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Washington University in St. Louis Washington University Open Scholarship Undergraduate Research Symposium Posters Undergraduate Research 5-22-2017 Isolation and Comparative Genomic Analysis of Final Third of Satis Genome Kelly Hartigan Nicole Curnutt Matthew McDermut Follow this and additional works at: http://openscholarship.wustl.edu/undergrad_research Part of the Bioinformatics Commons, Biology Commons, Computational Biology Commons, and the Genomics Commons Recommended Citation Hartigan, Kelly; Curnutt, Nicole; and McDermut, Matthew, "Isolation and Comparative Genomic Analysis of Final Third of Satis Genome" (2017). Undergraduate Research Symposium Posters. 104. http://openscholarship.wustl.edu/undergrad_research/104 This Unrestricted is brought to you for free and open access by the Undergraduate Research at Washington University Open Scholarship. It has been accepted for inclusion in Undergraduate Research Symposium Posters by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. Isolation and Comparative Genomic Analysis of Final Third of Satis Genome KellyHar)gan,NicoleCurnu3,Ma3hewMcDermut Mentors:ChristopherShafferandKathleenHafer Abstract A highly novel Streptomyces phage, Sa)s, was isolated from a direct environmental sample collected from outside Danforth House on the Washington University campus. Sa)s infects bacterial species Streptomyces lividans producing pinpoint, cloudy plaques less than 1mm in diameter. Electronmicroscopedatashowsrareatypicalphysicalfeatures.Ratherthan the common octahedral capsid shape, Sa)s has a prolate head with visible cross-linkedhexagonalproteinstructureandaveragemeasurementsof285 nmby47nmwithalong,flexibletailmeasuring268nm.Uponsequencing,it wasfoundthatSa)scontainsthelongestphagegenomediscoveredtodate throughtheSEA-PHAGEprogramat186,702basepairs.Thegenomeisquite novel in sequence, as its closest gene)c match, bacteriophage Chymera, is similaracrossonly0.2%ofthegenome.ThismeansthatSa)sbelongstono knownpreviouslycharacterizedclusterandisconsideredaSingletonphage. Thegenomecontains325proteincodinggenes,ofwhichourgroupanalyzed Gene 230 to the end of the genome. The vast majority of the genes in this sec)on run 3’ to 5’ and compared to the other two sec)ons, these genes seem to be the most unique in primary, secondary, and ter)ary structure. Due to the novelty of Sa)s, func)onal evidence from compara)ve genomic analysis is sparse. We are currently in the process of a more thurough compara)ve genomic analysis between Sa)s and other Streptomyces phages, par)cularly phage JustBecause, another Streptomyces phage isolatedbyWashingtonUniversityinSt.Louisstudentsin2016withsimilar morphologytoSa)s. Isola)onandPurifica)on Figure1:Isola)onsiteofPhageSa)s,outside DanforthHouseonthecampusof WashingtonUniversityinSt.Louis Figure2:Plate photoshowing plaque morphology.Sa)s createspinpoint cloudyplaques lessthan1mmin diameteras shownbydark spotscircledon plate. Characteriza)on TailLength HeadLength HeadWidth 268±3.8nm 285±5.3nm 47±2.1nm Table1:AveragesizeofSa)swithstandard devia)on.Calculatedusingasampleoffive TEMphotosandanalyzedusingImageJ. Figure3 Figure5:CloseupofSa)s headshellshowsinterlocking gridofhexagonalcapsid proteins. OrthologCaseComparisons Sa4s29vJustBecause25 Gene Start Stop Func)on 243 145055 144546 PhosphataseDomainof Polynucleo)deKinase 266 153436 152981 An)toxinDarG Table2:Func)onal annota)oncallsof finalthirdofSa)s genome.Shows highlyvariable regionofgenome Func)onalEvidence Gene243-PhosphataseDomain Figure7:Sa)sgene242 (blue)overlaidwiththe phosphatasedomainofT4 phagepolynucleo)de kinase.PhyreProtein Modelingshowed7of8 conservedac)vesites betweenthetwoproteins showninorange Figure10:Syntenymap ofSa)sand Streptomycesphage JustBecausealsofound thisyearshowing extremelyhighgene orderconserva)on betweenthetwophage. Sa)svs Sa)svs JustBecause Chymera GeneOrder Conserva)on ANI OrthologNumber AverageOrtholog Similarity AverageOrtholog