Whole genome sequence of two Rathayibacter toxicus strains reveals a tunicamycin biosynthetic

17 Pages·2017·3.44 MB·English

RESEARCHARTICLE
Whole genome sequence of two Rathayibacter
toxicus strains reveals a tunicamycin
biosynthetic cluster similar to Streptomyces
chartreusis
AaronJ.Sechler1,MatthewA.Tancos1,DavidJ.Schneider2¤a,JonasG.King1¤b,Christine
M.Fennessey1¤c,BrendaK.Schroeder3,TimothyD.Murray4,DouglasG.Luster1,William
L.Schneider1*,ElizabethE.Rogers1*
a1111111111
a1111111111 1 ForeignDisease/WeedScienceResearchUnit,AgriculturalResearchService,U.S.Dept.ofAgriculture,
Frederick,Maryland,UnitedStatesofAmerica,2 EmergingPestsandPathogensResearchUnit,Agricultural
a1111111111
ResearchService,U.S.Dept.ofAgriculture,Ithaca,NewYork,UnitedStatesofAmerica,3 Dept.of
a1111111111
Entomology,PlantPathologyandNematology,UniversityofIdaho,Moscow,Idaho,UnitedStatesofAmerica,
a1111111111 4 Dept.ofPlantPathology,WashingtonStateUniversity,Pullman,Washington,UnitedStatesofAmerica
¤a Currentaddress:GlobalInstituteforFoodSecurityandtheSchoolofEnvironmentandSustainability,
UniversityofSaskatchewan,Saskatoon,Saskatchewan,Canada
¤b Currentaddress:Dept.ofBiochemistry,MolecularBiology,EntomologyandPlantPathology,Mississippi
StateUniversity,MississippiState,Mississippi,UnitedStatesofAmerica
OPENACCESS ¤c Currentaddress:AIDSandCancerVirusProgram,FrederickNationalLaboratoryforCancerResearch
andLeidosBiomedicalResearch,Inc.,Frederick,Maryland,UnitedStatesofAmerica
Citation:SechlerAJ,TancosMA,SchneiderDJ,
*[email protected](EER);[email protected](WLS)
KingJG,FennesseyCM,SchroederBK,etal.
(2017)Wholegenomesequenceoftwo
Rathayibactertoxicusstrainsrevealsatunicamycin
biosyntheticclustersimilartoStreptomyces Abstract
chartreusis.PLoSONE12(8):e0183005.https://
doi.org/10.1371/journal.pone.0183005 RathayibactertoxicusisaforagegrassassociatedGram-positivebacteriumofmajorcon-
Editor:Chih-HorngKuo,AcademiaSinica,TAIWAN cerntofoodsafetyandagriculture.ThisspeciesislistedbyUSDA-APHISasaplantpatho-
genselectagentbecauseitproducesatunicamycin-liketoxinthatislethaltolivestockand
Received:May24,2017
maybevectoredbynematodespeciesnativetotheU.S.Thecompletegenomesoftwo
Accepted:July27,2017
strainsofR.toxicus,includingthetypestrainFH-79,weresequencedandanalyzedincom-
Published:August10,2017
parisonwithallavailable,completeR.toxicusgenomes.Genomesizesrangedfrom
Copyright:Thisisanopenaccessarticle,freeofall 2,343,780to2,394,755nucleotides,with2079to2137predictedopenreadingframes;all
copyright,andmaybefreelyreproduced,
fourstrainsshowedremarkablesyntenyovernearlytheentiregenome,withonlyasmall
distributed,transmitted,modified,builtupon,or
transposedregion.Aclusterofgeneswithsimilaritytothetunicamycinbiosyntheticcluster
otherwiseusedbyanyoneforanylawfulpurpose.
TheworkismadeavailableundertheCreative fromStreptomyceschartreusiswasidentified.Thetunicamycingenecluster(TGC)inR.
