Functional genome analysis of Alcanivorax borkumensis strain SK2 [Elektronische Ressource] : alkane metabolism, environmental adaptations and biotechnological potential / von Julia Sabirova

Isb

Sujets

• Biologie

Informations

• Publié par: technische_universitat_carolo-wilhelmina_zu_braunschweig
• Publié le: 01 janvier 2006
• Nombre de lectures: 53
• Poids de l'ouvrage: 5 Mo

Extrait

i
Functional genome analysis of Alcanivorax borkumensis strain
SK2: alkane metabolism, environmental adaptations and
biotechnological potential

VonderFakultätfürLebenswissenschaften
derTechnischenUniversitätCaroloWilhelmina
zuBraunschweig

zurErlangungdesGradeseiner
DoktorinderNaturwissenschaften
(Dr.rer.nat.)

genehmigte

Dissertation


vonJuliaSabirova
ausKasan,Russland




1.Referent:Prof.Dr.KennethN.Timmis
2.Referent:Prof.Dr.DieterJahn
eingereichtam:16.01.06
mündlichePrüfung(Disputation)am:12.04.06


2006ii
Vorveröffentlichungen der Dissertation
Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Gemeinsamen
Naturwissenschaftlichen Fakultät, vertreten durch die Mentorin oder den Mentor/die
BetreuerinoderdenBetreuerderArbeit,infolgendenBeiträgenvorabveröffentlicht:

Publikationen
Julia S. Sabirova, Manuel Ferrer, Daniela Regenhardt, Kenneth N. Timmis, and Peter N.
Golyshin.ProteomicinsightsintometabolicadaptationsinAlcanivorax borkumensis.
Journal of Bacteriology188:37633773(2006).
N.Golyshin,VitorA.P.MartinsDosSantos,OlafKaiser,ManuelFerrer,YuliaS.Sabirova,
H. Lünsdorf, Tatyana N. Chernikova, Olga V. Golyshina, Michail M. Yakimov,
Alfred Pühler, Kenneth N. Timmis. Genome sequence completed of Alcanivorax
borkumensis, a hydrocarbondegrading bacterium that plays a global role in oil
removalfrommarinesystems.Journal of Biotechnology106:215220(2003).
Patent
J. Sabirova, M. Ferrer, H. Lünsdorf, W.R. Abraham, K.N.Timmis, and P.N.Golyshin.
Extracellular polyhydroxyalkanoates produced by genetically engineered
thmicroorganisms.EPnumber:05017308.7(filedonAugust9 2005).

Tagungsbeiträge
M.Ferrer,J.Sabirova,D.Regenhardt,K.N.Timmis,andP.N.Golyshin.Alkanemetabolismin
Alcanivorax borkumensis: identification of the cytoplasmic and membrane proteins
nd
solely and differentially expressed on alkanes. 2 European Conference on
ProkaryoticGenomes,2326September,Bookofabstracts,p.102(2005).
Ioulia Sabirova, Tatyana Chernikova, Vitor Martins dos Santos, Peter N. Golyshin, and
Kenneth N. Timmis. Analysis of Alcanivorax borkumensis SK2 niche specificity
factors using Tn5 transposon mutagenesis. European Conference on Procaryotic
Genomes,58October,LastMinuteInformation,p.1(2003).
D.Regenhardt,J.S.Sabirova,H.Lünsdorf,S.Heim,P.N.GolyshinandK.N.Timmis.2003.
Bacteriameetsoil:genomeprofilingbyproteomicsandultrastructureanalysisofthe
interaction of Alcanivorax borkumensis strain SK2 with hydrocarbon droplets.
European Conference on Prokaryotic Genomes, 58 October, Book of abstracts,
Posters,p.69(2003).
iii
Table of contents

Preface v
Summary vi
Abbreviations viii

11. INTRODUCTION
1.1 Bacterialdegradationofalkanesanditsgeneticdeterminants 1
1.2 Alcanivorax borkumensisstrainSK2asanoligotrophic,oildegrading
marinebacterium 2
1.3 Functionalgenomeanalysisanditsmethods 4
1.4 Environmentaladaptationofmarinebacteria 6
1.4.1 Resistancetoultravioletradiation 6
1.4.2 Osmoadaptation 9
1.4.3 Resistancetolowtemperature 10
1.4.4 Bacterialbiofilmformation 11
1.5 Stateoftheartandobjectivesoftheproject 13

2. MATERIALS AND METHODS 14
2.1 Bacterialstrainsandgrowthconditions 14
2.2 Biofilmformationassay 14
2.2.1 Screeningformutantsdefectiveinbiofilmformation 14
2.2.2 Quantificationofbiofilmformation 14
2.3 PHAisolationandcompositionanalysis 15
2.4 Electronmicroscopy 16
2.5 ConstructionofaminiTn5transposonlibraryofA.borkumensisSK2 16
2.6 InversePCR 16
2.7 ReversetranscriptionPCR 17
2.8 CloningandexpressionoftesBlikeprotein 18
2.8.1 Constructionofthevector,growthofbacteriaandpreparation
ofcellextractsforthioesteraseassay 18
2.8.2 Synthesisof(R,S)3HydroxyacylCoAforthioesteraseassay 18
2.8.3 Thioesteraseassay 19
2.9 2DEofthecytoplasmicfraction 19
2.9.1 Preparationofthecytoplasmicproteinfractionfor2DE 19
2.9.2 Isoelectricfocusingandseconddimensionofthecytoplasmic 20
fraction
2.10 2DEofthemembranefraction 21
2.10.1 Preparationofthemembranefractionfor2DE 21
2.10.2 Isoelectricfocusingandseconddimensionofthemembrane 21
fraction
2.11 Predictionofputativeoperonstructuresandputativepromoters 22
2.12 Predictionofputativefunctionsofnovelproteinsbasedonsequence
homology 22

