Methods in Molecular Biology 2548 Paola Sperandeo Editor Lipopolysaccharide Transport Methods and Protocols 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. Lipopolysaccharide Transport Methods and Protocols Edited by Paola Sperandeo Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy Editor PaolaSperandeo DepartmentofPharmacologicaland BiomolecularSciences Universita`degliStudidiMilano Milan,Italy ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2580-4 ISBN978-1-0716-2581-1 (eBook) https://doi.org/10.1007/978-1-0716-2581-1 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2022 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,theauthorsandtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface ThedistinctivefeatureofGram-negativebacteriaisadoublemembranecellenvelopewhich greatly impacts on the physiology of this wide group of microorganisms. This cellular structure, involving an inner and an outer membrane, is essential for bacterial survival especially in the natural environment, where it represents the point of interaction with the external milieu and, in the case of pathogens, with the host immune system. The outer membrane not only contributes together with the peptidoglycan to give strength and rigiditytothecellwall,withstandingtheturgorpressure,butalsoprovidesaphysicalbarrier totheattackofnoxiousandpotentiallyharmfulmolecules,suchasmanyantibiotics.Infact, itcarriesapanoplyofeffluxpumpsthatactivelyextrudethetoxicmoleculesoutsidethecell preventingthemfromreachinghighenoughintracellularconcentrationstoexertabacteri- cidaleffect.Butmostimportantly,theoutermembraneservesitselfasapermeabilitybarrier toseveraldrugs,blockingthemfromdiffusingintotheperiplasmandthecytoplasmthanks to its peculiar asymmetric structure that, unlike the inner membrane, is not made by a phospholipidbilayer. The asymmetry of the outer membrane is guaranteed by the presence of a unique glycolipid,thelipopolysaccharides(LPS),intheouterleafletofthemembrane.Theamphi- pathic nature of LPS, made up of a lipid anchor linked to a core oligosaccharide that is furtherdecoratedbyavariablepolysaccharidechain,iscrucialforthedistinctivefunctionof the outer membrane as permeability barrier. Nevertheless, the chemical proprieties of LPS also pose several challenges to its biogenesis. In fact, like other components of the outer membrane (i.e., proteins and phospholipids), LPS precursors are also synthesized in the cytoplasm and need to be transported across subcellular compartments characterized by differentphysicochemicalproprieties(theinnermembraneandtheperiplasm),beforebeing assembled at the outer membrane. Importantly, periplasm and outer membrane are com- partments devoid of energy thus posing an additional burden for LPS biogenesis. For this reason, LPS trafficking across the cell envelope requires the intervention of proteins and multiprotein machineries that harness the energy from ATP to overcome the thermody- namicobstaclesandallowthepassageandassemblyofLPSprecursors. The integrity of the whole process of LPS biogenesis is fundamental for bacterial survival; therefore, molecules which interfere with any step of this process to inflict lethal damagetotheoutermembranewouldrepresentpromisingstartingpointstodevelopnovel antibacterialstrategies. Tremendous effort has therefore been made during the past 50 years to expand the knowledge on LPS biogenesis and identify unexplored mechanisms to be targeted by the rationaldesignorscreeningofnovelantibiotics. This volume is intended to bring together not only some of the most commonly used protocols in this field of study, such as membrane fractionation by sucrose gradient centri- fugationcoupledwithLPSprofiledetermination(describedinChap.4)and LPSchemical analysisbymassspectrometry(describedinChap.12),butalsobiochemicalandbiophysical techniques to study protein-protein (Chaps. 