Type IV pili (T4P) are very thin (5-8 nm in diameter) protein filaments that can be extended and retracted by certain classes of Gram-negative bacteria including P. aeruginosa . These bacteria use T4P to move across viscous interfaces, referred to as twitching motility. Twitching can occur for isolated cells or in a collective manner . We have used optical microscopy, together with a custom-built, temperature- and humidity-controlled environmental chamber, to study the expansion of twitching colonies across an agar-glass interface. The advancing front consists of finger-like protrusions (fingers) consisting of many bacteria, with the cells within the expanding colony arranged in a lattice-like pattern. The fingers consist of aligned bacteria 5 to 30 cells across, which move radially outward. Using particle image velocimetry (PIV) and Fourier analysis techniques, we have characterized the evolution of the advancing front of expanding colonies. In particular, we have characterized the average edge speed, average width and average bacterial cell orientation within the fingers for a range of agar concentrations 1.0 % w/v < C < 1.9% w/v. Interestingly, we observed a significant decrease in edge speed and a corresponding transition from monolayer to multilayer coverage within the fingers at a concentration of 1.6% w/v. We have studied this transition by characterizing multilayer formation and dissolution, and transient and stable multilayer regions within fingers. We observed that a minimum finger width is required for multilayer stability, and we have developed a simple nucleation model that describes the dependence of multilayer lifetime on finger width.
 Burrows, L.L. (2012) Annu. Rev. Microbiol. 66: 493–520.
 Semmler, A.B., Whitchurch, C.B., Mattick, J.S. (1999) Microbiology 145: 2863-2873.