Iden)ty 0.9774 0.0263 0.7440 266 0.5848 39 0.7467 0.5200 0.6450 0.3932 .639 .159 Table3&4:Comparisons ofgenomesofJustBecause andChymeraagainstSa)s showingcloserela)on betweenSa)sand JustBecausefarexceeding nextclosestrela)on Figure11(BoJom):Orthologmapofen)regenomeofSa)svsJustBecause. Orthologsareshownwithsamecolor,uniquegenesareblackedout WewouldliketothankourmentorsDrs.ChristopherShafferandKathleenHafer, our TA’s Ryan Smith, Kendra Woodruff, and Emily Moore, the Washington University in St. Louis Biology Department, the Hanull Lab, and the Howard HughsMedicalIns)tuteforsuppor)ngourresearch. Abedon,StephenT.“PhageEvolu)onandEcology.”AdvancesinApplied Microbiology,vol.67,Elsevier,2009,pp.1–45. SolwareUsed:DNAMaster,Glimmer,GeneMarkA,Phyre2,NCBIBLAST,PhagesDB BLAST,HHPRED,PECAAN,PyMol,Starterator,SplitsTree,GenBank,EMBOSS Stretcher Table5 Start Coordinate Stop Coordinate Top Result Descrip)on Sa)s29 16906 18219 pfam101 24 Mu-likeprophage majorheadsubunitgpT 100 56.06 6.6e-30 JustBecause 25 15649 16962 1ohg_a Majorcapsidprotein; BaceriophageHK97 97.75 62.01 6.6e-4 Evolu)onaryImplica)ons • Phagesdisplaywidegene)cdiversity:Sa)sandJustBecausehavegenomesthat arehighlyuniqueonboththeDNA,aminoacidsequence,andproteinlevel • Mul)plemechanismsforphagegenomicevolu)on o Sa)s,partofthesiphoviridaefamily,hasadoublestrandedDNAgenome meaningthatitcanreadilyrecombinewithandincorporatebacterialhost DNAthroughtransduc)on(thisDNAcouldbebacterialorphageinorigin) o Increasedgenevarietythroughbacterialvectors;canbothincorporateand donatephageDNAtoaninfec)ngphage • Highlyconservedgeneorder(synteny)andhighnumberoforthologssupport theexistenceofacommonancestorforSa)sandJustBecause,seenon Splitstree • LackofsyntenyandlownumberoforthologswithChymerasupportexistenceof amuchmoredistantcommonancestorbetweenSa)sanditsothermostclosely relatedStreptomycesphage,alsoshownonSplitstree • Viruseswithhighermuta)onratestendtohavephenotypesthatareless sensi)vetomuta)onalchange;thereforetheirgeneproductsareconserved o ExplainshighnumberoforthologsbetweenSa)sandJustBecause • Possibility:Sa)shasahighermuta)onratethanotherStreptomycesphagesdue toitssignificantlylargergenome o Fidelitymaybesacrificedforspeedofreplica)ontoimproveSa)s’ compe))vefitnessinout-replica)ngotherStreptomycesphages Probability Coverage E-Value Sa4s144vJustBecause144 Figure13&Table6:Sa)s 144(blue)overlaidwith JustBecause144using Phyreresultslel.Table belowshowstop HHPREDresultsforboth genes.Resultsshowvery conservedsecondaryand ter)arystructure. Sa)s JustBecause Chymera GeneCount 325 340 55 tRNACount 13 0 0 GenomeLength 186702bp 184281bp 34742bp GlobalAlignment Figure8:Sa)sgene266 (blue)overlaidwithDarG an)toxinfromM. tuberculosis.PhyreProtein Modelingshowed6outof 9conservedac)vesites betweenthetwoproteins showninorange Figure12&Table5:Sa)s 29(blue)overlaidwith JustBecause25using Phyreresultslel.Table belowshowstop HHPREDresultsforboth genes.Resultsshowvery conservedsecondaryand ter)arystructure. Figure9:Syntenymapof Sa)sandStreptomyces phageChymerashows verylowlevelofgene orderconserva)on. Chymeraistheclosest matchtoSa)scurrently published. Figure6:Thephameratormapforgenes231-325isshownabove.Themajorityofthe genesinthissec)onareorphamsmeaningtheydon’tfitintoanycurrentlyannotated proteinfamiliesintheSEA-PHAGEprogram. Acknowledgements&References Figure5 PhageComparison Sa4svsChymera Figure3:TEMphotooftwoSa)sphages showingprolateheadandlongflexibletail characteris)cofthesiphoviridaefamilyof phage.Takenat25000xmagnifica)on. Figure4:TEMphotoof JustBecauseatsame magnifica)on,showingvery similarsizeandmorphology betweenthetwophage. Figure4 Annota)onofGenes231-325 Table6 Start Coordinate Stop Coordinate TopResult Descrip)on Probability Coverage E-Value Sa)s144 90395 89094 2iut_A DNAtranslocase FTSK 100 94.67 0 JustBecause 144 90307 89009 2iut_A DNAtranslocase FTSK 100 99.3 4e-39 SplitsTreeMap Figures14:SplitsTree figuresshowsgene similaritygivinga roughevolu)onary mapofall Streptomycesphages publishedonGenBank