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Methods in Molecular Biology 1373

The Genetic Manipulation of Staphylococci

Methods and Protocols

M ETHODSIN M OLECULAR B IOLOGY

SchoolofLifeandMedicalSciences

UniversityofHertfordshire Hatfield,Hertfordshire,AL109AB,UK

Forfurthervolumes: http://www.springer.com/series/7651

TheGeneticManipulation ofStaphylococci

MethodsandProtocols

UniversityofKansasMedicalCenter,KansasCity,KS,USA

Editor JeffreyL.Bose

UniversityofKansasMedicalCenter

KansasCity,KS,USA

ISSN1064-3745ISSN1940-6029(electronic) MethodsinMolecularBiology

ISBN978-1-4939-3157-6ISBN978-1-4939-3158-3(eBook) DOI10.1007/978-1-4939-3158-3

LibraryofCongressControlNumber:2015951972

SpringerNewYorkHeidelbergDordrechtLondon # SpringerScience+BusinessMediaNewYork2016

Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthematerialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproduction onmicrofilmsorinanyotherphysicalway,andtransmissionorinformationstorageandretrieval,electronicadaptation, computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnot imply,evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsand regulationsandthereforefreeforgeneraluse.

Thepublisher,theauthorsandtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeenmade.

Printedonacid-freepaper

HumanaPressisabrandofSpringer

SpringerScience+BusinessMediaLLCNewYorkispartofSpringerScience+BusinessMedia(www.springer.com)

Preface

Theabilitytogeneticallymanipulatepathogenicbacteriaisessentialtounderstandingtheir physiology,responsestoenvironmentalchanges,andvirulence.Geneticmanipulationcan comeinmanyforms,includingbothdirectedandunbiasedapproaches.Historically, clinicalisolatesof Staphylococcus haveposedachallengeforresearcherstostudydueto theirinabilitytoacquireandmaintainplasmidsgeneratedinotherbacteriasuchas E.coli Thisbarrierinvolvesbacteria-specificDNArecognitionsystemsdesignedtopreventthe inclusionofforeignDNA,atopicdirectlyaddressedintheRestrictionModifications Systemschapterandhighlightedinmanyotherchapterswithinthisbook.Whilethese systemsarepotentbarrierstogeneticmanipulationofthestaphylococci,ourincreased understandingofthesesystemshasgreatlyenhancedourabilitytomoveplasmidsinto thesebacteria.ThesecondhurdletoovercomeinvolveswaysinwhichDNAistransferred into Staphylococcus cells.Thesebacteriaarenotnaturallycompetent,norcancompetence beinduced.Inaddition,whilepossible,conjugationisrarelyusedforsmallplasmids. Furthermore,unlike E.coli,staphylococcicannotbetransformedusingasimpleheatshockmethod.Togetplasmidsintostaphylococci,electrocompetentcellsmustbemade. Oftenthisisaccomplishedusingthe S.aureus laboratoryisolateRN4220,originally createdbyDr.RichardNovick’sresearchgroup,amutatedstrainthatreadilyaccepts DNAfrom E.coli.OnceinRN4220,plasmidsareoftenshuttledintostrainsofinterest usingbacteriophage,whichcaneffectivelybeusedtotransferbothplasmidsandchromosomalfragmentsbetweenstrains.Thisbookcontainsreviewchaptersandmethodswith vitalinformationtoaccomplishallofthesetasks.

Generatingmutantsof Staphylococcus canbeaccomplishedinmanyways,depending ontheintendedchanges.Toaidresearchers,thisbookcontainschaptersusingunbiased approachessuchaschemicalandtransposonmutagenesis,aswellasaprotocolforallelic exchangetomaketargetedmutants.Usingthesemethods,bothourlabandothershave beensuccessfulatmakingmutantsthatspanindividualsinglenucleotidechangesinthe chromosometowholegenomemutantlibraries.Thelatterstrategyprovidesausefultool forhigh-throughputscreening,whilesinglenucleotidechangesareanelegantwayof teasingaparttheimportanceofsinglenucleotidesingeneexpressionorcreatingspecific aminoacidsubstitutionstoexamineproteinfunction.

Tocomplementthechaptersdirectedtowardactualgeneticmanipulation,thisbook includesadditionalchaptersfornucleicacidanalysis.qRT-PCRisasensitivetoolthatcan beusedtoexaminespecificgeneexpressionchanges,whilenewlydevelopedRNAseq techniquesprovideapowerfulmeanstoexaminetheentiretranscriptome.Bothofthese toolsareessentialtounderstandinggeneexpressionandcanserveasabeginningtodirect mutantmakingorasameanstotesttheconsequencesofthegeneratedmutant.Furthermore,theincludedchaptersonRACEandEMSAsprovideusefulmethodsformapping RNAanddetectinginteractionsbetweenDNAandproteins,respectively.

Geneticmanipulationisoftenseenasadauntingtasktothoseunfamiliarwiththe topic.Indeed,nottoolongagoourcurrentcapabilitytomanipulateandstudy Staphylococcus wasmerelysciencefiction.Weareindebtedtothoseindividualsandresearchgroups whopioneeredthemethods/toolscoveredinthisbook.Thegoalforthisbookistoserve asaresourceandguidetoscientistsinthestaphylococcalcommunityastheypursuetheir studiesonthesebacteria.Whilemostprotocolswithinthisbookarewrittenwitha particularspeciesinmind,thesemethodsshouldbeadaptabletootherspecies.

KansasCity,KS,USAJeffreyL.Bose

UnderstandingStaphylococcalNomenclature .................................1

ChristinaN.KruteandJeffreyL.Bose

Restriction–ModificationSystemsasaBarrierforGeneticManipulation of Staphylococcusaureus ...................................................9

MaratR.Sadykov

ComplementationPlasmids,InducibleGene-ExpressionSystems, andReportersforStaphylococci. ............................................25

RalphBertram

DeNovoAssemblyofPlasmidsUsingYeastRecombinationalCloning

AmeyaA.MashruwalaandJeffreyM.Boyd

SplicingbyOverlapExtensionPCRtoObtainHybridDNAProducts ...........43

JustinA.Thornton

MethodforPreparationandElectroporationof S.aureus and S.epidermidis .........................................................51

MelindaR.GrosserandAnthonyR.Richardson

RapidIsolationofDNAfrom Staphylococcus .................................59

KelseyL.KrauszandJeffreyL.Bose

BacteriophageTransductionin Staphylococcusaureus:Broth-BasedMethod .....63

KelseyL.KrauszandJeffreyL.Bose

BacteriophageTransductionin Staphylococcusaureus..........................69

MichaelE.Olson

BatchTransductionofTransposonMutantLibrariesforRapid PhenotypeScreeningin Staphylococcus ......................................75

KatherineL.Maliszewski

ConjugativeTransferin Staphylococcusaureus ................................83

CortneyR.HalseyandPaulD.Fey

AllelicExchange ...........................................................89

McKenzieK.Lehman,JeffreyL.Bose,andKennethW.Bayles

CreationofStaphylococcalMutantLibrariesUsing TransposonTn917 .........................................................97

KellyC.Rice

GenerationofaTransposonMutantLibraryin Staphylococcusaureus and Staphylococcusepidermidis Using bursaaurealis...........................103 VijayaKumarYajjala,ToddJ.Widhelm,JenniferL.Endres, PaulD.Fey,andKennethW.Bayles

ChemicalandUVMutagenesis.. ............................................111

JeffreyL.Bose

PulseFieldGelElectrophoresis.. ............................................117

BatuK.Sharma-Kuinkel,ThomasH.Rude,andVanceG.FowlerJr.

RNA-Sequencingof Staphylococcusaureus MessengerRNA ....................131 RonanK.Carroll,AndyWeiss,andLindseyN.Shaw

QuantitativeReal-TimePCR(qPCR)WorkflowforAnalyzing Staphylococcusaureus GeneExpression.. ....................................143 AprilM.LewisandKellyC.Rice

ElectrophoreticMobilityShiftAssays .........................................155 SarahE.RoweandJamesP.O’Gara

RapidAmplificationofcDNAEndsforRNATranscriptSequencingin Staphylococcus. ...........................................................169 EricMiller Index

Contributors

KENNETH W.BAYLES CenterforStaphylococcalResearch,DepartmentofPathology andMicrobiology,UniversityofNebraskaMedicalCenter,Omaha,NE,USA

RALPH BERTRAM DepartmentofMicrobialGenetics,InterfacultyInstituteofMicrobiology andInfectionMedicineTu¨bingen(IMIT),UniversityofTu¨bingen,Tu¨bingen, Germany;EurofinsInstitutJagerGmbH,Tu¨bingen,Germany

JEFFREY L.BOSE DepartmentofMicrobiology,MolecularGeneticsandImmunology, TheUniversityofKansasMedicalCenter,KansasCity,KS,USA

JEFFREY M.BOYD DepartmentofBiochemistryandMicrobiology,RutgersUniversity, NewBrunswick,NJ,USA

RONAN K.CARROLL DepartmentofBiologicalSciences,OhioUniversity,Athens,OH,USA

JENNIFER L.ENDRES CenterforStaphylococcalResearch,DepartmentofPathologyand Microbiology,UniversityofNebraskaMedicalCenter,Omaha,NE,USA

PAUL D.FEY CenterforStaphylococcalResearch,DepartmentofPathology andMicrobiology,UniversityofNebraskaMedicalCenter,Omaha,NE,USA

VANCE G.FOWLER JR DepartmentofMedicine,DukeUniversityMedicalCenter, Durham,NC,USA

MELINDA R.GROSSER DepartmentofMicrobiologyandImmunology,UniversityofNorth CarolinaatChapelHill,ChapelHill,NC,USA

CORTNEY R.HALSEY CenterforStaphylococcalResearch,DepartmentofPathology andMicrobiology,UniversityofNebraskaMedicalCenter,Omaha,NE,USA

KELSEY L.KRAUSZ DepartmentofMicrobiology,MolecularGeneticsandImmunology, TheUniversityofKansasMedicalCenter,KansasCity,KS,USA

CHRISTINA N.KRUTE DepartmentofMicrobiology,MolecularGeneticsandImmunology, TheUniversityofKansasMedicalCenter,KansasCity,KS,USA

MCKENZIE K.LEHMAN CenterforStaphylococcalResearch,DepartmentofPathology andMicrobiology,UniversityofNebraskaMedicalCenter,Omaha,NE,USA

APRIL M.LEWIS DepartmentofMicrobiologyandCellScience,IFAS,Universityof Florida,Gainesville,FL,USA