CommonsCC0publicdomaindedication. toxicuscontained14genesintwotranscriptionalunits,withallofthefunctionalelementsfor
DataAvailabilityStatement:Genomedatahas tunicamycinbiosynthesispresent.TheTGChadasignificantlylowerGCcontent(52%)
beendepositedatGenBankunderthefollowing thantherestofthegenome(61.5%),suggestingthattheTGCmayhaveoriginatedfroma
accessionnumbers:R.toxicusFH-79BioProject
horizontaltransferevent.Furtheranalysisindicatednumerousremnantsofotherpotential
PRJNA312185andBioSampleSAMN04495682;R.
horizontaltransfereventsarepresentinthegenome.InadditiontotheTGC,genespoten-
toxicusFH-232BioProjectPRJNA312185and
BioSampleSAMN06040670. tiallyassociatedwithcarotenoidandexopolysaccharideproduction,bacteriocinsandsec-
ondarymetaboliteswereidentified.ACRISPRarrayisevident.Therewererelativelyfew
Funding:Fundingforthisworkwasfromthe
UnitedStatesDepartmentofAgriculture plant-associatedcell-wallhydrolyzingenzymes,buttherewerenumeroussecretedserine
AgriculturalResearchServiceappropriatedproject proteasesthatsharesequencehomologytothepathogenicity-associatedproteinPat-1of
8044-22000-040-00DtoDGL,WLS,andEER.No
Clavibactermichiganensis.Overall,thegenomeprovidesclearinsightintothepossible
grantwebsiteavailable.Fundingwasalsoreceived
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R.toxicustunicamycinbiosyntheticcluster
fromtwo2008FarmBillgrants,Section10201 mechanismsfortoxinproductioninR.toxicus,providingabasisforfuturegenetic
administeredthroughtheUnitedStates approaches.
DepartmentofAgriculture,AnimalandPlantHealth
InspectionService:13-8130-0247-CAand14-
8130-0367-CAtoBKS,TDM,DGL,andWLS.Grant
websiteishttp://www.aphis.usda.gov/aphis/
resources/farm-bill/!ut/p/z1/04_
iUlDg4tKPAFJABpSA0fpReYllmemJJZn5eYk5-
hH6kVFm8T7- Introduction
Therearefewphytobacteriawiththecapacitytodirectlyaffectthehealthofhumansorlive-
stock.Intherareinstanceswheretheycan,thepathogeniceffectsareoftenrelatedtothepro-
ductionoftoxins.Onesuchtoxin-produceristheGram-positivebacteriumRathayibacter
toxicus,thecausativeagentofannualryegrasstoxicity(ARGT)inAustralia.ARGTisan
often-fataltoxicosisofforageanimalscausedbyingestionofinfectedhayorgrain.Over10
millionhectaresofWesternAustralianfarmlandhasbeenaffectedandARGTcausedanesti-
mated$40millionAUDindirectlossesin2010[1].R.toxicusproducesahighlylethaltunica-
minyluracilclasscorynetoxin(LD 3–5mg/kginsheep)thatcausessevereandoftenfatal
50
neurologicalandhepaticdisease[2].Sub-lethaldosesarealsodamagingtolivestockand
diminishwoolqualityandquantity,meatquality,andcausefetalabortionsinsheep[1].Symp-
tomonsetcanoccurupto12weeksafteringestionandasingleexposurecancauselethality;
toxineffectsarecumulative[2].R.toxicuscorynetoxinswereidentifiedasanewmemberof
thetunicaminyluracilclassofantibiotics,whichinhibitanearlystageinprokaryoticpeptido-
glycancellwallassembly[3].Ineukaryotes,tunicamycinreducesproteinN-glycosylationby
inhibitinguridinediphospho-N-acetylglucoseamine:dolichol-N-acetylglucoseamine-1-phos-
phatetransferase[4].ThedangerstoU.S.agriculturepresentedbyR.toxicusandtunicamycin
productioninforageresultedinthebacteriumbeinglistedasaU.S.DepartmentofAgriculture
(USDA)PlantProtectionandQuarantineSelectAgentin2008andrelistedin2012(www.
selectagents.gov/SelectAgentsandToxinsList.html).
R.toxicusismostcommonlyfoundinannualryegrass(Loliumrigidum)inassociationwith
Anguinafunestaorotheranguinidseed-gallnematodes.TheinfectioncyclebeginswithR.tox-
icusadheringtothecuticleofcompatiblejuvenilenematodesinthesoilandbeingcarriedto
thegrowingpointoftheforagegrass.Onceinadevelopingseed,thenematodeandbacteria
competetoformeitheranematodeorabacterialgall.R.toxicusgrowthindevelopinggallscan
produceayellowexopolysaccharide“slime”orgummosis;therefore,theplantinfectionis
commonlycalledyellowslimedisease.Thetriggerfortoxinproductionisunknownbuttoxin
generallyappearslateinthegrowingseasonasseedaresenescing.Senescedseed,nematode
galls,andbacterialgallsdryandfalltothegroundtorepeatthediseasecyclethefollowing
year.HostrangeofR.toxicusappearstobedeterminedbythehostrangeofthevectoringnem-
atode[5,6].TunicamycinproductionisoftenassociatedwiththepresenceofanR.toxicus-
specificbacteriophageNCPPB3778,althoughtoxinproductionhasalsobeenmeasuredinthe
absenceofphage[7,8].TheNCPPB3778genomehasrecentlybeensequencedandissimilar
tosiphoviralgenomes[9].Althoughitsroleinnatureisunclear,NCPB3778infectionofR.tox-
icuscanrestoretunicamycinproductioninthelab,wheretheabilitytoproducetunicamycin
isotherwiserapidlylostinculture(A.J.Sechler,personalobservation).
