Terry J. McGenity Kenneth N. Timmis Balbina Nogales Editors Hydrocarbon and Lipid Microbiology Protocols Genetic, Genomic and System Analyses of Communities Springer Protocols Handbooks More information about this series at http://www.springer.com/series/8623 Terry J. McGenity Kenneth N. Timmis Balbina Nogales (cid:129) (cid:129) Editors Hydrocarbon and Lipid Microbiology Protocols Genetic, Genomic and System Analyses of Communities Scientific Advisory Board JackGilbert,IanHead,MandyJoye,VictordeLorenzo, JanRoelofvanderMeer,ColinMurrell,JoshNeufeld, RogerPrince,JuanLuisRamos,WilfredRo¨ling, HeinzWilkes,MichailYakimov Editors TerryJ.McGenity KennethN.Timmis SchoolofBiologicalSciences InstituteofMicrobiology UniversityofEssex TechnicalUniversityBraunschweig Colchester,Essex,UK Braunschweig,Germany BalbinaNogales DepartmentofBiology UniversityoftheBalearicIslands andMediterraneanInstituteforAdvanced Studies(IMEDEA,UIB-CSIC) PalmadeMallorca,Spain ISSN1949-2448 ISSN1949-2456 (electronic) SpringerProtocolsHandbooks ISBN978-3-662-50449-9 ISBN978-3-662-50450-5 (eBook) DOI10.1007/978-3-662-50450-5 LibraryofCongressControlNumber:2016938230 #Springer-VerlagBerlinHeidelberg2017 Thisworkis subjectto copyright.Allrightsarereservedbythe Publisher,whetherthe wholeorpartofthe materialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproductionon microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulationsand thereforefreeforgeneraluse. Thepublisher,theauthorsandtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedtobe trueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty,expressor implied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeenmade. Printedonacid-freepaper ThisSpringerimprintispublishedbySpringerNature TheregisteredcompanyisSpringer-VerlagGmbHBerlinHeidelberg Preface to Hydrocarbon and Lipid Microbiology Protocols1 Allactivecellularsystemsrequirewaterastheprincipalmediumandsolventfortheirmetabolicand ecophysiological activities. Hydrophobic compounds and structures, which tend to exclude water, althoughprovidinginteraliaexcellentsourcesofenergyandameansofbiologicalcompartmental- ization, present problems of cellular handling, poor bioavailability and, in some cases, toxicity. Microbesbothsynthesizeandexploitavastrangeofhydrophobicorganics,whichincludesbiogenic lipids, oils and volatile compounds, geochemically transformed organics of biological origin (i.e.petroleumandotherfossilhydrocarbons)andmanufacturedindustrialorganics.Theunderlying interactionsbetweenmicrobesandhydrophobiccompoundshavemajorconsequencesnotonlyfor thelifestylesofthemicrobesinvolvedbutalsoforbiogeochemistry,climatechange,environmental pollution, human health and a range of biotechnological applications. The significance of this “greasymicrobiology”isreflectedinboththescaleandbreadthofresearchonthevariousaspects ofthetopic.Despitethis,therewas,asfarasweknow,notreatiseavailablethatcoversthesubject. In an attempt to capture the essence of greasy microbiology, the Handbook of Hydrocarbon and Lipid Microbiology (http://www.springer.com/life+sciences/microbiology/book/978-3-540-77584- 3) was published by Springer in 2010 (Timmis 2010). This five-volume handbook is, we believe, uniqueandofconsiderableservicetothecommunityanditsresearchendeavours,asevidencedby the large number of chapter downloads. Volume 5 of the handbook, unlike volumes 1–4 which summarizecurrentknowledgeonhydrocarbonmicrobiology,consistsofacollectionofexperimen- talprotocolsandappendicespertinenttoresearchonthetopic. Asecondeditionofthehandbookisnowinpreparationandadecisionwastakentosplitoff themethodssectionandpublishitseparatelyaspartoftheSpringerProtocolsprogram(http:// www.springerprotocols.com/). The multi-volume work Hydrocarbon and Lipid Microbiology Protocols, while rooted in Volume 5 of the Handbook, has evolved significantly, in terms of range of topics, conceptual structure and protocol format. Research methods, as well as instrumentationandstrategicapproachestoproblemsandanalyses,areevolvingatanunprec- edented pace, which can be bewildering for newcomers to the field