KATHERINE L.MALISZEWSKI UniversityofNebraskaMedicalCenter,Omaha,NE,USA

AMEYA A.MASHRUWALA DepartmentofBiochemistryandMicrobiology,Rutgers University,NewBrunswick,NJ,USA

ERIC MILLER DepartmentofMicrobiologyandImmunology,SchoolofMedicine, UniversityofNevadaReno,Reno,NV,USA

JAMES P.O’GARA DepartmentofMicrobiology,SchoolofNaturalSciences,National UniversityofIreland,Galway,Ireland

MICHAEL E.OLSON DepartmentofMedicalMicrobiology,ImmunologyandCellBiology, SouthernIllinoisSchoolofMedicine,Springfield,IL,USA

KELLY C.RICE DepartmentofMicrobiologyandCellScience,IFAS,UniversityofFlorida, Gainesville,FL,USA

ANTHONY R.RICHARDSON DepartmentofMicrobiologyandImmunology, UniversityofNorthCarolinaatChapelHill,ChapelHill,NC,USA

SARAH E.ROWE AntimicrobialDiscoveryCenter,DepartmentofBiology, NortheasternUniversity,Boston,MA,USA

THOMAS H.RUDE DepartmentofMedicine,DukeUniversityMedicalCenter,Durham, NC,USA

MARAT R.SADYKOV CenterforStaphylococcalResearch,DepartmentofPathology andMicrobiology,UniversityofNebraskaMedicalCenter,Omaha,NE,USA

BATU K.SHARMA-KUINKEL DepartmentofMedicine,DukeUniversityMedicalCenter, Durham,NC,USA

LINDSEY N.SHAW DepartmentofCellBiology,Microbiology,andMolecularBiology, UniversityofSouthFlorida,Tampa,FL,USA

JUSTIN A.THORNTON DepartmentofBiologicalSciences,MississippiStateUniversity, MississippiState,MS,USA

ANDY WEISS DepartmentofCellBiology,Microbiology,andMolecularBiology, UniversityofSouthFlorida,Tampa,FL,USA

TODD J.WIDHELM CenterforStaphylococcalResearch,DepartmentofPathology andMicrobiology,UniversityofNebraskaMedicalCenter,Omaha,NE,USA

VIJAYA KUMAR YAJJALA CenterforStaphylococcalResearch,DepartmentofPathology andMicrobiology,UniversityofNebraskaMedicalCenter,Omaha,NE,USA

MethodsinMolecularBiology(2016)1373:1–7

UnderstandingStaphylococcalNomenclature

ChristinaN.KruteandJeffreyL.Bose

Abstract

Bacteriaareoftengroupedbyavarietyofproperties,includingbiochemicalactivity,appearance,andmore recently,nucleicacidsequencedifferences.Inthecaseofhumanpathogens,significantworkgoesinto “typing”strainstounderstandrelatedness.Thisisespeciallytruewhentryingtounderstandtheepidemiologyoftheseorganisms.InattemptstogroupStaphylococci,avarietyofmethodsandnomenclatureshave beenemployed,whichcanoftenserveasapointofconfusiontothoseenteringthefield.Therefore,the intentofthischapteristogiveabriefoverviewofsomecommonmethodsandassociatednomenclature usedtotypeStaphylococci,with S.aureus asanexample.

Keywords: Spatyping,MLST,PFGE,SCCmec,MLVA, Staphylococcus typing

1Introduction

Thegenus Staphylococcus wasoriginallygroupedwith Micrococcus, andwasfirstnamedin1883byAlexanderOgstonduringamicroscopicexaminationofpusassociatedwithmicrococcuspoisoning. Inhisexamination,Ogstondescribedtheexistenceoftwodistinct celltypes,chainedcocciandthosethatgroupedsimilartoabunch ofgrapes,whichhetermed Staphylococcus (Greek σταφυλη (staphule ´ ),abunchofgrapes)[1].Thefollowingyear,Anton J.Rosenbachisolatedandnamedtwo Staphylococcus species, S.aureus (gold)and S.albus (white),nowcalled S.epidermidis,basedontheir pigmentation[2].

Thegenus Staphylococcus acquireditsnameduetoitsappearanceasgrape-likeclusters,resultingfromcelldivisionoccurring alongtwoaxes.Aswithmostbacteria,theStaphylococciwere originally(andstillare)groupedbiochemically.Generallyspeaking, butwithexception,thegenusismetabolicallydiverseandare catalase-positive,oxidase-negativefacultativeanaerobes. Staphylococcus cellsarerelativelysmall(0.5–1.0 μmindiameter)grampositivecocciwithlow-GC-content(~30–40%DNA)genomes rangingfrom2.3to3.2Mb.Thiswiderangeinpossiblegenome sizeisduetocomplexdiversityinmobilegeneticelements,includingtransposons,bacteriophages,andantibioticresistancecassettes, amongdifferingStaphylococcistrains[3].

Therearecurrentlygreaterthan40recognizedspeciesof Staphylococcus andseveralsubspeciesthatgenerallyresideasnormal floraofmammalsandbirds(reviewedin[4]).Despitethisubiquity, themostwell-knownspeciesare S.epidermidis and S.aureus dueto theirimportanceinhumandisease.Becauseoftheirsignificancein humanhealth,muchworkhasgoneintoclassifyingortyping strains.Twocommon,yetnotcompletelyaccurate,waysofdiscerningbetweenthesetwospeciesaretheproductionofcoagulase, asecretedproteinwhichbindstohostprothrombin[5],orstaphyloxanthin,amembrane-associatedcarotenoid[6].Itisgenerally regardedthat S.aureus iscoagulase-positive,whilenon-aureus Staphylococcilackcoagulaseandarethereforecategoricallycalled coagulase-negative staphylococciorCoNS.Whilethisisusually true,some S.aureus isolatesdonotproducecoagulaseandsome otherspecieshavebeenshowntomakethisenzyme.Similarly, S. aureus isoftensingledoutbyitsgoldenpigment,theresultof staphyloxanthinproduction.However S.aureus isolatesmake greatlyvaryingamountsofpigment,andoftenpigmentationis onlyobviousafterprolongedgrowth.

Inadditiontotheaboveanalyses,thereisadizzyingarrayof methodstotype Staphylococcus isolates.Thefollowingsectionsare intendedtoserveasabriefintroductiontoorientatereadersto someofthecommonnomenclatureused,with S.aureus (themost complex)servingasanexample. S.aureus isolateshavecometobe groupedbyavarietyofdesignationswithnosingletypingmethod universallyused.Forexample,theimportantclinicalisolate FPR3757isaCA-MRSAUSA300ST8belongingtoclonalcomplexCC8withspatypet008andSCCmecIV.

2AntibioticResistanceStatus

Asisthecasewithmanyhumanpathogens,theemergenceor identificationofantibiotic-resistant S.aureus generallyappearsin ashortperiodoftimefollowinginitialclinicaloragriculturaluseof aparticularantibiotic.Thisisnotsurprisingsincebacteriaare efficientathorizontalgenetransferthatallowsfortheacquisition ofantibioticresistancegenes.Inaddition,whileantibioticsarenew tohumans,bacteriahavebeenengaginginthisformofchemical warfareformillennia.Thefirstmajordelineationof S.aureus strain designationbasedonanantibioticresistancephenotypewasin responsetotheantibioticmethicillin.Today,ifnoothercharacteristicisreportedonan S.aureus isolateitwillcertainlybe determinedtobe methicillin-resistant S. aureus (MRSA)or methicillin-susceptible S aureus (MSSA).Ofnote,MRSAisoften mistakenlycalled“multipleresistant.”Theantibioticmethicillin wasfirstintroducedin1959andjust2yearslater,in1961,the firstclinicalMRSAisolatewasidentifiedfromahospital[7].Itwas 2ChristinaN.KruteandJeffreyL.Bose

laterfoundthatmethicillinresistanceisduetotheacquisitionof the mecA gene,encodingthealternativepenicillin-bindingprotein PBP2a.Inaddition,PBP2aconfersresistancetoother β-lactam antibiotics.Sinceitsfirstemergence,MRSAhasbecomeasignificantglobalhealthproblem.

InresponsetotheemergenceandspreadofMRSA,vancomycin wasfast-trackedthroughtheFoodandDrugAdministrationin 1958,andbecamethedrugofchoiceforseriousandconfirmed MRSAinfections.In1997,thesecondmajorantibioticresistance designationfor S.aureus emergedwiththereportof vancomycinintermediateresistant S aureus (VISA)strainMu50inJapan[8]. VISAstrainsaremoderatelyresistanttovancomycin,whichisdue nottotheacquisitionofnewgenes,butinsteadcorrelateswith mutationsinexistinggenesthatareofteninvolvedinaffectingcell wallmetabolism[9–11].Thesemutantstypicallyhaveathickercell wallandmoreD-Ala-D-Alavancomycintargets,potentiallydiluting outthevancomycintosub-inhibitoryconcentrations.Itwasnot until2002thatthefirst vancomycin-resistant S aureus (VRSA) strainwasisolatedbytheMichiganStateDepartmentofHealth [12].ThisstrainhadanMIC ¼ 1024 μg/mlresultingfromthe acquisitionofthe vanA genefoundonTn1546 fromavancomycinresistantEnterococciduringacoinfection.In2006,theClinicaland LaboratoryStandardsInstituteupdatedthevancomycinsusceptibilitybreakpointas 2 μg/mlfor vancomycin-susceptible S. aureus (VSSA),4–8 μg/mlforVISA,and 16 μg/mlforVRSA[13].

3AcquisitionofInfection

Inadditiontoantibioticresistancestatus,pathogenicStaphylococci areoftendesignatedbasedonthesourceoftheinfection.Historically,Staphylococcispecies,including S.aureus,wereconsideredto beopportunisticpathogens,primarilyassociatedwithinfectionsof immunocompromisedindividualsorthosewhoundergosurgical proceduresinhospitals.Theseisolatesareoftencalled hospital acquiredor healthcare associated(HA).Incontrast,intheearly 1990s,strainsemergedthatcauseoutbreaksinthecommunity, independentofhospitalassociation.These community-associated (CA) S.aureus strainsareeasilytransmissible,morereadilyleadto dangerousinvasivediseases,andcaninfectotherwisehealthyindividuals.Theseinfectionsareespeciallyprevalentinmilitarypersonnel,athletes,students,children,andprisoninmatesastheyarein closecontactwithoneanother[14–17].Thisincreasingtrendof CA-MRSAinfectionsinotherwisehealthyindividualsleadstoquestioningoftheuseoftheterm“opportunistpathogen”for S.aureus Inaddition,someepidemicCA-MRSAstrainshavespreadquickly andaccountformanyhospital-acquiredinfections,andassuchare beginningtodisplaceHA-MRSAinclinicalsettings[18].