AlthoughcompletegenomesequencesarepublicallyavailablefortwoR.toxicusstrains,
FH-145(NZ_CP010848.1)andWAC3373(NZ_CP013292.1)[10],neithersequencehasbeen
carefullyannotated.Inaddition,thefullgeneticdiversityofR.toxicusisnotwellrepresented
bythesetwostrainsalone[10,11].Therefore,twoadditionalstrainsofR.toxicus,FH-79(the
typestrain)andFH-232weresequenced.Becauseanestablishedsystemforgeneticmodifica-
tionofR.toxicusisnotavailable,theanalysispresentedhereusescomparativeandstructural
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R.toxicustunicamycinbiosyntheticcluster
genomicstoidentifythegeneticbasisofseveralpreviouslydescribedphenotypesincludingthe
productionoftunicamycin.
Materialsandmethods
Bacterialstrains,culture,andDNAextraction
CulturesofR.toxicusFH-79andFH-232wereobtainedfromDr.IanRiley(UniversityofAde-
laide,SouthAustralia);additionalinformationabouttheiroriginsispresentedinTable1.
R.toxicuswasmaintainedonmodifiedYGM(mYGM)[12].Oneliterofthismodifiedmedia
containedyeastextract2g,glucose1.25g,K HPO 0.25g,KH PO 0.25g,MgSO (cid:1)7H O0.1
2 4 2 4 4 2
g,andagar16g.Cultureswereincubatedat25˚Cunlessotherwisenoted;cryogenicstocks
werestoredin15%glycerolat-80˚C.DNAwasextractedusingamodifiedMarmurmethod
[13]from3dayoldliquidcultures.DNAqualitywasestimatedbyOD ratioasmeasured
260/280
onaNanodrop2000(ThermoFisherScientific)andonlyDNAwitharatio>1.6wasusedfor
sequencing.PurityoftheculturesusedforDNAextractionwasconfirmedbyplating50μlon
mYGMandmonitoringforgrowthofnon-R.toxicuscolonies.16SrDNAwassequenced
usinganAppliedBiosystems3130XL(ThermoFisherScientific)totestpurityofextracted
DNApriortogenomicsequencing;onlyextractedDNAyieldingasingle16Ssequencewas
sequencedfurther.
Genomesequencingandassembly
ForR.toxicusFH-79,ashotgunDNAlibrarywasconstructedforthe454Junior(Roche)
accordingtothemanufacturer’sdirectionsandthreesequencingrunswereperformed.In
addition,alibraryFH-79wasalsoconstructedforthePacBioRSII(PacificBiosciences);three
SMRTcellsweresequencedforFH-79attheWashingtonStateUniversityGenomicsLab.The
454sequencedatawasassembledusingLasergeneNgenv12.0(DNAStar)andPacBioreads
usingPacificBioscience’sHierarchicalGenome-AssemblyProcess(HGAP)[14];consensus
sequencesfromthetwomethodswerecomparedusingNgen.ForR.toxicusFH-232,onlya
PacBiolibrarywasconstructedand3SMRTcellsweresequencedalsoattheWashingtonState
UniversityGenomicsLab;assemblywasperformedusingPacificBioscience’sHierarchical
Genome-AssemblyProcess(HGAP)[14].Theputativetunicamycingenecluster,vancomycin
resistancegenes,and16SrDNAfromFH-79andtheCRISPRregionfromFH-232were
Table1. GenomecomparisonsofsequencedRathayibactertoxicusstrains.
