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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
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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
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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