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Methods in Molecular Biology 2601 Peter Sass Editor Antibiotics Methods and Protocols Second Edition 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. Antibiotics Methods and Protocols Second Edition Edited by Peter Sass Interfaculty Institute for Microbiology and Infection Medicine, University of Tübingen, Tübingen, Germany Editor PeterSass InterfacultyInstituteforMicrobiology andInfectionMedicine UniversityofTu¨bingen Tu¨bingen,Germany ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2854-6 ISBN978-1-0716-2855-3 (eBook) https://doi.org/10.1007/978-1-0716-2855-3 ©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. 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Preface Antibioticsareamongthemostimportantdiscoveriesinmedicalhistory,savingmillionsof lives,sincetheyallowtheeffectivetreatmentofevencomplicated,life-threateningbacterial infections.Also,antibioticsrevolutionizedourpossibilitiesofmedicalintervention,thereby significantlyincreasingthequalityofhumanlife.Today,thebenefitsofantibioticinterven- tion are at elevated risk, and an increasing prevalence of antibiotic-resistant microbes challenges modern medicine, posing serious threats to human and animal health. A post- antibiotic era has already begun, and more than ever, we need new antibiotics with novel mechanismsofactionandresistance-breakingproperties. ThesecondeditionofAntibiotics:MethodsandProtocolsintheSpringerseriesMethods inMolecularBiologycontinuestoprovidestate-of-the-artandnovelmethodsonantibiotic isolation and purification, identification of antimicrobial killing mechanisms, and methods fortheanalysisanddetectionofmicrobialresponsesandadaptationstrategies.Accordingly, the chapters are organized under two major themes: (i) production and design, and (ii) mode of action and resistance. Following an overview on common antibiotics and examples for novel antibiotic modes of action (Chap. 1), the first part, on antibiotic production and design, includes methods to detect antibacterial activities in culture super- natants of actinomycetes (Chap. 2), to sample human microbiomes and screen them for antibiotic-producing commensals (Chap. 3), and to produce and isolate such compounds (Chaps. 4 and 5). With a new compound in hand, structure elucidation is important for compoundcharacterization(Chap.6)andprovidesthebasisfor furtheroptimization,that is, by structure- and ligand-based drug design (Chap. 7) to improve compound-target interactions. To be considered as a promising new therapeutic, the selected compound should have no or only low cytotoxic activity on eukaryotic cells. This can be monitored bymethodsprovidedinChap.8andshouldbeassessedbeforefurtherstepsaretaken. In the second part, the chapters lead the reader through methods to further explore antibioticmechanismsofactionaswellasrelatedbacterialresponsesandresistancemechan- isms. With a special focus on advanced microscopy techniques, which has greatly contrib- utedtotheelucidationofantibioticmodesofactioninthepast,Chaps.9to12describean assaytocharacterizebacterialphenotypesuponantibiotictreatmentviaamicroscopy-based multiwell assay (Chap. 9), explain a method for expansion microscopy to enable super- resolved visualization of specimen without the need of highly sophisticated and expensive optical instruments (Chap. 10), and show how to track the global and local changes in membrane fluidity through fluorescence spectroscopy and microscopy (Chap. 11). In addition, bioinformatic tools are presented to analyze microscopy image data qualitatively and quantitatively (Chap. 12). In the following chapters, further cell-based and in vitro assaysarepresentedthathelptocharacterizeantibioticmodesofactionandtheirimpacton certain biosynthesis pathways or cellular structures. Here, for example, the bacterial cell enveloperepresentsavalidatedtargetforantibiotics.Todetectantibioticsthatinterferewith cell wall integrity and synthesis, their ability to induce specific cell wall stress-responsive promoter fusions can be measured (Chap. 13). An impairment of the bacterial membrane can be determined by measuring the effect of antibiotics on membrane potential and potassium release from whole cells (Chap. 14). Also, antibiotics are frequently found to interfere with DNA replication and translation. Here, Chap. 15 provides a robust v vi Preface colorimetricassaythatenablestheidentificationandthevalidationofinhibitorsofbacterial