Strains R.toxicusFH-79 R.toxicusFH-232 R.toxicusFH-145 R.toxicusWAC3373
alternatenames FH-137;CS14;ATCC49908 FH-100;CS37;SE3 70137;CS30 WSM194
reference thisstudy thisstudy GenBankCP010848 [10]
chromosomesize(bases) 2,343,780 2,394,755 2,328,288 2,346,032
GCcontent(%) 61.5 61.4 61.5 61.5
foldcoverage 750 850 494 99
predictedORFs 2,079 2,137 2,118 2,083
tRNAs 46 45 45 45
yearisolated 1983 1991 1980 1978
location SouthAustralia SouthAustralia WesternAustralia WesternAustralia
host Loliumrigidum Polypogonmonspeliensis Avenasativa Phalarisparadoxa
ALFPsubgroup1 B C A ---
1ALFPsubgroupdesignationswerepreviouslyidentified[11].
https://doi.org/10.1371/journal.pone.0183005.t001
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R.toxicustunicamycinbiosyntheticcluster
resequencedbyprimerwalkingonanAppliedBiosystems3130XL(ThermoFisherScientific)
tovalidategenomeassembly.
ThegenomesequencespresentedherehavebeendepositedinGenBankunderthefollowing
accessionnumbers:R.toxicusFH-79BioProjectPRJNA312185andBioSampleSAMN04495682;
R.toxicusFH-232BioProjectPRJNA312185andBioSampleSAMN06040670.
Genomeannotationandanalysis
InitialautomatedgenomeannotationwasobtainedusingtheProkaryoticGenomeAnnotation
Pipeline(PGAP)atNationalCenterforBiotechnologyInformation(NCBI)[15].Customgene
modelswereconstructedasnecessarybyaligningtheselectedinputsequencesusingmuscle
(http://www.drive5.com/muscle/)[16],followedbyinvocationofhmmbuildfromtheHMMer
version3.1.b2package(http://hmmer.org/).ThehmmscantoolfromtheHMMersuitewas
usedfordatabasescans.Predictedchromosomaloriginofreplicationwasidentifiedusing
Ori-finder(http://tubic.tju.edu.cn/Ori-Finder/)[17].Standardproteinfamilyanddomain
modelswereobtainedfromTBLASTN(https://blast.ncbi.nlm.nih.gov/Blast),Pfam(http://
pfam.xfam.org/),TIGRFam(http://www.jcvi.org/cgi-bin/tigrfams/index.cgi)andTnpPred
[18].Alien_HunterandantiSMASHwereusedtoidentifyregionswithanomalousnucleotide
compositionandputativebiosyntheticclusters,respectively[19,20];identifiedregionswere
manuallyannotatedwithspecialattentionpaidtotransposasesandknownvirulencefactorsin
otherActinobacteria.WholegenomealignmentswereperformedwithMauve[21].CRISPR
analysiswasperformedusingCRISPRFinder[22].
Phylogenetictrees
FortheActinobacteriaphylogenetictree,sequencesforgyrB,secA1and16SrDNAgenes
wereobtainedfor15representativespeciesofActinobacteria.Sequenceswereconcatenated
andalignedusingthreeiterationsoftreesearchingandrealignmentwiththeClustalOmega
algorithminMegalignPro(Lasergene).MEGA6[23]wasthenusedtoconductmodeldeter-
minationandmaximumlikelihoodtreesearches(defaultsettings)with100iterationsof
bootstrappinganalyses.Aminimumbootstrapvalueof50wasusedasacut-offlevelofsup-
porttodeterminevalidbranches.Rubrobacterradiotolerans wassetastheoutgroup.
Fortheproteasetree,aminoacidsequencesofserineproteasesputativelysecretedfrom
R.toxicusFH-79andClavibactermichiganensissubsp.michiganensisNCPPB382werealigned
withMSAProbs[24,25].Alignedsequenceswereusedtogeneratemaximum-likelihoodtrees
basedontheJones-Taylor-Thornton(JTT)modelofMEGA7.0withbootstrappingrepetitions
of1,000[26,27].
GCcontentplotandstatistics
PercentageGCcontentwasplottedusingGCcontentcalculator(www.biologicscorp.com/
tools/GCContent)withaslidingwindowsizeof2,000bp.StatisticalsignificanceofGCcontent
differenceswascalculatedbyrepeatedrandomsamplingof100013.4kbregionsoftheR.toxi-
cusFH-79genomeexcludingrDNAandtheTGCitself.
Results
Whole-genomesequencing,assembly,andannotation
Sequencedataresolvedeachgenomeintoasinglecircularchromosomeof2,343,780and
2,394,755bpforR.toxicusFH-79andFH-232,respectively;noplasmidsorotherextra-chro-
mosomalsequenceswerefoundforeitherstrain.ThePacBioSMRTsequencingtechnology
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R.toxicustunicamycinbiosyntheticcluster
wasespeciallyimportantforevenlyclosingthesehigh-GCgenomes[28].Table1compares
thesetwogenomestothepreviouslyavailableR.toxicusFH-145(NZ_CP010848)and
WAC3373[10].AllfourR.toxicusstrainshaveanaverageGCcontentofapproximately61%.
