Table Of Content

Methods in Molecular Biology 2646 Tohru Minamino Makoto Miyata Keiichi Namba Editors Bacterial and Archaeal Motility M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. Bacterial and Archaeal Motility Edited by Tohru Minamino Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Makoto Miyata Graduate School of Science, Osaka Metropolitan University, Osaka, Japan Keiichi Namba Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Editors TohruMinamino MakotoMiyata GraduateSchoolofFrontierBiosciences GraduateSchoolofScience OsakaUniversity OsakaMetropolitanUniversity Suita,Osaka,Japan Osaka,Japan KeiichiNamba GraduateSchoolofFrontierBiosciences OsakaUniversity Suita,Osaka,Japan ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-3059-4 ISBN978-1-0716-3060-0 (eBook) https://doi.org/10.1007/978-1-0716-3060-0 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2023 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface Manybacteriaandarchaeamigratetowardmorefavorableenvironmentsfortheirsurvivalby using their own motility machinery. Motility is also required for effective infection of pathogenicbacteriaintotheirhostcells.Thus,bacterialandarchaealmotilityisanextremely intriguing topic. Many motile bacteria utilize flagella for their swimming motility in liquid environments and their swarming motility on solid surfaces. The bacterial flagellum is a motilityorganelledrivenbyarotarymotorpoweredbythetransmembraneelectrochemical gradient of ions such as protons (H+) and sodium ions (Na+). Motile archaea also have a flagellum-like structure called the archaellum, which looks very similar to the bacterial flagellum both in appearance and function. In contrast to bacterial flagella, however, ATP is the primary energy source to rotate the archaellum. Many bacteria also utilize the ATP-driven type IV pilus for twitching motility over surfaces. Interestingly, Mollicutes specieshaveneitherflagellanortypeIVpiliandusetheirownATP-drivenmotilitymachin- ery for gliding motility on surfaces and swimming motility in liquids. Thus, bacterial and archaealmotilitycanbedefinedasthecapabilityofindividualcellstoconvertelectrochemi- cal or chemical energy to mechanical work required for their locomotion under various environmentalconditions. In this volume, we have brought together a set of cutting-edge research protocols to study the structure and dynamics of bacterial and archaeal motility systems using bacterial genetics, molecular biology, biochemistry, biophysics, structural biology, cell biology, microscopy imaging, and molecular dynamics simulation. Our aim is to provide useful tools and pathways for the investigation of the supramolecular motility machines derived fromvariousbacterialandarchaealspeciesthroughtechniquesthatcanbeapplied.Sincethe principal goal of the book is to provide researchers with a comprehensive account of the practicalstepsofeachprotocol,theMethodssectionscontaindetailedstep-by-stepdescrip- tions of every protocol. The Notes sections complement the Methods to get the hang of each experiment based on the authors’ experiences and to figure out the best way to solve anyproblemanddifficultythatmightariseduringtheexperiment. Thebacterialflagellumisasupramolecularassemblycomposedof30differentproteins and consists of at least 3 structural parts: a basal body that acts as a bi-directional rotary motor,afilamentthatworksasamolecularscrewtoproducethrusttopropelthecellbody, and a hook that connects the basal body and filament and serves as a universal joint to transmittorqueproducedbythemotortothemolecularscrew.Bacteriaemploytheflagellar typeIIIsecretionsystems(fT3SS)toexportflagellarproteinstoconstructtheflagellaonthe cell surface. The fT3SS is composed of a transmembrane export gate complex and a