Flexibility of bacterial type IV pili determined using atomic force microscopy

Type IV pili (T4P) are very thin protein filaments extended and retracted from the surface of certain Gram-negative bacteria. Pili play a major role in processes such as adhesion, twitching motility and biofilm formation. We used atomic force microscopy (AFM) to perform force spectroscopy measurements on T4P of \textit{P. aeruginosa}. Bacteria were adhered to the end of an AFM cantilever that was brought into contact with a substrate, allowing the pili to adhere. Force-separation curves were collected by retracting the cantilever, corresponding to the stretching of the T4P that was well described by the worm-like chain (WLC) model. Distinct peaks were observed in the distributions of the best-fit values of the persistence length \textsl{L$_{p}$} on two different surfaces, providing strong evidence for close-packed bundling of very flexible T4P [1]. Surprisingly, the most prominent value of \textit{L$_{p}$} $\sim$ 1 nm is significantly less than the $\sim$ 8 nm length of the PilA subunit. We have investigated this intriguing result by refining our protocol to combine AFM with fluorescence microscopy to isolate a single bacterium on a colloidal probe, as well as critically examining the applicability of the WLC model. [1] S. Lu et al., Biophys. J. {\bf 108}, 2865 (2015).