmid: 11101896
references.citation: Science. 2001 May 18;292(5520):1360-3
references.pmid: 11359008
references.citation: Science. 1997 May 16;276(5315):1109-12
references.pmid: 9148804
references.citation: Proc Natl Acad Sci U S A. 1997 Jun 10;94(12):6185-90
references.pmid: 9177192
references.citation: J Mol Biol. 2003 Nov 7;333(5):993-1002
references.pmid: 14583195
references.citation: Nat Biotechnol. 1999 Oct;17(10):969-73
references.pmid: 10504696
references.citation: J Mol Biol. 1999 Feb 19;286(2):553-61
references.pmid: 9973570
references.citation: Science. 1988 Aug 12;241(4867):795-800
references.pmid: 17829174
references.citation: Biophys J. 1999 Feb;76(2):1072-9
references.pmid: 9916039
references.citation: Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2713-7
references.pmid: 1557377
references.citation: Proc Natl Acad Sci U S A. 1999 Nov 9;96(23):13170-4
references.pmid: 10557292
references.citation: Methods Enzymol. 1974;31:642-53
references.pmid: 4608978
references.citation: J Bacteriol. 1999 Jun;181(12):3688-94
references.pmid: 10368142
references.citation: Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3694-9
references.pmid: 10097099
references.citation: Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10444-8
references.pmid: 8248129
references.citation: Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8485-9
references.pmid: 11447284
references.citation: Annu Rev Microbiol. 1996;50:513-52
references.pmid: 8905090
references.citation: Science. 1997 May 16;276(5315):1112-6
references.pmid: 9148805
references.citation: Science. 1997 Feb 28;275(5304):1295-7
references.pmid: 9036852
references.citation: Q Rev Biophys. 2001 May;34(2):105-267
references.pmid: 11771120
references.citation: Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11288-92
references.pmid: 10500169
references.citation: Lett Appl Microbiol. 2003;37(3):254-8
references.pmid: 12904229
references.citation: Mol Microbiol. 2004 May;52(3):631-41
references.pmid: 15101971
references.citation: Nat Struct Biol. 1999 Nov;6(11):1025-8
references.pmid: 10542093