cytoplasmicATPaseringcomplex.ThefT3SSutilizesfreeenergyderivedfromATPhydro- lysis by the cytoplasmic ATPase complex and proton motive force across the cytoplasmic membrane to unfold and transport flagellar structural subunits from the cytoplasm to the distal end of the growing flagellar structure where each subunit is folded back and incorporatedintothestructure.ChaptersinPartIdescribehowtopurifythecorecomplex of the export gate complex (Chapter 1), how to quantitatively measure flagellar protein export in vitro (Chapter 2), how to visualize the assembly and ejection processes of the flagella(Chapter4),andhowtocarryouthigh-resolutioncryoEMstructuralanalysisofthe flagellarfilament(Chapter5).Moleculardynamicsimulationofdynamicopen-closedomain v vi Preface motionofatransmembraneexportgateproteinresponsibleforefficientandrobustenergy couplingmechanismofthefT3SS(Chapter3)isalsoincludedinPartI. Thebacterialflagellarmotorconsistsofarotorandadozenstatorunits.Theenergyfor motor rotation is supplied by ion influx along the electrochemical potential difference of specific ions, such as H+ and Na+, across the cytoplasmic membrane. The stator unit is composedoftwotransmembraneproteins,commonlyreferredtoasMotAandMotBinthe H+-driven motor, PomA and PomB in the Na+-driven motor of marine Vibrio, and MotP andMotSintheNa+-drivenmotorofextremelyalkalophilicBacillus.Thestatorunitserves also as a transmembrane ion channel complex to couple the ion flow through the channel withtorquegenerationviaelectrostaticinteractionsofeitherMotA,PomA,orMotPwitha rotor protein, FliG. The flagellar motor can spin in both counter-clockwise (CCW, viewed fromoutside)andclockwise(CW)directionswithoutchangingthedirectionoftheionflow. Several chemotaxis signaling molecules bind to the rotor and induce its highly cooperative conformational changes. As a result, the flagellar motor switches its rotational direction fromCCWtoCW.Whenthesignalingmoleculesdissociatefromtherotor,themotorspins againintheCCWdirection.ChaptersinPartIIwilldescribehowtoanalyzethestructural dynamics of FliG (Chapter 6), how to investigate stator-rotor interactions in vivo (Chapter 7), how to measure the ion channel activity of the stator unit (Chapters 8 and 9),howtovisualizestructuraldynamicsofthestatorunitinvitro(Chapter10),andhowto measureflagellarmotordynamicsatnearzeroload(Chapter11).Flagella-drivenmagneto- taxismotility(Chapter12),flagella-drivensurfacemotilityofSalmonella(Chapter13),and flagella-drivenmotilityofLeptospira(Chapters14and15)arealsoincludedinPartII. Motile archaea rotate long helical filaments called the archaella to swim in liquid environments. The archaella look like the bacterial flagella, but the archaellar structure has nothingincommonwiththebacterialflagella.Therotatingappendageisformedbyalong, helical filament, which is stably attached to a membrane-embedded rotary motor powered byATPhydrolysis.TheN-terminusofarchaellarfilamentproteinscontainsacleavablesignal peptide that is removed during secretion, and each filament subunit is incorporated at the proximal end of a nascent archaellar structure in a way similar to the type IV pilus. Furthermore,thecytoplasmicATPaseringcomplex,whichisresponsibleforbothassembly androtation,isstructurallyandfunctionallysimilartothetypeIVpilusATPases,suggesting that the archaellum may have been evolved from the type IV pilus. Chapters in Part III describe how to isolate the archaellar filaments (Chapter 16) and how to measure ATP-drivenarchaellar motor rotation(Chapter17). BacteriautilizetypeIVpilitomoveoversurfaces,andthiscrawlingbacterialmotilityis calledtwitchingmotility.EachtypeIVpilusiscomposedofabasalbodylocatedwithinthe cellenvelopeandapilusfilamentextendedfromthecellbody.Thepilusfilamentisahighly dynamic structure that undergoes cycles of extension and