primases, such as DnaG, which play essential roles in DNA replication, while Chap. 16 reports on the screening for inhibitors of the translation initiation pathway. A different targetareaiscoveredbyChap.17,whichdescribesinvitroandinvivoinhibitorscreensto search for modulators of bacterial histidine kinases, some of which either are essential for survival or were found to be involved in the development of antibiotic resistance. Under- standingbacterialresponsestoantibioticsaswellastheestablishedresistancemechanismsto clinically used drugs is mandatory to evaluate alternatives to commonly applied treatment strategies. To address theseaspects, global profiling methods to study the proteomeor metal ion homeostasis (Chaps. 18 and 19) of susceptible versus resistant strains (or untreated versus antibiotic-treatedstrains)havethepotentialtouncover theunderlyingantibioticmodesof action as well as resistance mechanisms. Chapter 20 goes further into this direction and characterizesalterationsinthestoichiometryandcompositionofribosomalandribosome- associated proteins during antibiotic stress, which impact on protein expression profiles or hibernating ribosomes. Heading further towards antibacterial resistance, functional meta- genomics emerged as a highly useful way to identify novel resistance genes from environ- mental samples (Chap. 21), which do not necessarily rely on the culturability of a specific straininthelaboratory,thusallowingtostudyantibioticresistanceinverydiversemicrobial communities such as soil-, marine-, human-, wastewater-, or animal- and agriculture- associatedcommunities. I would like to thank all contributing authors of the first and second edition of Anti- biotics: Methods and Protocols for sharing their expertise and protocols, thereby essentially contributingtothesuccessofthisbook.Antibioticresearchisamultidisciplinaryapproach, andthusAntibiotics:MethodsandProtocolsaddressesscientistsfromdiversefieldsinvolving microbiologists, cell biologists, molecular geneticists, pharmacists, immunologists, infec- tiologists,biochemists,biophysicists,bioinformaticians,andmanyothers.Wehopethatthe book will inspire your scientific work in the exciting field of antibiotic research, and we wouldbepleasedtoseethebookmoreofteninyourlabthaninthelibrary. Tu¨bingen,Germany PeterSass Contents Preface ..................................................................... v Contributors................................................................. ix PART I PRODUCTION AND DESIGN 1 Antibiotics:PreciousGoodsinChangingTimes ..... ........ ....... ........ 3 PeterSass 2 AWhole-CellAssayforDetectionofAntibacterialActivity inActinomyceteCultureSupernatants....... ....... ........ ....... ........ 27 AnikaRu¨tten,WolfgangWohlleben,LenaMitousis, andEwaMariaMusiol-Kroll 3 SamplingofHumanMicrobiomestoScreenforAntibiotic-Producing Commensals... .. ... ....... ........ ....... ....... ........ ....... ...... .. 39 BenjaminTorresSalazar,AnnaLange, LauraCamus,andSimonHeilbronner 4 ProductionofAntimicrobialCompoundsbyHomologous andHeterologousExpression ....... ....... ....... ........ ....... ........ 55 I.DewaM.Kresna,ZerlinaG.Wuisan,andTillF.Scha€berle 5 IsolationandPurificationofNaturalProductsfromMicrobialCultures........ 75 ThomasSchafhauserandAndreasKulik 6 StructureElucidationofAntibiotics......... ....... ........ ....... ........ 97 JuliaMoschny,GeorgiosDaletos,PeterProksch, andChambersC.Hughes 7 Computer-AidedDrugDesign:AnUpdate ......... ..... ... ...... .... ..... 123 WenboYu,DavidJ.Weber,andAlexanderD.MacKerellJr 8 CytotoxicityAssaysasPredictorsoftheSafetyandEfficacy ofAntimicrobialAgents..... ........ ....... ....... ........ ....... ........ 153 AlexanderZipperer,JasminScheurer,andDorotheeKretschmer PART II MODE OF ACTION AND RESISTANCE 9 Microscopy-BasedMultiwellAssaytoCharacterizeDisturbed BacterialMorphogenesisUponAntibioticAction.... ........ ....... ........ 171 CruzL.MatosdeOpitzandPeterSass 10 ExpansionMicroscopyofBacillussubtilis.... ....... ........ .. .... ......... 191 ViolaMiddelhauve,JanPeterSiebrasse,andUlrichKubitscheck 11 TrackingGlobalandLocalChangesinMembraneFluidity ThroughFluorescenceSpectroscopyandMicroscopy ........ ....... ........ 203 MadeleineHumphrey,IrenyAbdelmessehNekhala,KathiScheinpflug, OxanaKrylova,Ann-BrittScha€fer,JessicaA.Buttress, MichaelaWenzel,andHenrikStrahl vii viii Contents 12 QuantitativeAnalysisofMicroscopyDatatoEvaluateBacterial ResponsestoAntibioticTreatment.......... ....... ........ ....... ........ 231 DominikBrajtenbach,Jan-SamuelPuls,CruzL.MatosdeOpitz, PeterSass,UlrichKubitscheck,andFabianGrein 13 ApplicationofaBacillussubtilisWhole-CellBiosensor(P -lux) liaI for theIdentificationofCellWallActiveAntibacterialCompounds ........... 259 CarolinMartinaKobras,SaliMayMorris, ThorstenMascher,andSusanneGebhard 14 DeterminationofBacterialMembraneImpairment byAntimicrobialAgents.... ........ ....... ....... ........ ....... ........ 271 MiriamFuerst-WilmesandHans-GeorgSahl 15 AColorimetricAssaytoIdentifyandCharacterizeBacterial PrimaseInhibitors ......... .. ...... ...... .... .... ........ ....... ........ 283 AllanH.PangandOlegV.Tsodikov 16 Cell-BasedFluorescentScreenAmenabletoHTStoIdentify InhibitorsofBacterialTranslationInitiation......... ........ ....... ........ 303 MatteoRaneri,EmilioAlvarez-Ruiz,DanutaMossakovska, andFedericaBriani 17 BacterialTwoComponentSystems:OverexpressionandPurification: InVitroandInVivoInhibitorScreens ...... ....... ........ ....... ........ 313 AlinaDietrich,MikeGajdiss,MichaelTu¨rck, IanMonk,andGabrieleBierbaum 18 SamplePreparationforMassSpectrometry-BasedAbsolute QuantificationofBacterialProteinsinAntibioticStressResearch ..... ...... .. 335 SandraMaaß,MiniaAntelo-Varela,FlorianBonn, andDörteBecher 19 ElementalAnalysisfor theCharacterizationofAntimicrobialEffects .......... 349 ChristophH.R.SengesandJuliaE.Bandow 20 Label-FreeQuantitationofRibosomalProteinsfromBacillussubtilis forAntibioticResearch ..... ........ ....... ....... ........ ....... ........ 363 SinaScha€kermann,PascalDietze,andJuliaE.Bandow 21 FunctionalMetagenomicstoStudyAntibioticResistance..... ....... ........ 379 BejanMahmud,ManishBoolchandani,SanketPatel, andGautamDantas Index ...................................................................... 403 Contributors IRENYABDELMESSEHNEKHALA • DivisionofChemicalBiology,DepartmentofBiologyand BiologicalEngineering,ChalmersUniversityofTechnology,Gothenburg,Sweden EMILIOALVAREZ-RUIZ • GlaxoSmithKlinePlatform TechnologiesandScience,Parque TecnologicodeMadrid,Madrid,Spain MINIAANTELO-VARELA • DepartmentofMicrobialProteomics,UniversityofGreifswald, InstituteofMicrobiology,Greifswald,Germany;UniversityofBasel,Biozentrum,Focal AreaInfectionBiology,Basel,Switzerland JULIAE.BANDOW • AppliedMicrobiology,Ruhr-Universita€tBochum,Bochum,Germany; Ruhr-Universita€tBochum,AppliedMicrobiology,Bochum,Germany DO¨RTEBECHER • DepartmentofMicrobialProteomics,UniversityofGreifswald,Instituteof Microbiology,Greifswald,Germany GABRIELEBIERBAUM • InstituteofMedicalMicrobiology,ImmunologyandParasitology, UniversityofBonn,Bonn,Germany FLORIANBONN • DepartmentofMicrobialProteomics,UniversityofGreifswald,Instituteof Microbiology,Greifswald,Germany;ImmundiagnostikAG,Bensheim,Germany MANISHBOOLCHANDANI • TheEdisonFamilyCenterforGenomeSciences&SystemsBiology, WashingtonUniversitySchoolofMedicine,St.Louis,MO,USA DOMINIKBRAJTENBACH • InstituteofPhysicalandTheoreticalChemistry,UniversityofBonn, Bonn,Germany FEDERICA BRIANI • DipartimentodiBioscienze,UniversitàdegliStudidiMilano,Milan, Italy JESSICAA.BUTTRESS • CentreforBacterialCellBiology,BiosciencesInstitute,Newcastle University,NewcastleuponTyne,UK LAURA CAMUS • InterfacultyInstituteofMicrobiologyandInfectionMedicine,Institutefor MedicalMicrobiologyandHygiene,Tu¨bingen,Germany;(DFG)ClusterofExcellenceEXC 2124ControllingMicrobestoFightInfections,Tu¨bingen,Germany;GermanCentrefor InfectionResearch(DZIF),PartnerSiteTu¨bingen,Tu¨bingen,Germany GEORGIOSDALETOS • InstituteofPharmaceuticalBiologyandBiotechnology,Heinrich- Heine-University,Du¨sseldorf,Germany GAUTAMDANTAS • TheEdisonFamilyCenter forGenomeSciences&SystemsBiology, WashingtonUniversitySchoolofMedicine,St.Louis,MO,USA;DepartmentofPathology andImmunology,WashingtonUniversitySchoolofMedicine,St.Louis,MO,USA; DepartmentofBiomedicalEngineering,WashingtonUniversityinSt.Louis,St.Louis, MO,USA;DepartmentofMolecularMicrobiology,WashingtonUniversitySchoolof Medicine,St.Louis,MO,USA;DepartmentofPediatrics,WashingtonUniversitySchoolof Medicine,St.Louis,MO,USA ALINADIETRICH • InstituteofMedicalMicrobiology,ImmunologyandParasitology, UniversityofBonn,Bonn,Germany PASCALDIETZE • Ruhr-Universita€tBochum,AppliedMicrobiology,Bochum,Germany MIRIAMFUERST-WILMES • InstituteofMedicalMicrobiology,ImmunologyandParasitology, UniversityofBonn,Bonn,Germany MIKEGAJDISS • InstituteofMedicalMicrobiology,ImmunologyandParasitology,University ofBonn,Bonn,Germany ix