Flexibility of bacterial type IV pili determining using atomic force microscopy
Type IV pili (T4P) are very thin protein filaments extended and retracted from the surface of certain Gram-negative bacteria. T4P play a major role in processes such as adhesion, twitching motility and biofilm formation. We used atomic force microscopy (AFM) to improve on previous single cell force spectroscopy (SCFS) measurements of the mechanical properties of T4P of Pseudomonas aeruginosa. A protocol was adapted and developed to adhere a single bacterium to the end of a colloidal cantilever which is brought into contact with a substrate, allowing the pili to adhere. Force-distance curves are then collected by retracting the cantilever, corresponding to the stretching of the T4P. The persistence length, a parameter that characterizes the flexibility of a polymer, can be determined from the worm-like chain (WLC) model. The value of the persistence length measured by total internal reflection fluorescence (TIRF) microscopy is a factor of 1000 times greater than that measured in SCFS experiments. We show that the SCFS protocol is unlikely to be successful without rolling the bacteria beneath the cantilever, which could result in the T4P contacting the adhesive that coats the cantilever. We suggest that the persistence length discrepancy in the literature is due to a force-induced conformational change that occurs when pulling the T4P in the SCFS experiments.
- Dr. Robert Wickham, Chair
- Dr. John Dutcher, Advisor
- Dr. Leonid Brown
- Dr. Jacek Lipowski