PROJECT SUMMARY/ABSTRACT Tuberculosis (TB) is the leading cause of death due to an infectious disease worldwide. Although TB is treatable, treatments are prolonged, complex, and difficult for many people to tolerate. Mycobacterium tuberculosis (M. tb) also readily acquires drug resistance and global efforts to control TB are threatened by the increasing problem of drug resistance. M. tb is capable of assembling into biofilm communities capable of withstanding lethal doses of antibiotics and this phenomenon likely contributes to difficulties in treating and controlling TB. We have developed an in vitro system to study adaptation M. tb within biofilms. Leveraging analyses of these experimental populations with those of a well characterized natural population of M. tb, we discovered that M. tb strains separated by a small number of genetic differences exhibit a surprising array of phenotypic differences. Our preliminary data point to regulatory mechanisms underlying these changes. Contextualizing these data with our recent discoveries in M.tb evolutionary population genomics at regional and global scales, we hypothesize that M. tb adaptation occurs at the scale of individual hosts while large scale phenomena act primarily as a constraint on adaptation. Our major objective now is to delineate the mechanisms underlying observed phenotypic differences in these natural and experimental populations.