InadditiontoHA-MRSAandCA-MRSAdesignations,itisnot uncommontofindtheLA-MRSAabbreviationintheliterature, whereinLArefersto livestock-associatedstrains.Thishasbeen heightenedbyreportsofhumaninfectionsbyLA-MRSA,which insomecommunitiescanaccountfor15%ofalldeepskinandsoft tissueinfections[19].LA-MRSAinfectionstypicallycorrelatewith directcontactwithanimals;however,recentreportssuggestthat humansresidingclosetofarms,butwithoutdirectcontact,can becomecolonizedaswell.Inaddition,LA-MRSAstrainshavebeen foundinrawmeat[20]andmilk[21],whichcouldbecontributing factorstothespreadofthesestrainstohumans.

4PFGE

Pulsed-fieldgelelectrophoresis(PFGE)isahighlydiscriminatory waytotype Staphylococcus strains.Thistechnique(seechapterof thisbook)reliesondigestinggenomicDNAwiththeinfrequently cuttingendonucleaseSmaIandsubsequentseparationbyagarose electrophoresis.Unlikeconventionalelectrophoresis,thealternatingvoltageofPFGEallowsforseparationandresolutionoflarge DNAfragments.Separationoffragmentsresultsinauniquebandingpatternthatcanbeusedtoidentify Staphylococcus lineageswhen comparedtoknownstrains.Tobeconsistent,astrictprotocolis necessaryinordertocomparebetweenlaboratories.Thistechnique hasbeenwelladoptedintheUSAatthenationallevelandresultsin thereportingofaUSAtype,suchasUSA300.

5SpaLocus

WhilePFGEhasbeenusedextensivelyfortyping S.aureus strains, particularlyforepidemiologicalstudiesofoutbreaks,itdoeshave limitations.ComparingDNApatternsbygelelectrophoresisis complicatedifonedoesnothavetheappropriatereferencestrains available.Tocounterthis,single-locussequencingofthe spa gene wasproposedin1996[22].Spatypinginvolvessequencingthe30 X-regionofthe spa gene,whichcontainsavariablenumberof tandemrepeatsequences.Uponsequencing,repeatsareassigned anumericalcodebasedontheorderoftherepeats.Spatypingis curatedbySeqNet.org[23]viathe spa server(www.spaserver. ridom.de),whichharborsalargedatabase(asofwriting,14,883 Spatypesfromthousandsofstrainsand57countries).Sequencing ofthissinglelocusprovidesgooddiscriminatorypoweratan affordablecostandreasonableworkloadwhentypingmultiple strains.

Multilocus sequence typing(MLST)wasfirstsuggestedfor S.aureus asameanstomorediscriminatelydifferentiatebetween epidemic methicillin-resistant Staphylococcus aureus (EMRSA)and virulentMSSAstrains[24].Thetechniqueinvolvessequencing 402–516basepairsofsevenhousekeepinggenes, arc (carbamate kinase), aroE (shikimatedehydrogenase), glp (glycerolkinase), gmk (guanylatekinase), pta (phosphateacetyltransferase), tpi (triosephosphateisomerase),and yqiL (acetylcoenzymeAacetyltransferase). TheresultingsequencesarethendepositedintotheMultiLocus SequenceTypinghomepage(www.mlst.net).Bycomparingthe sequences,isolatesareassignedasequencetype(ST).Furthermore, STsthatshare6/7allelescanbeassignedtoclonalcomplexes(CC) usingeBURST(eburst.mlst.net)[25].Forconsistency,theMLST homepageprovidesdetailsonisolatingDNA,primersequences, andPCRconditions.

Muchlike S.aureus, S.epidermidis ismostoftentypedbyeither PFGEorMLST.Inthiscase,MLSTsequencingisperformedfor thefollowinggenes: arcC (carbamatekinase), aroE (shikimate dehydrogenase), gtr (anABCtransporter), mutS (DNArepair protein), pyrR (regulatorofpyrimidineoperon), tpiA (triosephosphateisomerase),and yqiL (acetylcoenzymeAacetyltransferase) [26].TheresultingdataisimportedintotheMLSThomepage (sepidermidis.mlst.net)andresultsintheassignmentofanSTtype.

The mecA geneisfoundonthemobilegeneticisland staphylococcal cassette chromosome mec,SCCmec.Whiletheoriginsof SCCmec areunclear,itisbelievedtohaveoriginatedinanonaureusStaphylococcus speciesandthenwasacquiredby S.aureus viahorizontalgenetransfer.SequencingoftheSCCmec ledtothe observationthatthisgeneticislandishighlyvariablebetween strains,andthuscouldserveasmeansoftypingSCCmec-positive strains.Todate,thereare11recognizedSCCmec typescontaining multiplesubtypesbasedonseveralcriteria.Duetotheoverall complexnatureofSCCmec typing,itwillnotbecoveredindetail heresincetheInternationalWorkingGroupontheStaphylococcal CassetteChromosomeelementswebsite(www.sccmec.org)providesawrittenandvisualoverviewofSCCmec typingwithstrain examples.

7SCCmec

Multilocus variable-numbertandemrepeat analysisseekstoprovide gooddiscriminatorypowerinahigh-throughputandlow-cost method.Thistypingmethodutilizesgenomicregionscontaining naturalvariablerepeats.Therepeatregionsareamplifiedbymultiplex PCRusingprimerstargeting sdrCDE, clfA, clfB, sspA, spa, mecA,and fnBP in S.aureus [27, 28].For S.epidermidis,primersamplifyregions of sdrG, sdrH,SERP0719,SERP2395, aap,and mecA [29, 30]. Followingamplification,theproductsareseparatedbygelelectrophoresis.MuchlikePFGE,theresultingbandingpatternis indicativeofstrainrelatedness.Oneadvantagetothismethodisthe abilityforautomation,makingitattractivetoclinicalepidemiology.

9LOOKINGFORWARD

Asisthecasewithmosttechniques,eachtypingmethodhasadvantagesanddisadvantagesincludingtime,cost,andfeasibility.Inaddition,dependingonones’need,onlyacertainlevelofidentityor phylogenymaybedesiredorneeded.Foraparticularstudyorepidemiologyanalysis,itmaynotbenecessarytohavesomeorallofthese typingdetails.Movingforward,thosestudiesneedingextremely detailedanalysiswilllikelytakeadvantageofthedecreasingcostsof whole-genomesequencing.Interestingly,arecentstudyofwholegenomesequencesrevealedthat S.aureus contains47mutational hotspotsconsistingof~2.5%ofthegenome[31].Sequencingof thesehotspotsshowedgoodresolutionofstrains,but,notsurprisingly,wasnotasstrongassequencingofthewholegenome.Ofnote, whilecostofwhole-genomesequencinghasrapidlydecreasedin recentyears,perhapsthelimitingfactorofperformingsuchdetailed studyisthebioinformaticstimeandcomputationalpowerneededto handleawhole-genomesequencingproject.

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3.MalachowaN,DeLeoFR(2010)Mobile geneticelementsof Staphylococcusaureus.Cell MolLifeSci67:3057–3071

4.MathemaB,MediavillaJR,ChenLetal(2009) EvolutionandtaxonomyofStaphylococci.In: CrossleyKB,JeffersonKK,ArcherGL,Fowler VGJr(eds)Staphylococciinhumandisease, 2ndedn.BlackwellPublishing,Hoboken,NJ, pp31–64

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MethodsinMolecularBiology(2016)1373:9–23

DOI10.1007/7651_2014_180 © SpringerScience+BusinessMediaNewYork2014

Publishedonline:03February2015

Restriction–ModificationSystemsasaBarrierforGenetic Manipulationof Staphylococcusaureus

MaratR.Sadykov

Abstract

Geneticmanipulationisapowerfulapproachtostudyfundamentalaspectsofbacterialphysiology, metabolism,andpathogenesis.Most Staphylococcusaureus strainsareremarkablydifficulttogenetically manipulateastheypossessstronghostdefensemechanismsthatprotectbacteriafromcellularinvasionby foreignDNA.In S.aureus thesebacterial“immunity”mechanismsagainstinvadinggenomesaremainly associatedwithrestriction–modificationsystems.Todate,prokaryoticrestriction–modificationsystemsare classifiedintofourdifferenttypes(TypeI–IV),allofwhichhavebeenfoundinthesequenced S.aureus genomes.Thischapterdescribestheroles,classification,mechanismsofactionofdifferenttypesof restriction–modificationsystemsandtherecentadvancesinthebiologyofrestrictionandmodificationin S.aureus.

Keywords: Staphylococcusaureus,RMsystems,REase,MTase,REBASE,Geneticmanipulation

1Introduction

Staphylococcusaureus isamajorhumanpathogenandaleading causeofbothnosocomialandcommunity-associatedinfections worldwide(1).Functionalgenomicsandgeneticmanipulationof S.aureus areessentialapproachesforstudyingthefundamental principlesofstaphylococcalphysiology,virulence,andpathogenesis,andforthediscoveryanddevelopmentofnewstrategiesto combatthesemultidrug-resistantbacteria.Themajorityofclinical isolatesof S.aureus arenotoriouslydifficulttomanipulategeneticallyastheypossessstronghostdefensemechanismslimitinghorizontalgenetransfer(HGT).Inrelationshiptothegenetic manipulationofstaphylococci,thistranslatestoadifficultyof transferringrecombinantDNAfromotherbacterialike Escherichia coli orligationproductsdirectlyintothetargetcells.In S.aureus thedefensemechanismsprotectingbacteriafrominvaderDNAare mainlyassociatedwithdifferenttypesofrestriction–modification (RM)systems.Understandingthebiologyofrestrictionandmodificationinvariousclinicalisolatesof S.aureus iscriticalforovercomingstrain-specificRMbarriersforDNAuptakeandfor developingbettergeneticengineeringtoolsandtechniques.