7, 9, and 10) and protein-LPS interaction (Chaps. 6, 8, and 13) that proved useful for the characterization of proteins and multi- v vi Preface protein machineries involved in LPS transport. Approaches to determine protein topology andstructurearealsofundamentaltoelucidateLPStransportmechanismsandareincluded (Chaps.5,11,14,and15). Importantly,thediscoveryoffactorsinvolvedinLPSbiogenesisisgenerallyhinderedby thelackofaspecificphenotypeandtheessentialityofmostofthegenes,whichhampersthe construction of knock-out mutants. Therefore, the research on LPS transport is strictly dependent on the development of clever genetic strategies to allow the analysis of this process. This aspect is addressed in Chaps. 1, 2, and 3, which describe protocols for the identification and the analysis of essential genes and crucial mutations in LPS transport- relatedgenes. Lastly, Chaps. 16 and 17 describe techniques to enable LPS visualization by confocal microscopyandatomicforcemicroscopy. Wehopethatthisvolumemightprovideanoverviewonhowmultidisciplinarythestudy ofLPStransportshouldbetoensureaholisticunderstandingofsuchacrucialprocessand toallowtheidentificationofnewpotentialAchille’sheelsoftheGram-negativebacterialcell for thedevelopmentofinnovativeantibacterialstrategies. I would like to express my deepest gratitude to all the authors contributing to this volume,whometiculouslysharedtheyexpertiseintheirchapters.Finally,aspecialthanksis due to the series Editor, Professor John Walker, for the great opportunity of editing this volume. Milan,Italy PaolaSperandeo Contents Preface ..................................................................... v Contributors................................................................. ix PART I GENETIC STRATEGIES TO STUDY ESSENTIAL LPS BIOGENESIS GENES 1 UseofMutagenesisandFunctionalScreenstoCharacterizeEssential GenesInvolvedinLipopolysaccharideTransport..... ........ ....... ........ 3 AndrewWilson,CarlosIniguez,andNatividadRuiz 2 GenerationofStableandUnmarkedConditionalMutants inPseudomonasaeruginosa.. ........ ....... ....... ........ ....... ........ 21 AlessandraLoSciuto,MariaConcettaSpinnato, MartinaPasqua,andFrancescoImperi 3 AnalyzingtheFunctionofEssentialGenesbyPlasmidShuffling...... ........ 37 FedericaAnnaFalchi PART II FUNCTIONAL ANALYSIS OF PROTEINS INVOLVED IN LPS TRANSPORT 4 MembraneFractionationbyIsopycnicSucroseDensityGradient CentrifugationforQualitativeAnalysisofLPSinEscherichiacoli...... ........ 53 ElisabeteC.CardosoMendesMoura,AlessandraPolissi, andPaolaSperandeo 5 ExploringtheTopologyofCytoplasmicMembraneProteinsInvolved inLipopolysaccharideBiosynthesisbyinSilicoandBiochemicalAnalyses ... ... 71 JuliaMonjara´sFeriaandMiguelA.Valvano 6 UseofSite-DirectedSpinLabelingEPRSpectroscopytoStudy Protein–LPSInteractions ........... ....... ....... ........ ....... ........ 83 KathrynM.SchultzandCandiceS.Klug 7 APhoto-CrosslinkingApproachtoMonitoringtheAssembly ofanLptDIntermediatewithLptEinaLivingCell.......... ....... ........ 97 RyojiMiyazaki,HiroyukiMori,andYoshinoriAkiyama 8 MassSpectrometryAnalysisofDynamicsandInteractionsoftheLPS TransloconLptDE......... ........ ....... ....... ........ ....... ... ..... 109 FrancescoFiorentinoandJaniR.Bolla 9 AffinityPurificationandCoimmunoprecipitationofTransenvelopeProtein ComplexesinGram-NegativeBacteria....... ....... ........ ....... ........ 