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Improved Classification and Identification of Acetic Acid Bacteria Based on Molecular Techniques

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Improved Classification and Identification of Acetic Acid Bacteria Based on Molecular Techniques FACULTY OF SCIENCES Faculty of Sciences Department of Biochemistry, Physiology and Microbiology Laboratory of Microbiology IMPROVED CLASSIFICATION AND IDENTIFICATION OF ACETIC ACID BACTERIA BASED ON MOLECULAR TECHNIQUES Ilse Cleenwerck Thesis submitted in fulfilment of the requirements for the degree of Doctor in Sciences, Biotechnology Academic year 2007-2008 Promotor: Prof. Dr. Paul De Vos EXAMINATION COMMITTEE Prof. Dr. Savvas Savvides (Chairman) Laboratory for Protein Biochemistry and Biomolecular Engineering Faculty of Sciences, Ghent University, Ghent, Belgium Prof. Dr. Paul De Vos (Promotor) Laboratory of Microbiology Faculty of Sciences, Ghent University, Ghent, Belgium Prof. Dr. ir. Luc De Vuyst Research Group of Industrial Microbiology and Food Biotechnology Faculty of Sciences and Bioscience Engineering, Vrije Universiteit Brussel, Brussels, Belgium Prof. Dr. Anne Willems Laboratory of Microbiology Faculty of Sciences, Ghent University, Ghent, Belgium Prof. Dr. Tom Coenye Laboratory of Pharmaceutical Microbiology Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium Prof. Dr. Yves Van de Peer Department of Molecular Genetics Faculty of Sciences, Ghent University, Ghent, Belgium Prof. Dr. ir. Jean Swings Laboratory of Microbiology Faculty of Sciences, Ghent University, Ghent, Belgium Dr. Danielle Janssens BCCMTM/LMG Bacteria Collection Faculty of Sciences, Ghent University, Ghent, Belgium Dr. Janja Trček Max von Pettenkofer-Institut Ludwig-Maximillians Universität, Munich, Germany DANKWOORD Het is dit jaar het tiende jaar dat ik werk voor de BCCMTM/LMG Bacterie Collectie en het belooft een bijzonder jaar te worden, onder andere daar ik dit doctoraatswerk zal verdedigen. Ik wil dan ook in eerste instantie Paul en Danny van harte bedanken, omdat ze me de kans hebben geboden te doctoreren aan de hand van onderzoeksresultaten die ik behaalde tijdens mijn werk voor de collectie. Ik wil Paul tevens bedanken dat hij erin slaagde om binnen zijn drukke agenda hier en daar wat tijd vrij te maken om mij met raad en advies bij te staan. Verder wens ik Jean te bedanken voor zijn enthousiasme tijdens zijn promotorschap van de BCCMTM/LMG Bacterie Collectie. Hij heeft mij op weg geholpen om te zoeken naar technieken die zowel voor de collectie als voor het Laboratorium voor Microbiologie van toepassing kunnen zijn. Onder zijn impuls werd de 'LAB' AFLP® databank gestart, die al zijn nut heeft bewezen. Tevens wil ik hem bedanken omdat hij mij warm gemaakt heeft mijn doctoraat toe te spitsen op azijnzuurbacteriën. Prof. Dr. ir. Luc De Vuyst wil ik van harte bedanken omdat hij mij de mogelijkheid heeft geboden mijn onderzoek op deze organismen weer extra leven in te blazen en mij sterk motiveerde tot het schrijven van publicaties hieromtrent. Angel en Nicholas wens ik te bedanken voor hun bijdrage in het onderzoek, en Peter voor zijn gerichte steun. Een special woordje van dank gaat naar Marjan en Katrien Engelbeen. Het zal hen zeker opgevallen zijn dat het schrijven van deze thesis, dat diende te gebeuren tussen al mijn taken voor de collectie in, zowat al mijn energie van het afgelopen jaar opslorpte. Ik wil hen bedanken voor hun steun en hun inzet in het kader van dit onderzoek, maar ook in 't algemeen. Ik vind het aangenaam om met jullie samen te werken en vind dat we samen al veel gerealiseerd hebben. Katrien Vandemeulebroecke, Leentje, Karen, Cindy en de ploeg van het fenolab wil ik bedanken voor hun technische ondersteuning. Geert wil ik bedanken voor de vele keren dat hij me uit de nood hielp als ik thuis weer eens tot de vaststelling kwam dat ik nog een file van het werk nodig had. Marc, Ariane, Nadine en de rest van het personeel van de collectie wens ik eveneens te bedanken. Zonder de inzet van jullie allemaal zouden we niet ver geraken. Claudine en Stefanie, wil ik in het bijzonder bedanken, voor hun luisterbereidheid en hun steun. Ook heel wat personen van het Labo voor Microbiologie hebben rechtstreeks en onrechtstreeks bijgedragen aan de verwezenlijking van dit onderzoeksproject, en dit dankwoord is dan ook aan hen gericht. Bart Hoste wil ik hierbij in het bijzonder bedanken. Bedankt voor je collegialiteit en je vele nuttige tips. Je inzicht in diverse technieken hebben mij meerdere keren goed geholpen. Katrien De Bruyne wil ik eveneens bedanken, voor haar diverse initiatieven op informaticavlak en voor haar bereidheid ons te adviseren bij de verwerking van MLSA data. Ilse Vandecandelaere, ook bedankt voor je administratieve tips i.v.m. doctoreren. Paul Segers, de laatste tijd heb ik je iets minder gezien en ik hoop binnenkort tijdens de lunch weer eens meer een gesprek