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Dissertationstefanschauer.Pdf (11.35Mb) Institut für Biologie Biofilmbildung bei Pflanzen-assoziierten Bakterien der Gattung Methylobacterium und molekulargenetisch-physiologische Charakterisierung eines neuen Marchantia-Isolats (Mtb. sp. JT1) Inaugural‐Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt im Fachbereich Naturwissenschaften der Universität Kassel von Dipl.‐Biol. Stefan Schauer Dekan: Prof. Dr. F. Herberg Gutachter: 1. Prof. Dr. U. Kutschera 2. Prof. Dr. H. Follmann eingereicht: 12. Januar 2009 Datum der Disputation: 6. Februar 2009 Inhaltsverzeichnis Inhaltsverzeichnis Inhaltsverzeichnis............................................................................................................... 2 Abkürzungsverzeichnis..................................................................................................... 7 1 Einleitung................................................................................................................... 10 1.1 Biofilme .............................................................................................................. 10 1.1.1 Vorteile von Biofilmen ............................................................................. 11 1.1.2 Matrixkomponenten bakterieller Biofilme............................................ 11 1.1.3 Biofilme auf Pflanzenoberflächen .......................................................... 12 1.2 Die Gattung Methylobacterium......................................................................... 13 1.2.1 Assoziation mit Pflanzen......................................................................... 13 1.2.2 Taxonomie ................................................................................................. 15 1.2.3 Identifikation............................................................................................. 17 1.3 Bakterielle Speziesdefinitionen....................................................................... 17 1.4 Fragestellung und Ziele der Arbeit................................................................ 19 2 Material und Methoden........................................................................................... 21 2.1 Verbrauchsmaterial .......................................................................................... 21 2.1.1 Chemikalien............................................................................................... 21 2.1.2 Enzyme....................................................................................................... 22 2.1.3 Oligonucleotide......................................................................................... 22 2.1.4 Größenmarker für DNA .......................................................................... 22 2.1.5 Fertigchemikalien (Kits)........................................................................... 23 2.1.6 Sonstige Verbrauchsmaterialien............................................................. 23 2.2 Software ............................................................................................................. 23 2.3 Geräte ................................................................................................................. 24 2.4 Mikroorganismen ............................................................................................. 25 2.5 Statistische Auswertung .................................................................................. 27 2.5.1 Mittelwert .................................................................................................. 27 2.5.2 Standardabweichung ............................................................................... 27 2.5.3 Standardfehler........................................................................................... 27 2.6 Sterilisation von Lösungen und Materialien ................................................ 27 2.6.1 Sterilfiltration ............................................................................................ 27 2.6.2 Dampfsterilisation .................................................................................... 27 2.6.3 Heißluftsterilisation.................................................................................. 27 2.7 Nährmedien....................................................................................................... 28 2.7.1 Minimalmedien......................................................................................... 28 2.7.1.1 ATCC Kulturmedium 784 (AMS‐Medium)...................................... 28 2.7.1.2 Methylobacterium Medium (DSMZ Medium 125)............................. 29 2.7.1.3 Medium nach Choi et al. (1989) .......................................................... 29 2.7.1.4 Medium 3 / Medium 4 (Bourque et al. 1995)..................................... 30 2.7.2 Komplexmedien........................................................................................ 31 2.7.2.1 Glycerol‐Pepton‐Medium (GP‐Medium).......................................... 31 2.7.2.2 R2A‐Medium (DSMZ Medium 830) .................................................. 31 2.7.2.3 Nutrient‐Agar (DSMZ Medium 1)..................................................... 32 2 Inhaltsverzeichnis 2.8 Mikroskopische Methoden.............................................................................. 32 2.8.1 Phasenkontrast‐ und Fluoreszenzmikroskopie.................................... 32 2.8.1.1 Lektinfärbung........................................................................................ 32 2.8.1.2 Calcofluor White‐Färbung .................................................................. 33 2.8.2 Rasterelektronenmikroskopie................................................................. 34 2.9 Mikrobiologische Methoden ........................................................................... 35 2.9.1 Photometrische Bestimmung der Trübung .......................................... 35 2.9.2 Herstellung einer Zellsuspension........................................................... 35 2.9.3 Herstellung von Dauerkulturen (Kryokonservierung)....................... 36 2.9.4 Zellzahlbestimmung mit einer Zählkammer nach Thoma................. 36 2.9.5 Kultivierung der Bakterien in Flüssigkultur ........................................ 36 2.9.6 Kultivierung der Bakterien auf Festmedium........................................ 37 2.9.7 Bestimmung der Koloniemorphologie auf Festmedium .................... 37 2.9.8 Bestimmung der Zelldimensionen......................................................... 37 2.9.9 Gram‐Färbung........................................................................................... 38 2.9.10 Sudan‐Schwarz Färbung (PHB‐Nachweis)........................................... 38 2.9.11 Kohlenstoffverwertung............................................................................ 39 2.9.12 Test auf assimilatorische Nitratreduktion ............................................ 39 2.9.13 Bestimmung von Temperatur‐Optima.................................................. 40 2.9.14 Bestimmung der Salztoleranz................................................................. 40 2.9.15 Oxidase‐Test.............................................................................................. 40 2.9.16 Katalase‐Test ............................................................................................. 41 2.9.17 Differenzierung der Bakterienstämme mittels Teststreifen ............... 41 2.9.17.1 Streifen‐Test (API 20 NE) ................................................................ 41 2.9.17.2 Stoffwechsel‐Test (API 50 CH) ....................................................... 42 2.9.18 CFW‐Platten .............................................................................................. 43 2.9.19 Flagellenfärbung nach Heimbrook et al. (1989).................................... 43 2.9.20 Carotinoidextraktion und in vivo Spektrum ......................................... 44 2.9.21 Motilitätstest.............................................................................................. 44 2.10 Molekularbiologische Methoden.................................................................... 45 2.10.1 DNA‐Isolation........................................................................................... 45 2.10.1.1 Quick & Dirty‐Methode................................................................... 46 2.10.1.2 DNA‐Isolation mit dem DNeasy Blood & Tissue Kit.................. 46 2.10.1.3 Phenol‐Chloroform‐Extraktion....................................................... 47 2.10.1.4 DNA‐Isolation nach Pitcher et al. (1989)........................................ 48 2.10.1.5 CTAB‐Methode ................................................................................. 48 2.10.1.6 DNA‐Isolation mit Anionenaustauschersäulen........................... 49 2.10.2 Spektrophotometrische Analyse genomischer DNA .......................... 50 2.10.3 Gelelektrophoretische Auftrennung von DNA.................................... 51 2.10.4 Vergleichende DNA‐Sequenzanalyse ................................................... 52 2.10.4.1 In vitro‐Amplifikation von DNA mittels PCR .............................. 53 2.10.4.1.1 Präamplifikation des 16S rRNA‐Gens....................................... 54 2.10.4.1.2 Präamplifikation des partiellen mxaF‐Gens.............................. 54 2.10.4.2 Aufreinigung von PCR‐ und Sequenzier‐Produkten.................. 55 3 Inhaltsverzeichnis 2.10.4.3 Sequenzierung der PCR‐Fragmente .............................................
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