and to experienced researchers desiring to take new approaches to problems. In attempting to be comprehensive – a one-stop source of protocols for research in greasy microbiology – the protocol volumes inevitablycontainbothsubject-specificandmoregenericprotocols,includingsamplinginthe field, chemical analyses, detection of specific functional groups of microorganisms and com- munity composition, isolation and cultivation of such organisms, biochemical analyses and activity measurements, ultrastructure and imaging methods, genetic and genomic analyses, 1AdaptedinpartfromthePrefacetoHandbookofHydrocarbonandLipidMicrobiology. v vi PrefacetoHydrocarbonandLipidMicrobiologyProtocols systems and synthetic biology tool usage, diverse applications, and the exploitation of bioin- formatic,statisticalandmodellingtools.Thus,whiletheworkisaimedatresearchersworking onthemicrobiologyofhydrocarbons,lipidsandotherhydrophobicorganics,muchofitwillbe equally applicable to research in environmental microbiology and, indeed, microbiology in general.This,webelieve,isasignificantstrengthofthesevolumes. We are extremely grateful to the members of our Scientific Advisory Board, who have made invaluable suggestions of topics and authors, as well as contributing protocols them- selves,andtogenerousadhocadvisorslikeWeiHuang,ManfredAuerandLarsBlank.Wealso express our appreciation of Jutta Lindenborn of Springer who steered this work with profes- sionalism,patienceandgoodhumour. Colchester,Essex,UK TerryJ.McGenity Braunschweig,Germany KennethN.Timmis PalmadeMallorca,Spain BalbinaNogales Reference TimmisKN(ed)(2010)Handbookofhydrocarbonandlipidmicrobiology.Springer,Berlin,Heidelberg Contents IntroductiontoGenetic,Genomic,andSystemAnalysesforCommunities. . . . . . . . . . 1 JackA.GilbertandNicoleM.Scott GenomicAnalysisofPureCulturesandCommunities. . . . . . . . . . . . . . . . . . . . . . . . . . 5 StepanV.Toshchakov,IlyaV.Kublanov,EnzoMessina, MichailM.Yakimov,andPeterN.Golyshin ProtocolsforMetagenomicLibraryGenerationandAnalysis inPetroleumHydrocarbonMicrobeSystems. . . . .. . . . . . . . .. . . . . . . . .. . . . . . . .. . 29 StephanieM.Moormann,JarradT.Hampton-Marcell, SarahM.Owens,andJackA.Gilbert PreparationandAnalysisofMetatranscriptomicLibraries inPetroleumHydrocarbonMicrobeSystems. . . . .. . . . . . . . .. . . . . . . . .. . . . . . . .. . 51 JarradT.Hampton-Marcell,AngelFrazier,StephanieM.Moormann, SarahM.Owens,andJackA.Gilbert ProkaryoticMetatranscriptomics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 DaniloPe´rez-PantojaandJavierTamames DefiningaPipelineforMetaproteomicAnalyses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 JosephA.Christie-Oleza,DespoinaSousoni,JeanArmengaud, ElizabethM.Wellington,andAlexandraM.E.Jones MetabolicProfilingandMetabolomicProceduresforInvestigating theBiodegradationofHydrocarbons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 VincentBonifay,EgemenAydin,DenizF.Aktas, JanSunner, andJosephM.Suflita GeneratingEnrichedMetagenomesfromActiveMicroorganisms withDNAStableIsotopeProbing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 CarolinaGrob,MartinTaubert,AlexandraM.Howat,OliverJ.Burns, YinChen,JoshD.Neufeld,andJ.ColinMurrell DNA-andRNA-BasedStableIsotopeProbingofHydrocarbonDegraders. . . . . . . . . . 181 TillmannLueders ProtocolforPerformingProteinStableIsotopeProbing(Protein-SIP) Experiments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 NicoJehmlichandMartinvonBergen vii viii Contents ProteinExtractionfromContaminatedSoilsandSediments. . . . . . . . . . . . . . . . . . . . . 