met je te kunnen aanknopen, want ik geniet ervan. Liesbeth en Evie, bedankt voor de %GC bepalingen. I would like to thank all the members of the reading and exam committee for the evaluation of this manuscript and their willingness to participate in the defence of this thesis. Speciale dank gaat tot slot naar mijn echtgenoot Geert voor de vele uren, weken en maanden die hij me toch wat heeft moeten missen, en mijn kinderen Jeroen en Lore die blij zullen zijn dat mama weer wat meer tijd zal hebben voor leuke uitstapjes en verre en minder verre reizen. Ik bedank ook de locale horeca die af en toe zorgden voor een aangenaam rustmoment. Ook mijn ouders en de rest van de familie bedank ik voor hun steun en hun vriendschap doorheen dit doctoraat. In de toekomst zal ik ook terug wat meer tijd kunnen vrijmaken voor de vrienden die ik de afgelopen tijd wat heb verwaarloosd. CONTENTS LIST OF ABBREVIATIONS PART I: BACKGROUND AND OBJECTIVES, LITERATURE STUDY, SHORT OVERVIEW OF THE EXPERIMENTAL WORK 1 Background and objectives 3 Chapter 1. Literature study 5 1.1. General characteristics of AAB 5 1.2. Habitats, metabolism, applications and concerns of AAB 6 1.2.1. Habitats 6 1.2.2. Metabolism 7 1.2.3. Applications 10 1.2.3.1. Vinegar 10 1.2.3.2. Other fermented beverages and food products 13 1.2.3.3. Organic chemicals 14 1.2.3.4. Bacterial cellulose 16 1.2.3.5. Plant growth promotion 18 1.2.4. Concerns 20 1.2.4.1. Spoilage of beverages 20 1.2.4.2. Bacterial rot in apples and pears and pink disease of pineapples 21 1.2.4.3. AAB as (opportunistic) human pathogens 22 1.3. The basic concepts of bacterial taxonomy 23 1.4. Taxonomy of AAB 25 1.5. Phylogeny of AAB based on 16S rRNA gene sequences 28 1.6. Identification of AAB 30 Short overview of the experimental work 35 PART II: EXPERIMENTAL WORK 39 Chapter 2. Re-examination of the genus Acetobacter, with descriptions of Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov. 41 Chapter 3. Identification and classification of AAB from different sources (rice, apple juice, wine and mango fruit) using a polyphasic approach 53 3.1. Natural association of Gluconacetobacter diazotrophicus and diazotrophic Acetobacter peroxydans with wetland rice 53 3.2. Description of Gluconacetobacter swingsii sp. nov. and Gluconacetobacter rhaeticus sp. nov., isolated from Italian apple fruit 67 3.3. Acetobacter oeni sp. nov., isolated from spoiled red wine 77 3.4. Acetobacter senegalensis sp. nov., a thermotolerant acetic acid bacterium isolated in Senegal (sub-Saharan Africa) from mango fruit (Mangifera indica L.) 83 Chapter 4. Evaluation of the DNA fingerprinting techniques rep-PCR and AFLP® for rapid and accurate identification and classification of AAB 91 4.1. Validation of the (GTG)5-rep-PCR fingerprinting technique for rapid classification and identification of AAB, with a focus on isolates from Ghanaian fermented cocoa beans 91 4.2. Differentiation of species of the family Acetobacteraceae by AFLP® DNA fingerprinting and reclassification of Gluconacetobacter kombuchae as Gluconacetobacter hansenii 107 Chapter 5. Descriptions of two novel Acetobacter species, both isolated from traditional heap fermentations of Ghanaian cocoa beans 131 5.1. Acetobacter ghanensis sp. nov., a novel acetic acid bacterium isolated from traditional heap fermentations of Ghanaian cocoa beans 131 5.2. Acetobacter fabarum sp. nov., a Ghanaian cocoa bean heap fermentation acetic acid bacterium 143 PART III: GENERAL DISCUSSION, PERSPECTIVES AND CONCLUSIONS 153 Chapter 6. General discussion, perspectives and conclusions 155 SUMMARY 165 SAMENVATTING 169 REFERENCE LIST 173 CURRICULUM VITAE 203 LIST OF ABBREVIATIONS 2KGADH 2-keto-D-gluconate dehydrogenase 2KGR 2-keto-D-gluconate reductase 5KGR 5-keto-D-gluconate reductase A. Acetobacter AAB acetic acid bacteria ABC transporter ATP-binding cassette transporter Ac. Acidomonas ADH membrane-bound alcohol dehydrogenase AFLP® amplified fragment length polymorphism ALDH membrane-bound acetaldehyde dehydrogenase As. Asaia ATCC American Type Culture Collection, USA BCCMTM/LMG Bacteria Collection of the Belgian Coordinated Collections of Microorganisms, Ghent University, Belgium BLAST basic local alignment search tool BMR Bio Molecular Research Center, University of Padua, Italy BNF biological nitrogen fixation CFU colony forming units CGD chronic granulomatous disease CTAB hexadecyltrimethylammonium bromide DNA deoxyribonucleic acid DSMZ German Collection of Microorganisms and Cell cultures, Germany EDTA ethylene diamine tetraacetic acid EMBL European Molecular Biology Laboratory, Cambridge, UK ERIC enterobacterial repetitive intergenic consensus FDA Food and Drug Administration, USA G. Gluconacetobacter GADH D-glucononate dehydrogenase GDH glucose dehydrogenase Gl. Gluconobacter Gr. Granulibacter HPAEC high pressure anion exchange chromatography HPLC high performance liquid chromatography HGT
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