AnnotationusingNCBI’sProkaryoticGenomeAnnotationPipelineyielded2,078openread-
ingframes(ORFs)forR.toxicusFH-79and2,137ORFsforFH-232(Table1).ThisPGAP
annotationalsocontainedalargenumberofgeneswiththe\pseudokeywordduetovariations
intheplacementofthestopcodon.Manualcomparisonwithcarefullyannotatedgenomes
suggestthattheobservedvariationsingenelengtharetypicalinActinobacteria;therefore,the
\pseudokeywordwasremoved.
ThetwosequencedgenomespresentedherewerealignedwiththetwoavailableR.toxicus
genomesusingMauve[21]afterrotatingand/orreversecomplementingsequencestoplace
dnaAasthefirstgeneonthepositivestrand.AsshowninFig1,thefourgenomesareessen-
tiallysyntenic.Thepink,yellow,andblueregionsrepresentthreelocallycollinearblocks
(LCBs).Thedistinctionbetweenthepinkandblueregionsisanartifactarisingfromcircular
genomesbeingtreatedaslinearbytheMauvealgorithm.Therefore,thereareonlytwophysi-
callydistinctLCBsseparatedbyshorttranspositions;locationoftranspositionregionis
markedbyagreenlineinFig1.R.toxicusFH-232has12insertionsnotpresentintheother
genomes;thisaccountsforitslargergenomesize(Fig1C).
Predictedandannotatedopenreadingframesspannedthetypicalrangeofnecessarybio-
logicalfunctions,metabolism,cellwallbiosynthesis,defense,etc.Importantly,noORFsanno-
tatedasphagegeneswerepresent,indicatingnoprophagesareincorporatedintothebacterial
genomeandthatsampleswerefreefromcontaminatingphage.R.toxicusFH-79andFH-232
bothhavetwo16SrDNAsequencesandhave46or45tRNAs,respectively(Table1).Because
oftheextensivesimilarityamongthefoursequencedR.toxicusstrains,furtheranalysisisonly
presentedforR.toxicusFH-79exceptforrarecaseswheresignificantdifferencesexist.
R.toxicusgroupswiththeMicrobacteriaceae
AphylogeneticanalysisbasedonthreeconservedgenesclearlydemonstratesthatR.toxicusis
amemberoftheMicrobacteriaceae,mostcloselyrelatedtoLeifsoniaxyliandClavibactermichi-
ganensis(Fig2).Thesethreegenes(gyrB,secA1,and16SrDNA)arefrequentlyusedforresolv-
ingsubfamilialrelationshipsinActinobacteriaduetoappropriatelevelsofwithinsubfamily
Fig1.CollinearityoffourcompleteR.toxicusgenomes.AMauvealignmentshowstwolargelocallycollinearblocksseparatedby
shorttranspositions.Greenlineconnectsshorttransposedregion.A)R.toxicusFH-79;B)R.toxicusFH-232;C)R.toxicusFH-145
(NZ_CP010848.1);D)R.toxicusWAC3373(NZ_CP013292.1).
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R.toxicustunicamycinbiosyntheticcluster
Fig2.R.toxicusgroupswiththeMicrobacteriaceae.Maximumlikelihoodbootstrapphylogramofrepresentative
ActinobacteriashowingstrongsupportforplacementofR.toxicusFH-79intheMicrobacteriaceae.Phylogenybased
onconcatenated16S,gyrB,andsecA1sequences.
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variation[29,30].AlthoughL.xyliandC.michiganensishaveslightlylargergenomesthanR.
toxicus(2.6Mband3.3Mb,respectively,vs.2.3Mb),allthreespecieshaveGC-richgenomes
andallareplant-associated[31,32].
Tunicamycingenecluster
Aputative13.4kbtunicamycingenecluster(TGC)wasidentifiedbasedonhomologytopro-
teinsencodedbytheTGCfromStreptomyceschartreusisNRRL3882[33].AsshowninFig3A,
theR.toxicusTGChasaGCcontentmarkedlylowerthanthegenomeasawhole(52%vs.
61%).Repeatedrandomsamplingofthegenomedemonstratedthatonly0.2%ofcomparably
sizedgenomesegmentshaveaGC-contentthatislowerthantheTGC(p-value<0.002).