Fig.1 Thehostdefensefunctionofrestriction–modificationsystems.RMsystemsrecognizethemethylation statusofDNAattheirspecifictargetsequence.FullymethylatedDNAisrecognizedtobeapartofthehost genomeandhemimethylatedDNAisrecognizedasnewlyreplicatedhostDNA.Themethylationstatusofthe genomicDNAismaintainedbythemethyltransferase(MTase).IncomingunmethylatedDNAsuchasthatof plasmidforexampleisrecognizedtobeforeignandcleavedbytherestrictionendonuclease(REase)

BacterialRMsystemstypicallyconsistofenzymesresponsible fortwoopposingactivitiesandmayfunctionasprimitiveprokaryoticimmunesystemsthatattackforeignDNAenteringthecell(2). Restrictionactivityisbasedontheactionofarestrictionendonuclease(REase),whilemodificationactivityiscarriedoutbyasitespecificDNAmethyltransferase(MTase).ClassicalREsesrecognize foreignDNAsuchasthatofbacteriophages,plasmids,andother mobilegeneticelementsbytheabsenceofpropermethylation withinspecificrecognitionsitesandtheninactivateinvadergenomesbyendonucleolyticcleavage(Fig. 1)(2–4).Theircognate site-specificMTasesprotectDNAfromcleavagebymethylating cytosineoradeninebaseswithinthesamerecognitionsequence (Fig. 1)(2, 3).Generally,thehemimethylatedstateofDNAatthe recognitionsitesconfersprotectiontothecleavage,andhostDNA isusuallyfullymethylatedbyaresidentMTasefollowingreplication whileinvadingforeignDNAisnot(Fig. 1).RMsystemsarewidely spreadamongprokaryoticorganisms,anddifferentbacterialspecies andstrainshavetheirowncombinationsofrestrictionandmodificationenzymes.Inaddition,asingleprokaryoticorganismoften retainsmultipleRMsystems(http://rebase.neb.com/rebase/ rebase.html).

TheindividualhighspecificityandtheprevalenceofRM enzymesinprokaryoticorganismsaswellastheoftenoccurrence oftightlinkagebetweencognaterestrictionandmodificationgenes suggesttheirdiversebiologicalfunctions.Inadditiontocellular defenseandimmigrationcontrolofDNAinbacteria(5),several importantbiologicalroleshavebeenattributedtoRMsystemsso far(seerecentreview(6)).Amongothers,theserolesincludethe functioningofRMsystemsasselfishmobilegeneticelements (7–9),stabilizationofgenomicislandsandothermobileelements inthecellpopulation(6, 10–13),involvementinrecombination andgenomerearrangements(14–19),andimplicationsoninstabilityandevolutionofprokaryoticgenomes(6, 20–23).

2TypesofRMSystems

Restriction–modificationsystemsarecurrentlyclassifiedintofour typesbasedontheirsubunitcomposition,cofactorrequirements, targetrecognition,andDNAcleavagemechanism(24).

2.1TypeISystems

TypeIRMsystemsarehetero-oligomericcomplexesthatarecomposedofthreedifferenttypesofsubunits(S,M,andR),whichare encodedbythe hsdS (hsd for host specificity determinant), hsdM, and hsdR genes(Table 1).Thespecificitysubunit(S)isrequiredfor therecognitionofaspecificDNAsequence,theMTasesubunit (M)catalyzesDNAmethylationandtheREasesubunit(R)is essentialforDNAcleavage(25, 26).TypeIRMsystemsexistas twofunctionalcomplexes:atrimerof1Sand2Msubunitsacts solelyasamethyltransferaseandapentamerof1S,2M,and2R subunitsthatexhibitsbothmethyltransferaseandendonuclease activities(27–29).DNAsequencespecificityofTypeIRMsystems isdeterminedbytwotargetrecognitiondomains(TRDs)intheS subunit.EachofthetwoTRDsrecognizesonehalfofabipartite targetsequenceseparatedbyagap.Forexample,thesequence recognizedbytheEcoKIisAAC(N6)GTGCwhereN ¼ anynucleotide(25, 30).DependinguponthemethylationstatusofDNAat therecognitionsequences,TypeIrestriction–modification enzymeshavetwomodesofaction.IfDNAisfullymethylated, theenzymecomplexdoesnotbindtotherecognitionsequenceand DNAisimmunetorestriction(31).Whenthetargetsiteishemimethylated,themethyltransferasecomplexcatalyzesmodification oftheotherstrandattheN6positionofadeninewithinthe recognitionsequence,usingS-adenosylmethionine(SAM)asa methylgroupdonor(32).WhenDNAisunmethylatedtherestrictionenzymecomplexbindstotherecognitionsequenceandtranslocatesDNAbidirectionallythroughtheboundcomplexina reactioncoupledtoATPhydrolysis(33–36).TherestrictionreactionrequiresATP,SAM(withtheexceptionforR.EcoR124I)and

CharacteristicsandorganizationofthegenesandsubunitsofthefourtypesofRMsystems

TypeITypeIITypeIIITypeIV

Methylation-dependentREase

Hetero-oligomeric REaseandMTase complex

SeparateREaseandMTaseor REase-MTaseinonepolypeptide (e.g.,subtypesIIC,IIG)

FeaturesHetero-oligomericREaseand MTasecomplex

ExampleEcoKIEcoRIEcoP1IEcoKMcrBC

hsdM , hsdR , hsdSecoRIR , ecoRIMmod , resmcrBC

Genes

Subunits3differentsubunits2differentsubunits2differentsubunits2differentsubunits (R,M,andS)(RandM)(ModandRes)(McrBandMcrC)

EnzymeactivitiesMTase:S+2M( 2R)OrthodoxMTase:MMTase:2M( 2R)REase:varies

REase:S+2M + 2ROrthodoxREase:2RREase:1or2R+2MGTPaseorATPase(requiredfor restriction)

ATPase(requiredforrestriction)ATPase(requiredfor restriction) CofactorsATP,SAM,Mg

Modified;Bipartiteand methylated;e.g.,EcoKMcrBC, 5 0 RmC(N 30–3000 )RmC a

Asymmetric;e.g., EcoP1I,5 0 AGACC

RecognitionsequenceAsymmetricandbipartite;e.g., EcoKI,5 0 AAC(N 6 )GTGC a Symmetricandasymmetric (methylatedforsubtypeIIM); e.g.,EcoRI,5 0 GAATTC

Variablelocationsfrommodified (e.g.,m6A,m4C,m5C,hm5C, ghm5C)recognitionsite

Fixedlocation24–28bp fromrecognitionsite

Fixedlocationwithinorcloseto recognitionsite

CleavagesiteVariablelocations,oftenfar (1,000bp)fromrecognition site

DNAtranslocationYesNoYesYes

Examplein S.aureus Sau1( 79 )Sau3AI( 68 )SauKLT6ORF138600P (REBASE)( 82 ); SauUSI( 83 , 77 )

Occurrencein S. aureus b 72422112 a N ¼ anynucleotide;R ¼ AorG b Currentnumberof S.aureus strainscontainingparticulartypeofRMsystemamong125isolateswhereRMsystemshavebeenidentified

2.2TypeIISystems

Mg2+ ions,andoccursatasitethatisdistantfromtherecognition sequence(25, 30, 37–41).Cleavageistriggeredbycollisionand stallingoftworestrictionenzymecomplexesduringtranslocation alongaDNAchainorbystallingofasingleenzymeonasingle-site circularsubstratefollowingDNAtranslocation(24, 30, 34, 42). Basedongeneticcomplementationandbiochemicalstudies,TypeI RMsystemsarecurrentlydividedintofivedifferentsubclasses (IA–IE)(24, 43, 44).TypeIRMsystemsarepresentinthevast majorityofsequencedbacterialgenomesandnearly6,000putative TypeIRMsystemshavebeenidentifiedsofar(http://rebase.neb. com/rebase/statlist.html).

TypeIIRMsystemsrepresentthemostabundantgroupofcharacterizedRMenzymesthatareclassifiedinto11overlappingsubtypes(http://rebase.neb.com/cgi-bin/sublist)(24).Themajority, butnotall,ofTypeIIRMsystemsconsistofseparateREaseand MTaseproteinsthatrecognizethesame,oftenpalindromic(4–8 basepairs),DNAsequence(Table 1)(24).TheMTasesshare severalconservedaminoacidmotifsandgenerallyactasmonomers tomodifyaspecificbaseattheirrecognitionsequences.TypeII MTasescatalyzemodificationofcytosineateithertheN4ortheC5 position,andmethylationofadenineattheN6positionusingSAM asamethylgroupdonor(41).TheREaseproteinsconsistofa distinctanddissimilaraminoacidsequencesandusuallyactindependentlyoftheircognateMTases(4, 24).TypeIIREasestypically requireMg2+ ionsasacofactorandcanactasmonomers,dimers,or eventetramers(4).TypeIIREasescleavebothDNAstrandsat fixedpositionseitherwithinorclose(generallywithin20bp)tothe recognitionsitegenerating50 -phosphatesand30 -hydroxyls(4). BecauseoftheirabilitytocleaveDNAmoleculesatprecisepositions,TypeIIREasesarewidelyusedastoolsinrecombinantDNA technology.TypeIIREasesencompassmanywell-knownrestrictionendonucleasessuchasBamHI,EcoRI,andHindIII.Nearly 4,000TypeIIrestrictionenzymeshavebeendiscoveredsofar,and manyofthemareavailablecommercially(45)(http://rebase.neb. com/rebase/statlist.html).

2.3TypeIIISystems

TypeIIIRMsystemsarehetero-oligomericcomplexescomposed ofMod(for modification)andRes(for restriction)proteinsubunits encodedby mod and res genes,respectively(Table 1)(24, 26).The ModsubunitisrequiredforsubstraterecognitionandDNAmethylationandtheRessubunitisessentialforDNAcleavage.TheMod subunitcanmethylatetherecognitionsiteindependently(asa dimerMod2)orincomplexwiththerestrictionendonuclease subunitRes(asanoligomerMod2Res1 orMod2Res2)(46, 47). TypeIIIMTasesrequireSAMasamethylgroupdonorandcatalyze methylationofonlyonestrandofanunmodified5–6bpasymmetricrecognitionsequenceattheN6positionofadeninetoproducea

2.4TypeIVSystems

hemimethylatedDNAmolecule(24, 47).ThismethylationissufficientforprotectionofhostDNAsinceonlycompletelyunmethylatedDNAcanbecutbyTypeIIIREases.Restrictionactivity requiresboththeModandRessubunits(Mod2Res1 orMod2Res2), Mg2+ ionsasacofactorandhasanabsoluterequirementforATP hydrolysis(24, 46, 47).InmostcasesDNAcleavagerequires interactionofTypeIIIRMenzymeswithtwoseparateinversely orientedasymmetricrecognitionsequencesinthesameDNAmoleculeandisprecededbyATPhydrolysis,whichisrequiredforthe long-distancecommunicationbetweentherecognitionsites (46–49).DNAcleavageisoccurredatadefinedlocationthatis 24–28bpdownstreamofoneoftherecognitionsites(47).The exactcleavagemechanismisnotyetclear,anddifferentmodels (e.g.,thecollisioncleavagemodelandtherandomwalksliding model)havebeenproposedtoexplainhowthelong-distanceinteractionbetweenthetworecognitionsitestakesplace(26, 47, 49–54).ThemoststudiedexamplesofTypeIIIenzymesare EcoP1IandEcoP15I(47).Todate,over2,000putativeTypeIII RMsystemshavebeenidentifiedthroughoutsequencedbacterial genomessuggestingthatthesesystemsarewidelyspreadinthese organisms(47)(http://rebase.neb.com/rebase/statlist.html).

