ABSTRACT Bacterial lifestyles can be extremely complex, manifested most strikingly in the formation of highly cooperativemulticellularstructureslikebiofilms.Beyondtheinherentbasicbiologicalinterest,thesecomplex traits have significant impacts on how bacteria relate to human health both as a defense response leading to increased drug resistance and as a source of clinically-??relevant antibiotics, which are often only produced in certainsocialcontexts.Inmypost-??doctoralworkIhaveusedthemodelbacteriumBacillussubtilistostudyhow multicellular behaviors affect the way bacteria interact with each other. I found that B. subtilis can judge whether or not neighboring cells are close relatives, a phenomenon called kin discrimination. They do this using the extensive and diverse complement of antimicrobial molecules that each strain produces. This kin discrimination behavior creates an evolutionary pressure to diversify, resulting in so much intraspecies variationthatevenverycloselyrelatedstrainscannotcoexistintheirnaturalhabitat. The first aim of this proposal will focus on the consequences of kin discrimination, specifically whether a populationcanbetterresistinvasionbynon-??kincellsthankin.Thiswillinformourunderstandingofnatural microbial populations living in and around us, and how they respond to outside influences. I will also learn important technical skills involved in experimental evolution, including genome sequencing, comparative genomics,andthebioinformaticandprogrammingskillsinherenttoworkingwithlargegenomicdatasets. The second aim seeks to gain a better understanding of the diversity lurking within the B. subtilis species by examining three routes of evolutionary change: amino acid substitutions, gain/loss of entire genes, and changes in gene transcription. By looking across multiple strains in multiple multicellular contexts, I will be abletoseewhichofthesechangesismostcommoninearlystraindivergenceandwhichgenesareunderthe mostpressuretochange.Thiswillgenerateacatalogofevolutionaryinformationthatwillbeusefulformany yearsandopenmanyavenuesofresearchformyownindependentlab.Thetechniquesinvolved,suchasRNA sequencingandbioinformaticanalyses,willbeinvaluableadditionstomyskillset. The third aim of this application will examine the generality of my findings in other bacteria. Given that B. subtilisuseantibioticmoleculestorecognizeeachother,Iwilllookinthephylumofbacteriathatproducesthe majorityofapprovedantibiotics:Actinobacteria.Examiningintraspeciesinteractionscanhelpshedlightonthe evolutionofantibioticproductionandhowbesttomineforclinicallyrelevantmolecules.Iwillinvestigatethe ability of the Actinobacteria Streptomyces to discriminate kin from non-??kin, and whether this is mediated throughantibioticdiversity.ThisprojectwillbemyintroductiontoActinobacteria,withthelong-??termgoalof studyingotheraspectsoftheirfascinatingmulticellularlifecycle. ThetrainingIwillcontinuetoreceiveunderDr.RobertoKolteratHarvardMedicalSchoolwillgivemeagreat foundation of microbiology experience on which to build my career. HMS provides a number of advantages thatwillfacilitatemyadvancement,includingcoresequencingfacilities,bioinformaticresources,andcourses dedicatedtocareerdevelopment.Additionally,Iwillreceivespecializedtrainingfromexpertsinthefieldsof microbial genomics and Actinobacteria biology to accomplish the goals of this proposal and broaden my developmentasascientist.TheknowledgeandtechniquesIwillacquireunderthisawardwillbeinstrumental inobtainingmycareergoalsandbecominganeffectiveindependentinvestigator.