AlthoughtheS.chartreusisTGCappearstobeasinglepolycystronicoperonconsistingof
either12(tunA-tunL)[34]or14(tunA-tunN)[33]genes,theR.toxicusTGCcontainstwo
operons,onemonocystronic(tunC)andonepolycystronic(tunA-tunF;Fig3B).R.toxicusalso
lacksthetunMmethyltransferaseandtunNNUDIXhydrolase;however,thesegenesarenot
essentialfortunicamycinbiosynthesis[34].TheTGCinR.toxicusdoescontaintwonovel
ORFs:tunO,ahypotheticalgeneuniquetoR.toxicus,andtunP,apolyketidesynthasewitha
beta-ketoacylsynthasedomain.AllthepredictedTGCgenesarepresentinthesameorderand
orientationinallfoursequencedstrains.R.toxicusFH-145andWAC3373areidenticalatthe
nucleotideleveltotheFH-79TGCexceptfortheadditionordeletionof2or3Gsinahighly
repetitive,G-richintergenicregionupstreamoftunC.TheFH-232TGCismorethan99%
identicaltotheotherTGCregions.FH-79hasbeenpreviouslyshowntoproducetunicamycin
[7];FH-232andFH-145alsoproducetoxin.WhiletunicamycinproductionbyWAC3373has
notbeenreported,biosynthesisislikelygiventhehighlyconservedTGC.Thehypothesized
tunicamycinbiosyntheticpathwayisshowninFig3C[33,34].
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R.toxicustunicamycinbiosyntheticcluster
Fig3.Structureofthetunicamycingenecluster(TGC)fromR.toxicusandoverviewoftunicamycin
biosyntheticpathway.A)GC-contentanalysisofa28-kbregionsurroundingtheTGC.B)R.toxicusFH-79
TGCcontains12geneswithhighhomologytotungenesfromS.chartreusis(tunA-L)andtwoadditional
genes(tunOandP)intwodivergentlytranscribedoperons.C)Hypothesizedtunicamycinbiosynthetic
pathway.Incorporatedfragmentsarehighlightedinlightblue.Adaptedfrom[33,34].
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Additionalsecondarymetabolites
Toidentifyregionswithanomalousnucleotidecompositionthatmayinterferewithstatisti-
callybasedgenecallingalgorithms,Alien_Hunter[19]wasusedtoquerytheR.toxicusFH-79
genome.Suchregionsarealsoofinterestbecausetheymayarisefromhorizontalgenetransfer
eventsandaremorelikelytocontainbiosyntheticgenesforsecondarymetabolitesorvirulence
factors.Forty-tworegions,includingtheTGCdescribedabove,wereidentifiedandarelisted
inS1Table.Tofurtheraidintheidentificationofsecondarymetabolitebiosyntheticclusters,
antiSMASHwasalsousedtoquerytheR.toxicusFH-79genome[20].AsshowninS2Table,
21ofthe42regionsidentifiedwithAlien_Hunterwerealsoidentifiedwithin14antiSMASH
regions.Regionsvaryfrom5.2–28.7kbandarepredictedtoencodeawidevarietyoffunctions:
bacteriocins(lantibiotic),typeIIIpolyketidesynthase(PKS)proteins,non-ribosomalpeptide
synthetase(NRPS)proteins,multidrugeffluxpermeases,serineproteases,exopolysaccharide-
relatedproteins,TypeVIIsecretionsystem(T7SS)proteins,andnumerousYD/RHS-like
repeat-associatedproteins.
Historically,R.toxicushasbeendefinedbasedonseveraldifferentbiochemicalcharacteris-
tics.Inadditiontotheproductionoftunicamycinasdescribedabove,theseincludeyellowcol-
onycolor,exopolysaccharide“slime”production,MK-10asthepredominantisoprenoid
quinone,andanon-mevalonatepathwayforisoprenoidbiosynthesis[35,36].Althoughthe
exactbiochemicalnatureoftheyellowpigmenthasnotbeendetermined,theonlycandidate
carotenoidbiosyntheticclusterinthegenomeisshowninFig4A.Itconsistsofsixpredicted
genes:crtEb(AYW78_09695,UbiA-likeprenyltransferase);crtYf(AYW78_09700,lycopene
cyclase);crtYe(AYW78_09705,lycopenecyclase);crtBI(AYW78_09710,bifunctionalphy-
toenesynthase/oxidoreductase);crtE(AYW78_09715,geranylgeranyldiphosphatesynthase);
andispH(AYW78_09720,isopentyl-diphosphatedelta-isomerase,typeI).Theonlypredicted
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R.toxicustunicamycinbiosyntheticcluster
Fig4.Pigmentandmenaquinonebiosyntheticclusters.GeneclustersfromR.toxicusFH-79appearing
toencodeacarotenoidpigment(A)andmenaquinoneMK-10(B).Scalebartickscorrespondto1kb.