retraction. Two distinct ATP-drivenmotorslocatedatthebaseofthefilamentaredirectlyinvolvedinsuchdynamic cycles of the pilus filament motion. One ATP-driven motor facilitates the extension of the pilusfilament,allowingthefilamenttoattachtothesurfaceofthehostcellsandsubstrates, and then the other ATP-driven motor promotes pilus retraction, resulting in the forward movement of the cell body. Chapters in Part IV describe how to perform structural (Chapter 18) and functional analyses (Chapters 19 and 20) of type IV pili of twitching- capablebacteria. Some bacterial species have their own specialized motility machinery to drive gliding motility over surfaces. This type of bacterial motility does not involve a conventional bacterial motility machinery such as flagella and type IV pili. Bacteroidota adhere to solid Preface vii surfaces through their own adhesions and move back and forth by the movement of the adhesinsonhelicalfilamentousstructureslocatedintheperiplasm.Adhesivemovementsare dependentonprotonmotiveforceacrossthecytoplasmicmembrane.Themechanicalforce required for adhesive migration should occur around the cytoplasmic membrane and be transmitted to the outer membranesurface via the peptidoglycan layer. Chapters in PartV describehowtoperformstructural(Chapter21)andfunctionalanalyses(Chapters22and 23)oftheglidingmotilitymachineryofBacteroidota.Achapterdescribinghowtovisualize thepeptidoglycanlayerstructures(Chapter24)isalsoincludedinPartV. Mycoplasmaspeciesutilizeadhesinstocatch,pull,andreleasesialylatedoligosaccharides onthehostcellsurfacestodriveglidingmotilityover thecellsurfaces.Theglidingmotility of Mycoplasma is powered by ATP hydrolysis. Thus, the gliding motility machinery of MycoplasmaisstructurallyandfunctionallydifferentfromthatofBacteroidota.Interestingly, the structure and locomotion mechanism of the ATP-driven gliding motility machinery of Mycoplasma pneumoniae and Mycoplasma mobile, both of which belong to the class Molli- cutes,seemtobedifferentfromeachother.Furthermore,Spiroplasma,whichalsobelongto the class Mollicutes, utilize a unique motility machinery to swim in liquid environments. Chapters in Part VI describe how to carry out structural (Chapter 25) and functional analysesof themotilitymachineryof Mollicutes(Chapters 29and 30)and how to directly measure the velocity and force generated by the motility machinery of Mollicutes using high-resolutionopticalmicroscopictechniques(Chapters26,27,28,and31).Chloroflexus has its own gliding motility machinery, but the machinery is not yet identified. A chapter describing how to characterize the gliding motility of Chloroflexus (Chapter 32) is also includedinPartVI. Allthecontributorsareleadingresearchersinthefieldofbacterialandarchaealmotility, and we would like to acknowledge them for providing their comprehensive protocols and techniquesforthisvolume.WewouldliketothankDr.JohnWalker,Editor-in-Chiefofthe MethodsinMolecularBiologyseries,forgivingusagreatopportunitytoeditthisvolumeand hiscontinuoussupportandencouragement. WehopeyouallenjoythesechaptersandbenefitfromtheminthisvolumeofMethodsin MolecularBiology. Osaka,Japan TohruMinamino Osaka,Japan MakotoMiyata Osaka,Japan KeiichiNamba Contents Preface ..................................................................... v Contributors................................................................. xiii PART I BACTERIAL FLAGELLAR PROTEIN EXPORTAND ASSEMBLY 1 PurificationoftheTransmembranePolypeptideChannelComplex oftheSalmonellaFlagellarTypeIIISecretionSystem ........ ....... ........ 3 MikiKinoshita,KeiichiNamba,andTohruMinamino 2 InVitroFlagellarTypeIIIProteinTransportAssayUsingInverted MembraneVesicles......... ........ ....... ....... ........ ....... ........ 