129 AlessandraM.Martorana,CarloSantambrogio, andAlessandraPolissi 10 ABacterialTwo-HybridSystemforInVivoAssaysofProtein-Protein InteractionsandDrugDiscovery .... ....... ....... ........ ....... ........ 145 DanielLadant 11 DetectingLipoproteinsSneakingOutoftheLipopolysaccharideLeaflet....... 169 NaemiCsoma,DidierColau,andJean-Franc¸oisCollet vii viii Contents PARTIII TOOLSFORSTRUCTURALANALYSISOFLPSANDLPSTRANSPORTPROTEINS 12 DissectingLipopolysaccharideCompositionandStructurebyGC-MS andMALDISpectrometry.......... ....... ....... ........ ....... ........ 181 PilarGarcia-Vello,ImmacolataSpeciale,FlavianaDiLorenzo, AntonioMolinaro,andCristinaDeCastro 13 ProbingtheFunctionalInteractionInterfaceofLipopolysaccharide andAntimicrobialPeptides:ASolution-StateNMRPerspective ...... ........ 211 KarishmaBiswasandAnirbanBhunia 14 Cryo-EMAnalysisoftheLipopolysaccharideFlippaseMsbA......... ........ 233 Franc¸oisA.The´lotandMaofuLiao 15 ProteinCrystallizationofTwoRecombinantLptProteins .... ....... ........ 249 MichelaBollatiandLouiseJ.Gourlay PART IV MICROSCOPY-BASED APPROACHES 16 MetabolicIncorporationofAzido-SugarsintoLPStoEnableLive-Cell FluorescenceImaging ...... ... .... ........ ....... ........ ....... ........ 267 IngaNilssonandDavidA.Six 17 UseofAtomicForceMicroscopytoCharacterizeLPSPerturbations.......... 279 YooJinOh Index ...................................................................... 289 Contributors YOSHINORIAKIYAMA • InstituteforFrontierLifeandMedicalSciences,KyotoUniversity, Kyoto,Japan ANIRBANBHUNIA • DepartmentofBiophysics,BoseInstitute,Kolkata,India KARISHMABISWAS • DepartmentofBiophysics,BoseInstitute,Kolkata,India JANIR.BOLLA • TheKavliInstituteforNanoscienceDiscovery,Oxford,UK;Departmentof PlantSciences,UniversityofOxford,Oxford,UK MICHELA BOLLATI • DepartmentofBiosciences,Universit`adegliStudidiMilano,Milan, Italy;InstituteofBiophysics-CNR,Milan,Italy ELISABETEC.CARDOSOMENDES MOURA • DepartmentofPharmacologicaland BiomolecularSciences,Universit`adegliStudidiMilano,Milan,Italy DIDIERCOLAU • deDuveInstitute,Universite´catholiquedeLouvainandWELBIO,Brussels, Belgium JEAN-FRANC¸OISCOLLET • deDuveInstitute,Universite´catholiquedeLouvainandWELBIO, Brussels,Belgium NAEMICSOMA • deDuveInstitute,Universite´catholiquedeLouvainandWELBIO,Brussels, Belgium CRISTINADECASTRO • DepartmentofAgriculturalSciences,UniversityofNaples,Portici, (NA),Italy FLAVIANADILORENZO • DepartmentofChemicalSciences,UniversityofNaples,Naples, Italy FEDERICA ANNAFALCHI • DepartmentofBiosciences,Universit`adegliStudidiMilano, Milan,Italy FRANCESCOFIORENTINO • DepartmentofDrugChemistryandTechnologies,Sapienza UniversityofRome,Rome,Italy PILARGARCIA-VELLO • DepartmentofChemicalSciences,UniversityofNaples,Naples,Italy LOUISEJ.GOURLAY • DepartmentofBiosciences,Universit`adegliStudidiMilano,Milan, Italy FRANCESCOIMPERI • DepartmentofScience,UniversityRomaTre,Rome,Italy;IRCCS FondazioneSantaLucia,Rome,Italy CARLOSINIGUEZ • DepartmentofMicrobiology,TheOhioStateUniversity,Columbus,OH, USA CANDICES.KLUG • DepartmentofBiophysics,MedicalCollegeofWisconsin,Milwaukee,WI, USA DANIELLADANT • Unite´deBiochimiedesInteractionsMacromole´culaires,De´partementde BiologieStructuraleetChimie,CNRSUMR3528,InstitutPasteur,Paris,France MAOFULIAO • DepartmentofCellBiology,HarvardMedicalSchool,Boston,MA,USA ALESSANDRA LOSCIUTO • DepartmentofScience,UniversityRomaTre,Rome,Italy ALESSANDRA M.MARTORANA • DepartmentofPharmacologicalandBiomolecularSciences, Universit`adegliStudidiMilano,Milan,Italy RYOJI MIYAZAKI • NaraInstituteofScienceandTechnology,Ikoma,Nara,Japan ANTONIOMOLINARO • DepartmentofChemicalSciences,UniversityofNaples,Naples,Italy JULIAMONJARA´SFERIA • Wellcome-WolfsonInstituteforExperimentalMedicine,Queen’s UniversityBelfast,Belfast,UK ix