https://doi.org/10.1371/journal.pone.0183005.g004
exopolysaccharidebiosyntheticclusterintheR.toxicusgenomeispresentonantiSMASHclus-
terAS-8(S2TableandS1AFig).Thisclusterwasidentifiedbasedonsimilaritytoproteinsin
thewcm,wcn,wco,andwcqexopolysaccharidebiosyntheticclustersinClavibactermichiganen-
sissubsp.nebraskensisNCPPB2581(NC_020891.1).Thecarotenoidpigmentandthesecreted
exopolysaccharidemayaccountfortheyellowslimeobservedduringplantinfection.
Themenaquinoneprofile,alongwith16SrDNAsequenceandcellwallaminoacidcompo-
sition,wasusedtojustifymovingthetypestrainfromClavibactertoRathayibacter[36].The
predominantmenaquinoneidentifiedbySasakietal.,MK-10,isalsotheexpectedproductofa
geneclusterfromantiSMASHclusterAS-5(Fig4B).Thisclustercontainsgeneswithsimilarity
tomenB-menFandubiE,thecoremenaquinonebiosyntheticgenesfirstidentifiedinE.coli
[37],aswellasseveraladditionalgenes.TheORFlabeledidsAispredictedtoencodeageranyl-
geranylpyrophosphatesynthasethatmaybeinvolvedinbothmenaquinoneandcarotenoid
production[38].
Mostorganismsuseoneoftwodifferentpathwaystosynthesizetheimportantisoprenoid
buildingblocksisopentenylpyrophosphateanditsisomerdimethylallylpyrophosphate,either
theclassicalmevalonicacid(MVA)pathwayorthenon-mevalonate/methylerythritolphos-
phate(MEP)pathway[39].AlthoughGram-negativebacteriaonlyusetheMEPpathway,sev-
eralGram-positiveorganisms,includingmanyintheMicrobacteriaceaefamily,usetheMVA
pathway[35,39].Studiesusingtheisoprenoidbiosyntheticinhibitorfosmidomycinareconsis-
tentwithuseoftheMEPpathwaybyseveralRathayibacterspecies[35].TheR.toxicusgenome
containsORFssimilartothecoreMEPpathwayproteinsfromE.coli:DXS1-deoxy-D-xylu-
lose5-phosphatesynthase,AYW78_05260;DXR/IspC1-deoxy-D-xylulose5-phosphate
reductoisomerase,AYW78_03715;IspE4-diphosphocytidyl-2-C-methylerythritolkinase,
AYW78_07950;andabifunctionalIspD/IspF4-diphosphocytidyl-2-C-methylerythritolsyn-
thetaseand2-C-methylerythritol2,4-cyclodiphosphatesynthase,AYW78_08320.TheMVA
pathwayappearstobeabsentfromR.toxicus.
antiSMASHclusterAS-18ispredictedtoencodealantibioticorclassIbacteriocin,a
heavilymodified,ribosomallysynthesizedanti-microbialpeptide[40].Thepredictedprepro-
peptideisencodedbythegenewithlocustagAYW78_09457andisserineandalaninerich.
NeighboringORFsAYW78_09425andAYW78_09430encodeproteinscontaininglantibiotic
dehydratasedomainswhileAYW78_09455encodesaputativepeptidecyclodehydratase(S1B
Fig).AYW78_09440andAYW78_09445encodeFMN-dependentoxidasesthatmayacton
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R.toxicustunicamycinbiosyntheticcluster
thecyclizedthioesters.TheonlyR.toxicusgenethatexhibitsanysignificantsimilaritytothe
LanP-typepeptidasesinvolvedincleavinglantibioticleaderpeptidesisnotpartofthiscluster
(AYW78_08500).
AlthoughnotidentifiedbyeitherAlien_HunterorantiSMASH,itisnotablethattheR.toxi-
cusgenomeencodesthreepredictedvancomycinresistanceproteins:VanHpyruvatedehydro-
genase,AYW78_09940;VanAD-lactatedehydrogenase,AYW78_09945;andVanXD-ala-D-
alapeptidase,AYW78_09950.R.toxicusFH-79isresistanttovancomycinexperimentally.