TypeIVsystemsaremodification-dependentREasesthatcleave DNAsubstratesonlywhentheirrecognitionsitesattheN4or theC5positionsofcytosineorattheN6positionofadeninehave beenmodified,e.g.,methylated(m),hydroxymethylated(hm),or glucosyl-hydroxymethylated(ghm)(Table 1)(24).TypeIVREases recognizemodifiedDNAwithlowsequencespecificity,andtheir recognitionsiteshaveusuallynotbeenwelldefined(24, 55).Type IVenzymescurrentlycompriseahighlydiversefamily,andonlya fewofthemhavebeencharacterizedinanydetail(44).Thebest studiedexamplewithinthisgroupisEcoKMcrBC(55–58),which iscomposedoftwosubunits,McrBandMcrC.TheMcrBsubunit isresponsibleforspecificDNAbindingandGTPhydrolysis, whereastheMcrCharborsthecatalyticcenterforDNAcleavage (55, 58–60).EcoKMcrBCrecognizestwocopiesofadinucleotide sequence,consistingofapurinefollowedbyamodifiedcytosineat eithertheN4ortheC5position,whichareseparatedbyanywhere from30to3,000bases(2, 24, 55, 61).Therecognitionsitesmay beoneitherDNAstrandandthusdonotneedtobeinaparticular orientation(57, 62).LikeTypeIRMsystems,theEcoKMcrBC endonucleasetranslocatesDNAandremainsboundtoitsrecognitionsites(63).DNAtranslocationandcleavagebythe EcoKMcrBCenzymerequiresGTPhydrolysisinthepresenceof Mg2+ ions(57, 59, 63).DNAcleavageresultsinadouble-strand breakandpreferentiallytakesplace~30–35bpawayfromoneof themodifiedRmCsites(R ¼ AorG)(55, 62, 64).Cleavage requirescooperationoftwositesandtakesplacewhentranslocation

isblockedbycollisionoftranslocatingcomplexesorbytopological barrierssuchasnucleoproteincomplexesforexample(55, 61–63). Cleavagewillalsooccurwhenrecognitionelementsarelocatedon oppositesidesofaDNAreplicationfork(65).TypeIV modification-dependentrestrictionendonucleasesprovidepowerfultoolstodeterminethemethylationstatusofCGislandsandto studyepigeneticmechanismsinmammalsandplants(55, 66, 67). Todate,over5,000putativeTypeIVenzymeshavebeenidentified amongsequencedprokaryoticgenomes(http://rebase.neb.com/ rebase/statlist.html).

3RMSystemsin S.aureus

RMsystemsarewidelyspreadin S.aureus. Todate,allfourtypes ofRMsystemshavebeenidentifiedthroughout125wholeand partlysequenced S.aureus isolates(http://tools.neb.com/ ~vincze/genomes/enz_count.php).SomeoftheseRMsystems havebeencharacterizedindetail(22, 68–77).Itisnoteworthythat themajorityof S.aureus isolatescontainmorethanoneRMsystem (http://tools.neb.com/~vincze/genomes/enz_count.php).Whole genomesequencingof48 S.aureus strainsrevealedsimultaneous presenceofatleasttwoRMsystemsofdifferenttypesintheir genomeswiththemostfrequentco-occurrenceofTypeIandType IVsystems(http://tools.neb.com/~vincze/genomes/index.php? page¼S).

TypeIRMsystemsrepresentoneofthemostabundantgroups ofRMenzymesin S.aureus.CurrentlyTypeIRMsystemshave beenfoundinall48completed S.aureus genomes(http://tools. neb.com/~vincze/genomes/index.php?page¼S)andinthe majority(72outof125)ofthe S.aureus isolateswhereRM enzymeshavebeenidentified(http://tools.neb.com/~vincze/ genomes/enz_count.php)(Table 1).Manyofthe S.aureus strains, e.g.,LGA251,M013,andMSSA476,containmorethanoneType IRMsystemintheirgenomes(78)(http://tools.neb.com/ ~vincze/genomes/index.php?page¼S).ThemostfrequentTypeI RMsystemfoundin S.aureus wasgiventhegenericnameofSau1 (22, 79).Among48 S.aureus isolateswithcompletelysequenced genomes40strainscontainSau1system(http://tools.neb.com/ ~vincze/genomes/index.php?page¼S).TheSau1systemis unusualasitconsistsofasingle hsdR geneandtwocopiesofboth hsdM and hsdS genes(10, 22, 79).Thetwosetsof hsdMS genesof theSau1RMsystemarelyingwithintwodifferentgenomicislands andtheyaredistantlylocatedonthechromosomefromeachother andfromthe hsdR gene(10, 79).TheRandMsubunitsofthe Sau1RMsystemexhibithomologytotheRandMsubunitsofthe EcoR124IsystemwhichbelongstotheTypeICfamily(10, 22). However,thelevelsofidentitybetweenthemwereconsiderednot

beingsufficientenoughforthemembersofthesameTypeIC groupanditwassuggestedthatSau1RMsystemsmightform theirownfamily(22).TheSau1systemistightlyassociatedwith thelineagesorclonalcomplexes(CC)intowhich S.aureus strains aredivided(22, 79).TheHsdRandHsdMproteinsoftheSau1 systemarenearlyidenticalbetweendifferentCCgroups,whilethe HsdSproteinsarehighlyconservedamongstrainsofthesame clonalcomplexanddiffersubstantiallybetweenlineages(79, 80). Also,twocopiesofSau1 hsdS geneswithinthesamegenome usuallyhavedifferentsequencesthatalloweachlineagetorecognizetwodifferentDNAtargetsites(22).Thelineagespecific sequencevariationoftheHsdSproteinsislocalizedtothetwo TRDdomainsthatflankedbyhighlyconservedaminoacid sequences(22).RecentlythespecificDNAtargetsitesrecognized bytheSau1RMsystemhavebeenidentifiedforthedominant MRSAlineages(CC1,CC5,CC8,andST239)(22).Interestingly, the hsdS genesofCC8areidenticaltothoseofST239andthesetwo lineagesshareone hsdS sequencewithCC1isolates,andtheother withCC5(22, 80).

TypeIIRMsystemsarelessabundantamong S.aureus isolates. Todate,genesencodingTypeIIRMsystemsand/orTypeII orphanDNAmethyltransferaseshavebeenidentifiedin42outof 125S.aureus isolateswithpartlysequencedgenomesandin27out of48completed S.aureus genomes(Table 1)(http://tools.neb. com/~vincze/genomes/enz_count.php; http://tools.neb.com/ ~vincze/genomes/index.php?page¼S).Interestingly,severalof thosestrains,e.g.,NRS100,M1,andCOL,carryinactiveType IIGRMenzymeswhichcontainframeshiftmutationswithintheir genes.ThemoststudiedTypeIIRMsystemsin S.aureus are Sau3AIandSau96I.Sau3AIREasebelongstothesubtypesE,P (http://rebase.neb.com/cgi-bin/sublist)andcleavesbothDNA strandsoftheunmodifiedpalindromicGATCsequencejust50 to theguaninenucleotides(68).ItscognateMTase,M.Sau3AI,protectsDNAfromcleavagebymethylatingtherecognitionsiteatthe C5positionofcytosine(71).Sau96IREasebelongstothesubtype P(http://rebase.neb.com/cgi-bin/sublist)andcleavesboth DNAstrandsoftheunmodifiedpalindromicGGNCCsequences betweentwoguaninenucleotides,whileitscognateMTase, M.Sau96I,protectsthesesitesbymethylatingtheinternalcytosine attheC5position(69, 72).

TypeIIIRMsystemsappeartobeextremelyrarein S.aureus. Todate,genesencodingputativeResandModsubunitsofTypeIII RMsystemshaveonlybeenidentifiedintwo S.aureus isolates,i.e., KLT6and118(Table 1)(81, 82)(http://tools.neb.com/~vincze/ genomes/enz_count.php).Noneofthe S.aureus TypeIIIRM systemshavebeencharacterizedyet.

TypeIVmodification-dependentREasesrepresentanothermost abundantgroupofRMenzymesin S.aureus.CurrentlyTypeIV

systemshavebeenfoundin44outof48 S.aureus isolateswithfully sequencedgenomesandin112outof125strainswhereRMenzymes havebeenidentified(http://tools.neb.com/~vincze/genomes/ enz_count.php; http://tools.neb.com/~vincze/genomes/index. php?page¼S)(Table 1).Approximatelyonethirdofthosestrains carrymorethanoneTypeIVREseintheirgenomes.Theonly well-characterizedTypeIVsystemin S.aureus isSauUSI(77, 83). SauUSIREaseisamulti-domainproteinthatcontainsaPLD-family endonucleasecatalyticsiteattheN-terminus,anATPaseandDNA helicasemotifsinthemiddlepartoftheprotein,andapresumed m5C/hm5CTRDdomainlocatedattheC-terminus(77).SauUSI REasepredominantlycleavesmodifiedDNAcontainingmethylated and/orhydroxymethylatedcytosinesattheC5position(77).The preferredrecognitionsitesforSauUSIareSm5CNGS(whereS ¼ C orG)(77).SimilartoMcrBC,efficientcleavagebySauUSIrequires interactionoftheenzymewithmorethanonerecognitionsite(77). DNAcleavageusuallytakesplace~2–18bpawayfromoneofthe modifiedrecognitionsequences(77).Theendonucleaseactivity requiresATPordATPhydrolysisinthepresenceofdivalentcations suchasMg2+,Mn2+,Ca2+,orCo2+ (77).SauUSIREaseishighly conservedandwidelyspreadamong S.aureus isolates,BLASTp analysisofSauUSIto S.aureus proteinsrevealedpresenceofthe SauUSIhomologswithatleast97%ofaminoacidsidentityin40 different S.aureus isolates.Interestingly,someofthe S.aureus isolates,e.g.,N315,Mu3,andMu50,containanonsensemutationin themiddleofthe sauUSI gene(83).