17 KatsumiImadaandHiroyukiTerashima 3 MolecularSimulationtoInvestigateOpen–CloseMotionofaFlagellar ExportApparatusProteinFlhA ..... ....... ....... ........ ....... ..... ... 27 C AkioKitao 4 Live-CellImagingoftheAssemblyandEjectionProcessesoftheBacterial FlagellabyFluorescenceMicroscopy ... ..... ....... ........ ....... ........ 35 Xiang-YuZhuang,Chao-KaiTseng,andChien-JungLo 5 PurificationandCryoEMImageAnalysisoftheBacterial FlagellarFilament.......... ........ ....... ....... ........ ....... ........ 43 TomokoYamaguchi,TomokoMiyata,FumiakiMakino, andKeiichiNamba PART II FLAGELLA-DRIVEN MOTILITY OF BACTERIA 6 Site-SpecificIsotopeLabelingofFliGforStudyingStructuralDynamics UsingNuclearMagneticResonanceSpectroscopy.... ........ ....... ...... .. 57 TatsuroNishikinoandYoheiMiyanoiri 7 Site-DirectedCross-LinkingBetweenBacterialFlagellarMotorProteins InVivo .... ........ ....... ........ ....... ....... ........ ....... ........ 71 HiroyukiTerashima,MichioHomma,andSeijiKojima 8 MeasurementsoftheIonChannelActivityoftheTransmembrane StatorComplexintheBacterialFlagellarMotor ..... ........ ....... ........ 83 YusukeV.MorimotoandTohruMinamino 9 PurificationoftheNa+-DrivenPomABStatorComplexandItsAnalysis UsingATR-FTIRSpectroscopy...... ....... ...... ......... ....... ........ 95 SeijiKojima,MichioHomma,andHidekiKandori 10 PurificationofNa+-DrivenMotPSStatorComplexesandSingle-Molecule ImagingbyHigh-SpeedAtomicForceMicroscopy .......... ....... ........ 109 NaoyaTeraharaandNoriyukiKodera 11 High-ResolutionRotationAssayoftheBacterialFlagellarMotorNear ZeroLoadsUsingaMutantHavingaRod-LikeStraightHook ...... ........ 125 ShuichiNakamuraandTohruMinamino ix x Contents 12 Live-CellFluorescenceImagingofMagnetosomeOrganelle forMagnetotaxisMotility........... ....... ....... ........ ....... ........ 133 YukakoEguchiandAzumaTaoka 13 SwarmingMotilityAssaysinSalmonella ..... ....... ........ ....... ........ 147 JonathanD.PartridgeandRasikaM.Harshey 14 AnalysisofAdhesionandSurfaceMotilityofaSpirocheteBacterium.......... 159 ShuichiNakamura,JunXu,andNobuoKoizumi 15 ForceMeasurementofBacterialSwimmingUsingOpticalTweezers .......... 169 KeigoAbe,KyosukeTakabe,andShuichiNakamura PART III ARCHAELLA-DRIVEN MOTILITY OF ARCHAEA 16 ArchaellaIsolation ...... ... ...... .. ...... .... .... ........ ....... ........ 183 ShamphaviSivabalasarma,JoaõN.deSousaMachado, Sonja-VerenaAlbers,andKenF.Jarrell 17 DirectObservationofArchaellarMotorRotationbySingle-Molecular ImagingTechniques........ ........ ....... ....... ........ ....... ........ 197 YoshiakiKinosita PART IV TYPE IV-DRIVEN TWITCHING MOTILITY OF BACTERIA 18 InSituStructureDeterminationofBacterialSurfaceNanomachines UsingCryo-ElectronTomography.......... ....... ... ..... ...... .... ..... 211 LongshengLai,Yee-WaiCheung,MatthewMartinez, KathrynKixmoeller,LeonPalaoIII,StefanSteimle, Meng-ChiaoHo,BenE.Black,Erh-MinLai,andYi-WeiChang 19 TwitchingMotilityAssaysofLysobacterenzymogenesOH11UnderaLight Microscope ........ ....... ........ ....... ....... ........ ....... ........ 249 BingxinWang,XiaolongShao,andGuoliangQian 20 LiveCellImagingoftheTwitchingMotilityofCyanobacteria byHigh-ResolutionMicroscopy..... ....... ....... ........ ....... ........ 255 DaisukeNakane PART V ADHESION-BASED GLIDING MOTILITY OF BACTERIA 21 IsolationandVisualizationofGlidingMotilityMachineryinBacteroidota ..... 267 SatoshiShibataandDaisukeNakane 22 LiveCellImagingofGlidingMotilityofFlavobacteriumjohnsoniae UnderHigh-ResolutionMicroscopy ........ ....... ........ ....... ........ 277 DaisukeNakaneandSatoshiShibata 23 SocialMotilityAssaysofFlavobacteriumjohnsoniae .. ........ ....... ........ 287 ChikaraSatoandKeikoSato 24 VisualizationofPeptidoglycanStructuresofEscherichiacoli byQuick-FreezeDeep-EtchElectronMicroscopy ........... ....... ........ 299 YuheiO.TaharaandMakotoMiyata