Bacteria are able to detect changes in their environment and adapt accordingly. As bacteria mostly exist within complex matrix-encased structures, termed biofilms, the behaviour of the entire bacterial community is influenced by signals originating from within the biofilm as well as from the external environment. The formation of biofilms by the opportunistic pathogen, Pseudomonas aeruginosa, is associated with its successful colonisation of a variety of hosts, infection of implanted medical devices, as well as the chronic nature of infections in cystic fibrosis and immunocompromised individuals. The ability of P. aeruginosa to undergo a form of type IV pili (tfp)-mediated surface translocation, referred to as twitching motility, is required for biofilm expansion. A number of regulatory systems, including the two-component sensor/regulator pairs FimS/AlgR and PilS/PilR as well as the Chp chemosensory system, are known to be involved in regulating expression, assembly and function of tfp, however the environmental signals that these systems respond to are not well characterised. In the current study we demonstrate that extracellular ATP (eATP) is an environmental signal which controls twitching motility-mediated expansion within interstitial biofilms, via modulation of surface tfp levels. Testing the effect of eATP on twitching motility of 40 mutants of candidate genes has not identified the component(s) responsible for sensing eATP. We are currently screening a high density transposon mutant library to identify the putative sensory system(s) involved. Our results thus far suggest that twitching motility-mediated biofilm expansion in P. aeruginosa is controlled by a novel mechanism which relies upon sensing eATP derived from within the biofilm itself.