Examination of twitching motility-mediated biofilm expansion in response to eATP by wildtypeP. aeruginosastrains and mutants ofcyaA,cyaB,cpdAor the Chp chemosensory system. The twitching motility response of P. aeruginosa strains to eATP at the interstitial space between a solidified nutrient media-coated microscope slide and a coverslip after incubation for 15 h at 37°C. (A) Phase-contrast microscopy of interstitial biofilms of wildtype P. aeruginosa strains PAK, PAO1, PA14 and PA103 in the presence of a H2O-saturated disc (upper images) or ATP-saturated disc (lower images). Scalebar is 50 μm and the arrows indicate the direction of expansion towards the disc. Images are representative of three independent experiments. (B) Surface areas of interstitial biofilms formed in the absence of eATP (black bars) or a constant concentration of 7.5 mM eATP (grey bars); (C) the distances expanded towards a H2O-saturated disc (black bars) or ATP-saturated disc (grey bars). The data are represented as the mean ± SD for three independent experiments.