4OvercomingRestrictionBarriersin S.aureus

Thebigquestion,asitpertainstothisbook,iswhatdoesallofthese meanintheabilitytogeneticallymanipulate Staphylococcus andhow doesonecircumventsuchabarrier?Aspreviouslystated,RM enzymesareabundantin S.aureus andmostofthe S.aureus isolates containmultipleRMsystemsofdifferenttypeswithintheirgenomes.Consequently,thenumberanddiversityoftheRMsystems withinparticular S.aureus strainswillhavealargeimpactonthe choiceofDNAtransferapproachandthetransformationsuccess rate.Thecommonstrategiestoovercomerestrictionbarriersin bacteriaincludedeletionoftherestrictionsitesfromrecombinant DNA,theusageofhelperplasmidsthatcarryspecificormultiple MTases,invitrosite-specificmethylationbycell-freeextractsorby specificMTases,propagationofplasmidsinmethylationdeficient E.coli strainsandconstructionofmutantstrainslackingREases (83–93).Thelastapproachwassuccessfullyappliedforgenetic manipulationofstaphylococciover30yearsagobygeneration (viaUVandnitrosoguanidinemutagenesisof8325-4strain)of therestriction-deficientbutmodification-proficientstrain

designatedasRN4220(94).Sinceitsconstruction,thisstrainhas beenoneofthemostextensivelyusedinstaphylococcalstudies becauseofitsabilitytoacceptandmodifyDNAfrom E.coli cells thatallowsitsapplicationasanintermediatecloningstrain(85).In spiteofthat,theuseofRN4220asanintermediatealsohasits limitationsasitprovidesanefficientDNAtransferonlyintoa limitednumberofcloselyrelatedstrains(CC8lineage),whereas transferofDNAintomanyclinicalisolatesof S.aureus,suchas UAMS-1(CC30)forexample,stillremainsquiteadifficulttask (80, 95).Whole-genomesequencingofRN4220revealedamultitudeofsinglenucleotidepolymorphisms(SNPs)bywhichitdiffers fromtheparental8325strain,andanonsensemutationwithinthe hsdR geneoflineage-specificSau1TypeIRMsystemwasoneof them(96, 97).InsupportoftheimportanceoftheSau1systemas oneofthemajorrestrictionbarrierslimitingHGTin S.aureus, complementationofRN4220strainwithfull-length sau1hsdR genepreventedtransformationofplasmidsfrom E.coli,inhibited transductionwithphagesisolatedfromdistantlineages,and reducedconjugativetransferfromenterococci(79).Laterexperiments,however,showedthatinactivationofthe sau1hsdR genein threedifferent S.aureus strains(8325-4,SH100,andCOL)was notsufficienttomakethemreadilytransformable,suggestingthe presenceofanadditionalrestrictionbarrierin S.aureus (98).This secondmajorbarrierpreventingtransformationofplasmidsfrom E.coli,asithasbeenrecentlydemonstrated,isbasedonthepresenceofaTypeIVmodification-dependentREase,SauUSI(77, 83).Importantly,inadditiontothemutationwithinthe sau1hsdR gene,RN4220strainalsocontainsprematurestopcodoninthe sauUSI genewhichexplainsitsefficienttransformationbyplasmids propagatedin E.coli background(96).Moreover,restorationof thewildtypealleleofthe sauUSI geneinRN4220significantly reducedtransformationefficiencyofthisstrain(99).Asitwasnoted above,SauUSIREaserecognizesandcleavesmodifiedDNAcontainingmethylatedcytosinesattheC5position(77).Thus,propagationoftheplasmidsinbacterialackingcytosinemethylation willfacilitateDNAtransferinto S.aureus isolatescontaining SauUSIREase.Correspondingly,forovercomingTypeIVbarrier in S.aureus isolates,animprovedtransformationprotocolthat includespropagationofplasmidsinaDNAcytosinemethyltransferasemutant(dcm )ofthehigh-efficiency E.coli cloningstrain DH10BdesignatedasDC10Bhasbeenrecentlydeveloped(99). Asitisclearnow,themajorrestrictionbarrierslimitingDNA transferin S.aureus isolatesarebasedonthepresenceoftwo differenttypesofRMsystems,i.e.,alineage-specificTypeIRM systemSau1andTypeIVmodification-dependentREaseSauUSI. Alineage-specificSau1RMsystemrestrictsHGTbetweendifferent clonalcomplexesof S.aureus andlimitstransferofunmodified recombinantDNA,whileSauUSIREaseconfinestransferofmodifiedrecombinantDNAfrom E.coli andotherbacteriaandmight

limitHGTfromcertain S.aureus isolates,containingSau96IType IIRMsystemforexample.TheSau1andSauUSIRMsystemsare themostabundantin S.aureus andoftensimultaneouslyoccur withinthesameisolates.TheabilityoftheseRMsystemstocoexist withinthesamecellisbasedonthedifferencesintherecognitionof thesitespecificmethylationpatterns,i.e.,Sau1systemsmethylate itsrecognitionsitesattheN6positionofadeninewhileSauUSI cleavesDNAwithmethylatedcytosinesattheC5position.Consequently,inactivationofthe sau1hsdR geneandpropagationofthe recombinantDNAinthem5Cmodificationdeficientbackground willbeoftensufficientforthesuccessfulDNAtransferinto S. aureus isolatescontainingthesetwoRMsystems.

5OutlookandConcludingRemarks

RMsystemsareaverydiversegroupofenzymesthatarewidespread among S.aureus isolates.Thepresenceofvariousandoftenmultiplerestriction–modificationsystemsinmostofthe S.aureus isolates createsstrongstrain-specificbarriersthatlimitfunctionalgenomic analysesofthisbacterium.ForalongtimeaconfinedHGT betweendifferentclonalcomplexesof S.aureus andadifficultyof transferringrecombinantDNAfromotherbacteriaintomostofthe S.aureus isolateshadrestrictedgeneticmanipulationofthisbacteriumtoalimitednumberofstrains.Majoradvancesinunderstandingthebiologyofstaphylococcalrestrictionandmodificationhave occurredinpost-genomicerawithcharacterizationofalinagespecificTypeIRMsystem(Sau1)andatypeIVmodificationdependentREase(SauUSI),whichwereshowntobedominant barrierslimitingforeignDNAuptakeby S.aureus.Constructionof theSauIrestrictiondeficientmutantsindifferent S.aureus lineages andusageof dcm negative E.coli strainsforplasmidspropagation willfacilitateHGTbetweendifferentclonalcomplexesaswellas DNAtransferintomanypreviouslyuntransformablestrains.Thisin turnwillallowrapidandmoresophisticatedgeneticmanipulation ofclinicallyrelevant S.aureus isolates,whichwillsignificantly improveourknowledgeofthismedicallyimportantpathogen.

References

1.LowyFD(1998) Staphylococcusaureus infections.NEnglJMed339:520–532

2.TockMR,DrydenDT(2005)Thebiologyof restrictionandanti-restriction.CurrOpin Microbiol8:466–472

3.WilsonGG,MurrayNE(1991)Restriction andmodificationsystems.AnnuRevGenet 25:585–627

4.PingoudA,JeltschA(2001)Structureand functionoftypeIIrestrictionendonucleases. NucleicAcidsRes29:3705–3727

5.MurrayNE(2002)2001FredGriffithreview lecture.ImmigrationcontrolofDNAinbacteria:selfversusnon-self.Microbiology148:3–20

6.VasuK,NagarajaV(2013)Diversefunctions ofrestriction-modificationsystemsinaddition

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Launcelot.—Nay, indeed, if you had your eyes, you might fail of the knowing me: it is a wise father that knows his own child. Well, old man, I will tell you news of your son: give me your blessing. Truth will come to light; murder cannot be hid long,—a man’s son may; but, in the end, truth will out.

Gobbo.—Pray you, sir, stand up: I am sure you are not Launcelot, my boy.

Launcelot.—Pray you, let’s have no more fooling about it, but give me your blessing: I am Launcelot, your boy that was, your son that is, your child that shall be.

Gobbo.—I cannot think you are my son.

Launcelot.—I know not what I shall think of that: but I am Launcelot, the Jew’s man; and I am sure Margery your wife is my mother

Gobbo.—Her name is Margery, indeed: I’ll be sworn, if thou be Launcelot, thou art mine own flesh and blood. Lord, worship’d might he be! What a beard hast thou got! thou hast got more hair on thy chin than Dobbin, my fill-horse, has on his tail.

Launcelot.—It should seem, then, that Dobbin’s tail grows backward: I am sure he had more hair of his tail than I have on my face, when I last saw him.

Gobbo.—Lord, how art thou chang’d! How dost thou and thy master agree? I have brought him a present. How ’gree you now?

Launcelot.—Well, well; but, for mine own part, as I have set up my rest to run away, so I will not rest till I have run some ground. My master’s a very Jew: give him a present! give him a halter: I am famish’d in his service; you may tell every finger I have with my ribs. Father, I am glad you are come: give me your present to one Master Bassanio, who, indeed, gives rare new liveries: if I serve not him, I will run as far as God has any ground.—O rare fortune! here comes the man:—to him, father, for I am a Jew, if I serve the Jew any longer.

—Act II, Scene II, Lines 29-104.

HAMLET’S DECLARATION

Hamlet. What ho! Horatio!

OF FRIENDSHIP

Horatio. Here, sweet lord, at your service.

Hamlet. Horatio, thou art e’en as just a man As e’er my conversation coped withal.

Horatio. O, my dear lord,—

Hamlet. Nay, do not think I flatter; For what advancement may I hope from thee That no revenue hast, but thy good spirits, To feed and clothe thee? Why should the poor be flatter’d? No, let the candied tongue lick absurd pomp, And crook the pregnant hinges of the knee Where thrift may follow fawning. Dost thou hear? Since my dear soul was mistress of her choice And could of men distinguish, her election Hath sealed thee for herself; for thou hast been As one, in suffering all, that suffers nothing, A man that fortune’s buffets and rewards Hast ta’en with equal thanks: and blest are those Whose blood and judgment are so well commingled That they are not a pipe for Fortune’s finger To sound what stop she pleases. Give me that man That is not passion’s slave, and I will wear him In my heart’s core, ay, in my heart of hearts, As I do thee.

—From Act III, Scene 2.

OTHELLO’S APOLOGY

[The speech calls for great dignity, ease, and power, in both speech and manner.]

Most potent, grave, and reverend signiors, My very noble and approved good masters, That I have ta’en away this old man’s daughter, It is most true; true, I have married her: The very head and front of my offending Hath this extent, no more. Rude am I in my speech, And little bless’d with the soft phrase of peace; For since these arms of mine had seven years’ pith, Till now some nine moons wasted, they have used Their dearest action in the tented field, And little of this great world can I speak, More than pertains to feats of broil and battle, And therefore little shall I grace my cause In speaking for myself. Yet, by your gracious patience, I will a round unvarnish’d tale deliver Of my whole course of love; what drugs, what charms, What conjuration, and what mighty magic,— For such proceeding I am charg’d withal,— I won his daughter.

