Date of Award
Doctor of Philosophy
Mitchel J. Doktycz
David P. Allison, Barry D. Bruce, David C. Joy, Engin H. Serpersu
Atomic force microscopy (AFM) holds a unique position in microbiology because of its potential for nanometer (nm) scale imaging and piconewton (pN) force detection. These features can be exploited to characterize bacteria from the cellular down to the molecular level. In order to pursue such characterization studies, reliable sample preparation techniques must be developed. Spheroplasts are bacteria which have been treated with enzymes to remove cell wall components. Because the cytoplasmic membrane is exposed in spheroplasts, they are suitable for localizing transporters and other membrane proteins using AFM techniques. Constituents on the surface of intact bacteria are responsible for their adhesion to various substrates in vivo. The absence of these constituents in spheroplasts necessitates specialized immobilization strategies. This study presents a technique in which spheroplasts are immobilized by cross linking them with glutaraldehyde to mica surfaces pretreated with aminopropyltriethoxysilane. As suggested by the AFM images, this approach facilitates stable imaging in appropriate buffers. Because the sample preparation strategies presented are compatible with optical and atomic force microscopies, investigations in which molecular system components are monitored can be targeted.
Evidence that the cells retain membrane integrity, continue glucose uptake, increase in diameter and initiate protein synthesis after immobilization is also presented. Based on this data, it is concluded that metabolic processes continue in immobilized spheroplasts. As a result of this finding, spheroplasts are proposed to be a platform for various imaging-based investigations.
Elasticity and indentation measurements on intact bacteria and spheroplasts revealed significant differences between the two forms. They also provided the justification for using glutaraldehyde fixed spheroplasts for molecular recognition experiments designed to locate the glucose transporter on the surface of spheroplasts. An avidin-biotin system was used in which biotin was tethered to the AFM tip using a polyethylene glycol linker, When this functionalized tip probed spheroplast surfaces previously immunolabeled with a biotinylated antibody and avidin, molecular recognition was demonstrated. The fact that the biotin functionalized tips can be used in multiple applications is an attractive feature of this strategy. That results from AFM experiments can be validated with optical microscopy techniques is also an advantage.
Sullivan, Claretta Jackson, "Advanced Microscopy Techniques for the Molecular Scale Analysis and Physical Characterization of Escherichia coli Spheroplasts. " PhD diss., University of Tennessee, 2007.