215 MercedesV.DelPozo,Mo´nicaMart´ınez-Mart´ınez,andManuelFerrer AnalysisoftheRegulationoftheRateofHydrocarbonandNutrient FlowThroughMicrobialCommunities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 WilfredF.M.Ro¨ling,LucasFillinger,andUlissesNunesdaRocha ConstructingandAnalyzingMetabolicFluxModelsofMicrobial Communities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 Jose´ P.Faria,TahminehKhazaei,JanakaN.Edirisinghe,PamelaWeisenhorn, SamuelM.D.Seaver,NealConrad,NomiHarris,MatthewDeJongh, andChristopherS.Henry ProtocolforEvaluatingthePermissivenessofBacterialCommunities TowardConjugalPlasmidsbyQuantificationandIsolation ofTransconjugants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 UliKlu¨mper,ArnaudDechesne,andBarthF.Smets About the Editors Terry J. McGenity is a Reader at the University of Essex, UK. His Ph.D., investigating the microbial ecology of ancient salt deposits (University of Leicester), was followed by postdoctoral positions at the Japan Marine Science and Technology Centre (JAMSTEC, Yoko- suka)andthePostgraduateResearchInstituteforSedimentology(Uni- versityofReading). Hisoverarching researchinterest istounderstand how microbial communities function and interact to influence major biogeochemicalprocesses.HeworkedasapostdocwithKenTimmisat theUniversityofEssex,wherehewasinspiredtoinvestigatemicrobial interactionswithhydrocarbonsatmultiplescales,fromcommunitiestocells,andasbothasourceof foodandstress.Hehasbroadinterestsinmicrobialecologyanddiversity,particularlywithrespect tocarboncycling(especiallythesecondmostabundantlyproducedhydrocarbonintheatmosphere, isoprene), and is driven to better understand how microbes cope with, or flourish in hypersaline, desiccatedandpoly-extremeenvironments. Kenneth N. Timmis read microbiology and obtained his Ph.D. at Bristol University, where he became fascinated with the topics of environmental microbiology and microbial pathogenesis, and their interfacepathogenecology.Heundertookpostdoctoraltrainingatthe Ruhr-University Bochum with Uli Winkler, Yale with Don Marvin, andStanfordwithStanCohen,atthelattertwoinstitutionsasaFellow oftheHelenHayWhitneyFoundation,whereheacquiredthetoolsand strategiesofgeneticapproachestoinvestigatemechanismsandcausal relationships underlying microbial activities. He was subsequently appointedHeadofanIndependentResearchGroupattheMaxPlanck InstituteforMolecularGeneticsinBerlin,thenProfessorofBiochem- istry in the University of Geneva Faculty of Medicine. Thereafter, he became Director of the Division of Microbiology at the National Research Centre for Biotechnology (GBF)/now the Helmholtz Centre for Infection Research (HZI) and Professor of Microbiology at the Technical UniversityBraunschweig.Hisgrouphasworkedformanyyears,interalia,onthebiodegradationof oil hydrocarbons, especially the genetics and regulation of toluene degradation, pioneered the genetic designand experimental evolutionof novel catabolic activities,discovered the new group of marine hydrocarbonoclastic bacteria, and conducted early genome sequencing of bacteria that ix x AbouttheEditors becameparadigmsofmicrobesthatdegradeorganiccompounds(PseudomonasputidaandAlcani- voraxborkumensis).Hehashadtheprivilegeandpleasureofworkingwithandlearningfromsome of the most talented young scientists in environmental microbiology, a considerable number of whicharecontributingauthorstothisseries,andinparticularBalbinaandTerry.HeisFellowofthe RoyalSociety,MemberoftheEMBO,RecipientoftheErwinSchro¨dingerPrize,andFellowofthe AmericanAcademyofMicrobiologyandtheEuropeanAcademyofMicrobiology.Hefoundedthe journals Environmental Microbiology, Environmental Microbiology Reports and Microbial Bio- technology.KennethTimmisiscurrentlyEmeritusProfessorintheInstituteofMicrobiologyatthe TechnicalUniversityofBraunschweig. BalbinaNogalesisaLecturerattheUniversityoftheBalearicIslands, Spain. Her Ph.D. at the Autonomous University of Barcelona (Spain) investigated antagonistic relationshipsinanoxygenicsulphur photosyn- thetic bacteria. This was followed by postdoctoral positions in the researchgroupsofKen Timmisatthe GermanNationalBiotechnology Institute (GBF, Braunschweig, Germany) and the University of Essex, where she joinedTerryMcGenity aspostdoctoralscientist.Duringthat time, she worked in different researchprojects on community diversity analysisofpollutedenvironments.Aftermovingtohercurrentposition, herresearchisfocusedonunderstandingmicrobialcommunitiesinchronicallyhydrocarbon-polluted marineenvironments,andelucidatingtheroleinthedegradationofhydrocarbonsofcertaingroupsof marinebacterianotrecognizedastypicaldegraders.