...

Her father loved me; oft invited me; Still question’d me the story of my life, From year to year,—the battles, sieges, fortunes, That I have pass’d. I ran it through, even from my boyish days, To the very moment that he bade me tell it: Wherein I spake of most disastrous chances, Of moving accidents by flood and field, Of hair-breadth scapes i’ the imminent deadly breach, Of being taken by the insolent foe And sold to slavery, of my redemption thence And portance in my travels’ history:

This to hear

Would Desdemona seriously incline: But still the house-affairs would draw her thence;

Which ever as she could with haste despatch, She’d come again, and with a greedy ear

Devour up my discourse: which I observing, Took once a pliant hour, and found good means To draw from her a prayer of earnest heart

That I would all my pilgrimage dilate, Whereof by parcels she had something heard, But not intentively: I did consent, And often did beguile her of her tears, When I did speak of some distressful stroke That my youth suffer’d. My story being done, She gave me for my pains a world of sighs: She swore, in faith, ’twas strange, ’twas passing strange, ’Twas pitiful, ’twas wondrous pitiful: She wish’d she had not heard it, yet she wish’d That heaven had made her such a man: she thank’d me, And bade me, if I had a friend that loved her, I should but teach him how to tell my story, And that would woo her. Upon this hint I spake: She loved me for the dangers I had pass’d; And I lov’d her that she did pity them. This only is the witchcraft I have used.

THE SEVEN AGES

[This is a succession of purely imaginative ideas which the voice should touch lightly. In this speech one meets always the question of impersonation: shall the mewling infant, the whining schoolboy, the sighing lover and the rest be imitated by the reader? It is in better taste not to impersonate these seven characters beyond certain almost imperceptible hints which the gayety of Jaques’s mind might naturally throw off.]

All the world’s a stage, And all the men and women merely players: They have their exits and their entrances; And one man in his time plays many parts,

His acts being seven ages. At first the infant, Mewling and puking in the nurse’s arms: And then the whining schoolboy, with his satchel And shining morning face, creeping like snail Unwillingly to school. And then the lover, Sighing like furnace, with a woeful ballad Made to his mistress’ eyebrow. Then a soldier, Full of strange oaths and bearded like the pard, Jealous in honor, sudden and quick in quarrel, Seeking the bubble reputation Even in the cannon’s mouth. And then the justice, In fair round belly with good capon lined, With eyes severe and beard of formal cut, Full of wise saws and modern instances; And so he plays his part. The sixth age shifts Into the lean and slipper’d pantaloon, With spectacles on nose and pouch on side; His youthful hose, well saved, a world too wide For his shrunk shank; and his big manly voice, Turning again toward childish treble, pipes And whistles in his sound. Last scene of all, That ends this strange eventful history, Is second childishness and mere oblivion, Sans teeth, sans eyes, sans taste, sans everything.

—“As You Like it,” Act II, Scene 7.

SOLITUDE PREFERRED TO COURT LIFE

Duke S. Now, my co-mates and brothers in exile, Hath not old custom made this life more sweet Than that of painted pomp? Are not these woods More free from peril than the envious court? Here feel we but the penalty of Adam. The season’s difference, as the icy fang And churlish chiding of the winter’s wind, Which, when it bite and blows upon my body,

Even till I shrink with cold, I smile and say ’Tis no flattery; these are counselors That feelingly persuade me what I am. Sweet are the uses of adversity, Which, like the toad, ugly and venomous, Wears yet a precious jewel in his head; And this our life, exempt from public haunt, Finds tongues in trees, books in the running brooks, Sermons in stones, and good in everything. I would not change it.

Amiens. Happy is your grace, That can translate the stubbornness of fortune Into so quiet and so sweet a style.

Duke S. Come, shall we go and kill us venison? And yet it irks me the poor dappled fools, Being native burghers of this desert city, Should in their own confines with forked heads Have their round haunches gor’d.

—“As You Like It,” Act II.

THE POTION SCENE

S: J’ C

(Enter Juliet and Nurse, who bears wedding garments.)

Juliet (looking at garments).

Ay, those attires are best; but, gentle nurse, I pray thee, leave me to myself to-night; For I have need of many orisons To move the heavens to smile upon my state, Which, well thou knowest, is cross and full of sin.

(Enter Lady Capulet.)

Lady Capulet.

What are you busy, ho? need you my help?

Juliet.

No, madam; we have cull’d such necessaries

As are behoveful for our state to-morrow: So please you, let me now be left alone, And let the nurse this night sit up with you; For, I am sure, you have your hands full all, In this so sudden business.

Lady Capulet (crossing and kissing Juliet on the forehead).

Good night; Get thee to bed and rest, for thou hast need.

(Exit Lady Capulet with nurse.)

Juliet (looking after them).

Farewell! God knows when we shall meet again. I have a faint cold fear thrills through my veins, That almost freezes up the heat of life: I’ll call them back again to comfort me. (Runs to R.) Nurse! What should she do there?

My dismal scene I needs must act alone. Come, vial. (Takes vial from bosom.)

What if this mixture do not work at all?

Shall I be married then to-morrow morning? No, no! (draws dagger) this shall forbid it.

(Lays dagger on table.)

Lie you there. (To vial.)

What if it be a poison, which the friar

Subtly hath ministered to have me dead, Lest in this marriage he should be dishonored Because he married me before to Romeo? I fear it is; and yet, methinks, it should not, For he hath still been tried a holy man.

(Puts vial in bosom.)

How if, when I am laid into the tomb, I wake before the time that Romeo Come to redeem me? there’s a fearful point! Shall I not then be stifled in the vault, To whose foul mouth no healthsome air breathes in, And there die strangled ere my Romeo comes?

Or, if I live, is it not very like, The horrible conceit of death and night, Together with the terror of the place,— As in a vault, an ancient receptacle, Where, for these many hundred years, the bones Of all my buried ancestors are packed; Where bloody Tybalt, yet but green in earth, Lies festering in his shroud; where as they say, At some hours in the night spirits resort; ... O, if I wake, shall I not be distraught, Environed with all these hideous fears? And madly play with my forefathers’ joints? And pluck the mangled Tybalt from his shroud? And, in this rage, with some great kinsman’s bone, As with a club, dash out my desperate brains? O, look! methinks I see my cousin’s ghost Seeking out Romeo, ...

Stay, Tybalt, stay!— Romeo, I come! (Drawing out vial—then cork.)

This do I drink to thee.

(Throws away vial. She is overcome and sinks to the floor.)

—From “Romeo and Juliet,” Act IV, Scene 3.

BANISHMENT SCENE

SCENE III, A ROOM IN THE PALACE

(Enter Celia and Rosalind.)

Cel. Why, cousin; why Rosalind;—Cupid have mercy;—Not a word?

Ros. Not one to throw to a dog.

Cel. No, thy words are too precious to be cast away upon curs, throw some of them at me; come, lame me with reasons.

Ros. Then there were two cousins laid up; when the one should be lamed with reasons, and the other mad without any.

Cel. But is all this for your father?

Ros. No, some of it for my father’s child: O, how full of briars is this working-day world!

Cel. They are but burrs, cousin, thrown upon thee in holiday foolery; if we walk not in the trodden paths, our very coats will catch them.

Ros. I could shake them off my coat; these burrs are in my heart.

Cel. Hem them away.

Ros. I would try; if I could cry hem, and have him.

Cel. Come, come, wrestle with thy affections.

Ros. O, they take the part of a better wrestler than myself.

Cel. Is it possible, on such a sudden, you should fall into so strong a liking with old Sir Rowland’s youngest son?

Ros. The duke my father lov’d his father dearly.

Cel. Doth it therefore ensue, that you should love his son dearly? By this kind of chase, I should hate him, for my father hated his father dearly; yet I hate not Orlando.

Ros. No ’faith, hate him not, for my sake.

Cel. Why should I not? Doth he not deserve well?

Ros. Let me love him for that; and do you love him, because I do: Look, here comes the duke.

Cel. With his eyes full of anger

(Enter Duke Frederick, with Lords.)

Duke F. Mistress, despatch you with your safest haste, and get you from our Court.

Ros. Me, uncle?

Duke F. You, cousin, within these ten days if thou be’st found so near our public court as twenty miles, thou diest for it.

Ros. I do beseech your grace, let me the knowledge of my fault bear with me: if with myself I hold intelligence, or have acquaintance with mine own desires; if that I do not dream, or be not frantic (as I do trust I am not), then, dear uncle, never so much as in a thought unborn, did I offend your highness.

Duke F. Thus do all traitors, if their purgation did consist in words, they are as innocent as grace itself: let it suffice thee, that I trust thee not.

Ros. Yet your mistrust cannot make me a traitor: tell me, whereon the likelihood depends.

Duke F. Thou art thy father’s daughter, there’s enough.

Ros. So was I, when your highness took his dukedom; so was I, when your highness banish’d him: treason is not inherited, my lord: or, if we did derive it from our friends, what’s that to me? my father was no traitor: then, good my liege, mistake me not so much, to think my poverty is treacherous.

Cel. Dear sovereign, hear me speak.

Duke F. Aye, Celia; we stay’d here for your sake. Else had she with her father rang’d along.

Cel. I did not then entreat to have her stay, it was your pleasure, and your own remorse; I was too young that time to value her, but

now I know her; if she be a traitor, so am I: we still have slept together; rose at an instant, learn’d, play’d, eat together;

And wheresoe’er we went, like Juno’s swans, Still we went coupled, and inseparable.

Duke F She is too subtle for thee; and her smoothness, Her very silence, and her patience, Speak to the people and they pity her.

Thou art a fool: she robs thee of thy name; And thou wilt show more bright, and seem more virtuous, When she is gone: then open not thy lips; Firm and irrevocable is my doom Which I have pass’d upon her; she is banish’d.

Cel. Pronounce that sentence then on me, my liege; I cannot live out of her company

Duke F. You are a fool:—You, niece, provide yourself; If you outstay the time, upon my honor, And in the greatness of my word, you die.

(Exeunt Duke Frederick and Lords.)

Cel. O my poor Rosalind: whither wilt thou go? Wilt thou change fathers? I will give thee mine. I charge thee, be not thou more griev’d than I am.

Ros. I have more cause.

Cel. Thou hast not, cousin, Pr’ythee, be cheerful: know’st thou not, the duke Hath banish’d me his daughter?

