Project Summary/AbstractPhysiological profiling of Pseudomonas aeruginosa biofilm subpopulationsThe association of microorganisms into biofilms produces functionally organized microbial structuresthat promote community survival in a wide range of environments. Much like individual cells within amulticellular organism express different genes from the same DNA base, individual microbial cellslocated within different regions of a biofilm structure can exhibit distinct genetic programs. Thesespatially-defined regions of physiologically-differentiated cells are reminiscent of the role of tissues inmulticellular organisms, with specific subpopulations in the microbial community serving defined rolesto promote the overall health of the biofilm. In this proposal, we seek to assess the function of thesubpopulations that we recently identified within the biofilm communities of the pathogenic bacteriumPseudomonas aeruginosa. Based on previous knowledge of the physiological functions of biofilmsubpopulations gleaned from studies focused on model microorganisms such as Bacillus subtilis, it islikely that these populations serve one of two major functions: (1) individual resistance to anenvironmental stressor, enabling subsequent proliferation of single microbial cells within the communityto ensure ultimate survival of the microbial strain or (2) the production of shared resources whichpromote survival of the entire community. The study of subpopulation functionality is particularlyimportant in the context of P. aeruginosa biofilm communities because most infections caused by thisorganism are mediated by biofilm formation (e.g. infections of wounds, the urinary tract, and the cysticfibrosis lung). The long-term goal of this project is to identify drugs targeting specific biofilmsubpopulations. The rationale of this application is that the recent discovery of new P. aeruginosabiofilm subpopulations enables physiological profiling of these populations to identify targetableweaknesses. Our overall hypothesis is that the targeting of subpopulations responsible for producingshared resources can disrupt the entire community to promote clearance of the biofilm. Theexperiments outlined in this proposal will not only define the community functions of P. aeruginosasubpopulations but will reveal metabolic susceptibilities (the ?Achilles heel?) of these communities,which can be exploited for subsequent therapeutic targeting.