CRISPRarrays
R.toxicuspossessesacompleteTypeI-ECRISPR-Cassystem(E.coli-type)[41]witheightcas
genesandanadjacentapproximately8.9kbCRISPRspacerarray(Fig5A).Thefourdifferent
sequencedstrainshaveslightlydifferentnumbersofnon-repetitivespacersequencesand
conserveddirectrepeats.R.toxicusFH-79andFH-145bothhave145non-repetitivespacer
sequencesand146conserveddirectrepeatswhileWAC3373has144and145andFH-232has
139and140,respectively.Non-repetitivespacersequencesrevealednoidentitytoknownplas-
midorphagesequences.
Predictedpathogenicity-relatedgenes
RelativetotherelatedphytopathogenClavibactermichiganensissubsp.michiganensis,R.toxi-
cuspossessesalimitedarsenalofplant-associatedcell-wallhydrolyzingenzymes,consistingof
onlyasinglepolygalacturonase(AYW78_01285)andpectatelyase(AYW78_01485).Thisis
consistentwiththelifestrategyofR.toxicus,whichapparentlycannotinfectplantleavesor
stemsbutmostacquirenutrientsinseedgallsinitiatedbynematodeinfestation.However,R.
toxicusdoespossessnumeroussecretedserineproteasesthatsharesequencehomologytothe
pathogenicity-associatedproteinPat-1ofC.michiganensissubsp.michiganensis.Atotalof11
secretedserineproteaseswereidentifiedwithanadditionalconservedpseudogene;allcontain
predictedsignalpeptidessuggestingextracellularlocalizationasdescribedinC.michiganensis
subsp.michiganensis.ThecorrespondinggenesweredesignatedchpA-K(chromosomal
homologytopat-1)andsbtA(subtilisin-likeserineprotease).IncontrasttoC.michiganensis
subsp.michiganensis,thesecretedserineproteasesaredispersedthroughoutthechromosome,
butseveraloftheproteasesarelocatedincloseproximityincluding:(i)chpG,chpH,chpK
(pseudogene)and(ii)chpB,chpC.
Phylogenetically,theserineproteasesofR.toxicusandC.michiganensissubsp.michiganen-
sisappeardistinctwiththemajorityofR.toxicusproteases(ChpB-E,ChpI-J)formingasub-
group(Fig6).NoR.toxicusserineproteasesclusteredwiththeC.michiganensissubsp.
michiganensisPpafamilyorplasmid-associated(PhpA-B)serineproteases.Thesubtilisin-like
serineproteasesofR.toxicusandC.michiganensissubsp.michiganensisweretheonlysecreted
proteasestoclusteracrossspecies(Fig6).
Discussion
ThekeyfeatureofR.toxicusisitsabilitytoexploitaprotectedenvironmentalniche,thedevel-
opinggrassseed,andproducetunicamycin,apotenttoxinforgrazinglivestock.Priortothe
workpresentedhere,verylittlewasknownaboutthebiosynthesisoftunicamycinbyR.toxicus.
UntilthepublicationofthephageNCPPB3778sequence[9],itwashypothesizedthattunica-
mycinproductioncouldresideinthephageratherthanonthebacterialchromosome.How-
ever,noORFswithsimilaritytoknowntunicamycinbiosyntheticgeneswerefoundinthe
phagegenome[9].Thediscoveryofatunicamycingenecluster(TGC)inR.toxicus(Fig3)
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R.toxicustunicamycinbiosyntheticcluster
Fig5.MaximumlikelihoodphylogenetictreeofputativelysecretedserineproteasesforR.toxicusFH-79andC.michiganensis
subsp.michiganensisNCPPB382.Percentageoftreesinwhichtheassociatedtaxaclusteredtogetherisshownnexttothebranches;
valueslessthan70havebeenomitted.R.toxicusFH-79isdesignatedwithblackdiamonds;genenameandaccessionnumbersare
displayedinparentheses.
https://doi.org/10.1371/journal.pone.0183005.g005
withsimilaritytothepreviouslycharacterizedclusterfromS.chartreusisisanimportantfirst
stepinunderstandingtoxinproductioninthisbacterium.BoththelowerGCcontentofthe
TGCanditssimilaritytoStreptomycesindicatethatR.toxicusprobablyacquiredtheabilityto
synthesizetunicamycinviaahorizontalgenetransferevent;however,theTGCdoesnot
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