Ros. That he hath not.

Cel. No? hath not? Rosalind lacks then the love Which teaches thee that thou and I art one:

Shall we be sunder’d? shall we part, sweet girl? No; let my father seek another heir. Therefore devise with me, how we may fly, Whither to go, and what to bear with us: And do not seek to take your charge upon you, To bear your griefs yourself, and leave me out; For by this heaven, now at our sorrows pale, Say what thou can’st, I’ll go along with thee.

Ros. Why, whither shall we go?

Cel. To seek my uncle.

Ros. Alas, what danger will it be to us, Maids as we are, to travel so far?

Beauty provoketh thieves sooner than gold.

Cel. I’ll put myself in poor and mean attire, And with a kind of umber smirch my face; The like do you; so shall we pass along, And never stir assailants.

Ros. Were it not better, Because that I am more than common tall, That I did suit me in all points like a man? A boar-spear in my hand; and in my heart Lie there what hidden woman’s fear there will, We’ll have a swashing and a martial outside; As many other mannish cowards have, That do outface it with their semblances.

Cel. What shall I call thee when thou art a man?

Ros. I’ll have no other worse than Jove’s own page, And therefore, look you, call me Ganymede. But what will you be call’d?

Cel. Something that hath a reference to my state:

No longer Celia, but Aliena.

Ros. But, cousin, what if we assayed to steal The clownish fool out of your father’s court? Would he not be a comfort to our travel?

Cel. He’ll go along o’er the wide world with me; Leave me alone to woo him: Let’s away And get our jewels and our wealth together; Devise the fittest time, and safest way To hide us from pursuit that will be made After my flight: Now go we in content, To liberty, and not to banishment.

—From “As You Like It,” Act I.

CORYDON

B T B A

SCENE, A ROAD-SIDE IN ARCADY

Shepherd. Good sir, have you seen pass this way A mischief straight from market-day?

You’d know her at a glance, I think; Her eyes are blue, her lips are pink; She has a way of looking back Over her shoulder, and alack! Who gets that look one time, good sir, Has naught to do but follow.

Pilgrim. I have not seen this maid methinks, Though she that passed had lips like pinks.

Shepherd. Or like two strawberries made one By some sly trick of dew and sun.

Pilgrim. A poet.

Shepherd. Nay, a simple swain That tends his flocks on yonder plain Naught else I swear by book and bell. But she that passed you marked her well Was she not smooth as any be That dwells here—in Arcady?

Pilgrim. Her skin was the satin bark of birches.

Shepherd. Light or dark?

Pilgrim. Quite dark.

Shepherd. Then ’twas not she.

Pilgrim. The peaches side That next the sun is not so dyed As was her cheek. Her hair hung down Like summer twilight falling brown; And when the breeze swept by, I wist Her face was in a somber twist.

Shepherd. No that is not the maid I seek; Her hair lies gold against her cheek, Her yellow tresses take the morn, Like silken tassels of the corn, And yet brown-locks are far from bad.

Pilgrim. Now I bethink me this one had A figure like the willow tree Which, slight and supple, wondrously Inclines to droop with pensive grace, And still retain its proper place. A foot so arched and very small The marvel was she walked at all; Her hand in sooth, I lack for words— Her hand, five slender snow-white birds, Her voice, tho’ she but said “God Speed”—

Was melody blown through a reed; The girl Pan changed into a pipe

Had not a note so full and rife. And then her eye—my lad, her eye!

Discreet, inviting, candid, shy, An outward ice, an inward fire, And lashes to the heart’s desire. Soft fringes blacker than the sloe—

Shepherd. Good sir, which way did this one go?

Pilgrim. So he is off! The silly youth Knoweth not love in sober sooth, He loves—thus lads at first are blind— No woman, only womankind. I needs must laugh, for by the mass No maid at all did this way pass.

PART FOUR

Oratoric Reading and the Art of Public Speech

Discussion of forceful speech in making history. Value of forceful speech. Practice selections.

HAMLET’S INSTRUCTION TO THE PLAYERS

Speak the speech, I pray you, as I pronounced it to you,— trippingly on the tongue; but if you mouth it, as many of our players do, I had as lief the town-crier spake my lines. Nor do not saw the air too much with your hand, thus, but use all gently; for in the very torrent, tempest, and, as I may say, whirlwind of your passion, you must acquire and beget a temperance, that may give it smoothness. Oh! it offends me to the soul to hear a robustious periwig-pated fellow tear a passion to tatters,—to very rags,—to split the ears of the groundlings; who, for the most part, are capable of nothing but inexplicable dumb show and noise. I would have such a fellow whipped for o’erdoing Termagant; it out-herods Herod. Pray you avoid it.

S.

CHAPTER XIII

ORATORIC READING AND THE ART OF PUBLIC SPEECH

Upon this important subject of public speaking, and the interpretation of the addresses made by others, great men have thus expressed themselves: Dr. Charles W. Eliot, formerly President of Harvard University, says: “Have we not all seen, in recent years, that leading men of business have a great need of a highly trained power of clear and convincing expression? Business men seem to me to need, in speech and writing, all the Roman terseness and the French clearness. That one attainment is sufficient reward for the whole long course of twelve years spent in liberal study.” Abraham Lincoln likewise said: “Extemporaneous speaking should be practiced and cultivated. It is the lawyer’s avenue to the public. However able and faithful he may be in other respects, people are slow to bring him business if he can not make a speech.”

Every thinker knows what a vital part eloquence plays in national as well as individual welfare. If at first thought effective speaking seems a simple thing and a superficial part of education, on mature thought and consideration it will be found to be one of the most complex, vital and difficult problems that education has to meet. And yet, notwithstanding this complexity of the problem, the teacher is cheered by the delightful assurance of giving the student a consciousness of his latent talents and the ability to reveal and make use of them for the proper influencing of his fellow men.

There is a belief fairly commonly held that only a limited few need study the art of public speaking. Never was there a greater error or a more fatal mistake—especially in a republic like ours, where every man should be vitally interested in public affairs. No single citizen can afford not to be able to stand before his fellows and clearly, pleasingly and convincingly present his ideas upon any subject of local, state, or national importance. It is no more an ornamental accomplishment than is grammar, penmanship or simple arithmetic. It should be as universal as “the three r’s.” The hints and selections

that follow are carefully chosen to incite every good citizen to the acquirement of this useful and practical aid for his own benefit as well as that of his fellows. All the lessons and analyses that have gone before in these pages will materially aid in the elucidation of these brief lessons.

The basis for development in Effective Speaking rests upon one’s bodily, emotional and mental agencies of expression, and a knowledge of their respective importance and efficient use. That which counts most for development is conscientious practice; without which, progress is impossible.

There are three definite means of communicating thought and feeling to others: (a) Pantomime: face, hands, body; (b) Vocal: tone sound; (c) Verbal: words, which are conventional symbols manifesting mental and emotional states.

The problem, then, is to obtain a harmonious coördination of these three languages. In other words, the content of the word when spoken should be reflected in the tone and in the body. Thus speech becomes effective merely because it receives its just and fair consideration.

With this general understanding let us take up and master the successive steps which ultimately lead to a realization of the desired end.

The first important essential of effective speaking is the Spirit of Directness. By this is meant natural, unaffected speech. Nothing can be more important than that the person speaking use in public address the ordinary elements of Conversation.

Hence, the first step is practice in natural speaking. Commit to memory Hamlet’s Instructions to the Players given on a preceding page. Do this not line by line, but the entire selection as a whole. First: Read it through silently three times to familiarize yourself with the subject-matter. Second: Read it aloud at least five times. Third: Speak it conversationally at least five times from memory In this practice always be intensely conscious that you are addressing an individual and not an audience.

Now take any of the prose or poetic selections from the earlier pages of this book, memorize them, after studying them as the instructions require, and speak them directly and naturally, in the ordinary conversational style.

Sufficient practice in this is the necessary preparation for the next step, viz., the acquiring of a natural elevated conversational style, which is merely another name for the higher type of public speaking.

Commit all, or a part, of the following selections, keeping in mind that in speaking them you are addressing a group of people.

THE GETTYSBURG ADDRESS

B A L

Fourscore and seven years ago our fathers brought forth upon this continent a new nation, conceived in liberty, and dedicated to the proposition that all men are created equal. Now we are engaged in a great Civil War, testing whether that nation, or any nation, so conceived and so dedicated, can long endure. We are met on a great battlefield of that war. We are met to dedicate a portion of it as the final resting place of those who here gave their lives that that nation might live.

It is altogether fitting and proper that we should do this. But in a larger sense we cannot dedicate, we cannot consecrate, we cannot hallow this ground. The brave men, living and dead, who struggled here, have consecrated it far above our power to add or detract. The world will little note, nor long remember, what we say here, but it can never forget what they did here.

It is for us, the living, rather to be dedicated here to the unfinished work they have thus far so nobly carried on. It is rather for us to be here dedicated to the great task remaining before us, that from these honored dead we take increased devotion to the cause for which they gave their last full measure of devotion; that we here highly resolve that these dead shall not have died in vain, that the Union shall, under God, have a new birth of freedom, and that the

government of the people, by the people, and for the people, shall not perish from the earth.

By this time you should have mastered Ordinary Conversational Style; Elevated Conversational Style; and Abandon and Flexibility of Speech. The next consideration is the importance of Clearness. Clearness in speech means making prominent central words and subordinating unimportant words, or phrases. In other words, the logical sequence of thought must be clearly shown. This is brought about by a variety of inflections, changes of pitch, pause, etc. Clearness in speech is dependent upon clearness of Thinking.

It is important now to give full consideration to the subject of Emphasis. There are more ways than one of emphasizing your thought. The most common way is by merely increasing the stress of voice upon a word. This, however, is the most undignified form of emphasis. It is common to ranters and “soap-box” orators and is one mark of an undisciplined and uncultured man. Remember that loudness is a purely physical element, and does not manifest thought. Such emphasis is an appeal to the brute instinct, and is only expressive of the lower emotions. But Inflection, Changes of Pitch, Pause, Movement and Tone-Color—as have been fully explained in preceding pages—all appeal to the exalted nature of man.

In proportion to the nobleness of an emotion or thought, we find a tendency to accentuate these above-named elements. Such methods of emphasis are appropriate to the most disciplined and cultured man. More than that, they are the surest evidence of a great personality.

Commit, then make clear to the hearer, the vital thought in the following:

He have arbitrary power! My lords, the East India Company have not arbitrary power to give him; the King has no arbitrary power to give him; your Lordships have not; nor the Commons; nor the whole legislature. We have no arbitrary power to give, because arbitrary power is a thing which

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