233. RESULTS
3.1 Identificationofgeneticdeterminantsofalkanemetabolismin
AlcanivoraxstrainSK2byproteomics 23
3.2 Identificationofgeneticdeterminantsofenvironmentaladaptationof
Alcanivorax strainSK2bytransposonmutagenesis 32
3.2.1 IsolationandidentificationofUVsensitivemutants 34 iv
3.2.2 Isolationandidentificationofmutantssensitivetolow
temperature 42
3.2.3 Isolationandidentificationofsaltsensitivemutants 46
3.2.4 Isolationandidentificationofbiofilmdeficientmutants 50
3.3 Production,overproduction,andexcretionofpolyhydroxyalkanoates
byAlcanivoraxSK2anditsminiTn5mutant 57
3.3.1 Productionofpolyhydroxyalkanoateby Alcanivorax strain
SK2 57
3.3.2 Isolationofamutantstrainshowinghyperproductionand
excretionofPHA 59
3.3.3 PhysiologicalcharacteristicsoftheC9mutant 61
3.3.4 GeneticbasisoftheC9mutation:knockoutofatesBlike
gene 62
3.3.5 CharacterizationofA.borkumensistesBlikegeneexpressed
inE.coli 63

4. DISCUSSION 65
4.1 Alcanivoraxinthepresenceofoilspill:terminaloxidationofalkanes,
intracellularcarbonfluxes,changesinthemembranecompositionand
cofactorsynthesis 65
4.2 Alcanivoraxinitsmarineenvironment:adaptationtoUV,salt,low
temperature,andbiofilmformation 74
4.3 ArationalforhyperproductionandexcretionofPHAintheC9
mutant 81
4.4 BiotechnologicalpotentialofAlcanivorax:PHAproductionasraw
materialforbioplastics 84
4.5 Outlook 85

865. REFERENCES



















v
Preface

This thesis is presented to obtain the Ph.D. degree from the Technical University of
Braunschweig.TheworkwasperformedattheGesellschaftfürBiotechnologischeForschung
mbH(GBF)andattheTechnicalUniversityofBraunschweig,InstituteofMicrobiologywith
Dr. Peter Golyshin and Pr.Dr K.Timmis as supervisors. I would like to express my great
gratitudetomysupervisorsDr.PeterGolyshinandDr.K.Timmisforthescientificsupervision
duringmyPh.D.
Iwouldliketoacknowledgethefollowingsourcesofresearchfunding:GermanMinistryfor
EducationandResearch(BMBF)andEuropeanCommunityProjectCOMODE.
IwouldliketothankProf.Dr.SigmundLangandProf.Dr.DieterJahnforparticipatingin
myPh.D.commission.
SpecialthankstoDr.DanielaRegenhardtandespeciallytoDr.ManuelFerrerforcollaboration
inproteomicpartoftheresearchandforchemicalanalysisofpolyhydroxyalkanoate.Iwould
also like to express my gratitude to Prof. Dr. Sigmund Lang for a fruitful discussion
concerningtheC9mutant.IamalsoverymuchthankfultoDr.SabinaHeimforthehelpwith
apatentapplication.
IamverygratefultoDr.HeinrichLunsdorfforhishelpwithelectronmicroscopy.Thanksto
Dr. WA. Abraham for MS analysis. I would also like to thank Angelika Arnscheidt and
RamonaJoachimfortechnicalassistance.Thankstoallmylabmembersandallotherpeople
oftheMicrobiologyDepartmentattheGBFfortheirsupportandcooperation.
Finally,mydeepestthankstomysweetheartandmymum,whoconstantlyencouragedand
motivatedmethroughoutthewholeperiodofPhDstudyandfinallymadethisworkpossible.






Braunschweig,January2006




JuliaSabirova

vi
Summary

A comprehensive functional genomic analysis of the alkane metabolism of the marine oil
degradingbacteriumAlcanivorax borkumensisSK2anditsconcomitantmetabolicadaptations
is presented. 99 cytoplasmic and membraneassociated proteins of alkanegrown cells were
foundtobedifferentiallyexpressedascomparedtopyruvategrowncellsascontrols.Among
these,46putativeoperonstructureswereidentified.Whilecytoplasmicproteinsfoundtobe
upregulatedinhexadecanegrowncellsmostlyrepresentenzymesoftheglyoxylatebypass,
ofthegluconeogenesispathway,ofthebiosynthesisandoftheβoxidationoffattyacids,up
regulatedmembraneproteinsweremostlycharacteristicforspecificresponsesrelatedtothe
terminal oxidation and further degradation of alkanes or other functions related to alkane
metabolism.Threedifferentenzymaticapproachestotheterminaloxidationofalkaneswere
identified,i.e.enzymesencodedbythepreviouslydescribedalkB1geneclusteraswellastwo
newgeneclustersapparentlycodingforalternativealkanehydroxylatingsystems,comprising
a p450 cytochromecontaining monooxygenase and another putative monooxygenase,
predictedtobeinvolvedinthemetabolismofcycloalkanes.Thelattertwosystemsofterminal
54 70
oxidation of alkanes are preceeded by different sigma and sigma dependent promoters,
correspondinglyandthereforearelikelytobedifferentlyregulated.

Transposon mutagenesis was used for functional genome analysis of Alcanivorax SK2 to
identify the genetic basis of environmentally relevant phenotypes, such osmotolerance, UV
and low temperature adaptation, and biofilm formation. 48 relevant transposon mutants
deficient in any of these environmentally responsive functions were isolated, and the
correspond