Biosynthesis of aminocoumarin antibiotics in Streptomyces [Elektronische Ressource] : investigations on the regulation of novobiocin production = Biosynthese von Aminocoumarin-Antibiotika in Streptomyces / vorgelegt von Volker Dangel

Ute Weinmann

Sujets

• Gesundheit

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2009
• Nombre de lectures: 133
• Poids de l'ouvrage: 1 Mo

Extrait

Biosynthesis of aminocoumarin antibiotics in
Streptomyces: Investigations on the regulation
of novobiocin production


Biosynthese von Aminocoumarin-Antibiotika in
Streptomyces: Untersuchungen zur Regulation
der Novobiocinproduktion



DISSERTATION


der Fakultät für Chemie und Pharmazie
der Eberhard Karls Universität Tübingen

zur Erlangung des Grades eines Doktors
der Naturwissenschaften





2009





vorgelegt von

Volker Dangel
































Tag der mündlichen Prüfung: 08.06.2009

Dekan: Prof. Dr. L. Wesemann
1. Berichterstatter: Prof. Heide
2. PD Dr. C. Wolz CONTENTS I
CONTENTS

PUBLICATIONS AND PRESENTATIONS………………………………………………IV
ABBREVIATIONS………………………………………………………………………….VI
SUMMARY……………………………………..…………………………………………….1
ZUSAMMENFASSUNG…………………………………..………………………………...4
I. INTRODUCTION............................................................................................................................... 7
I.1. STREPTOMYCES – THE LARGEST ANTIBIOTIC-PRODUCING GENUS............................................................ 7
I.2. REGULATION AND OVERPRODUCTION OF ANTIBIOTICS PRODUCTION IN STREPTOMYCES......................... 8
I.3. AMINOCOUMARIN ANTIBIOTICS ........................................................................................................... 10
I.3.1. Chemical structure ........................................................................................................................ 11
I.3.2. Mechanism of action and clinical application .............................................................................. 12
I.3.3. Structure-activity relationships ..................................................................................................... 14
I.3.4. Biosynthetic gene clusters ............................................................................................................. 15
I.3.5. Resistance genes............................................................................................................................ 17
I.3.6. Regulation of aminocoumarin-antibiotic production .................................................................... 18
I.4. AIMS OF THIS STUDY............................................................................................................................ 23
II. MATERIALS AND METHODS...................................................................................................... 25
II.1. CHEMICALS.......................................................................................................................................... 25
II.2. MATERIALS FOR CHROMATOGRAPHY .................................................................................................. 26
II.3. ENZYMES AND KITS.............................................................................................................................. 26
II.4. MEDIA, BUFFERS AND SOLUTIONS........................................................................................................ 27
II.4.1. Media for bacterial cultivation...................................................................................................... 27
II.4.1.1. Cultivation of E. coli ............................................................................................................................. 27
II.4.1.2. tion of Streptomyces .................................................................................................................. 28
II.4.1.3. Novobiocin production medium............................................................................................................ 29
II.4.1.4. Protoplast transformation of Streptomyces............................................................................................ 30
II.4.2. Antibiotic solutions........................................................................................................................ 31
II.4.3. Buffers and solutions..................................................................................................................... 31
II.4.3.1. Buffers and Solutions for DNA isolation .............................................................................................. 31
II.4.3.2. Buffers for DNA gel electrophoresis......... 33
II.4.3.3. Buffers and solutions for Southern blot analysis................................................................................... 33
II.4.3.4. Solutions for blue/white selection of E. coli.......................................................................................... 34
II.4.3.5. Buffers for preparation of protoplasts and transformation of Streptomyces .......................................... 34
II.4.3.6. Solution for isolation of RNA from Streptomyces................................................................................. 35
II.5. PLASMIDS, BACTERIAL STRAINS, PRIMERS AND PROBES....................................................................... 35
II.5.1. Vectors, cosmids and plasmids...................................................................................................... 35
II.5.2. PCR primers used for construction of plasmids............................................................................ 37
II.5.3. Primers used for RT-PCR experiments ......................................................................................... 38
II.5.4. Primers used for qRT-PCR exnts ....................................................................................... 38
II.5.5. Bacterial strains............................................................................................................................ 39
II.5.6. Probe used in Southern blot analysis ............................................................................................40
II.6. CULTURE CONDITIONS ......................................................................................................................... 40
II.6.1. Cultivation of E. coli ..................................................................................................................... 40
II.6.2. Cultivation of Streptomyces coelicolor 41
II.6.2.1. General cultivation................................................................................................................................ 41
II.6.2.2. Production of secondary metabolites..................................................................................................... 41
II.6.2.3. Preparation of homogenized and frozen inoculum 42
II.6.2.4. Preparation of mycelia for storage and spore suspensions of Streptomyces .......................................... 42
II.7. METHODS OF MOLECULAR BIOLOGY .................................................................................................... 43
II.7.1. Purification, concentration and quantification of DNA ................................................................ 43
II.7.2. Agarose gel electophoresis of DNA...............................................................................................43 CONTENTS II
II.7.3. DNA manipulation with enzymes .................................................................................................. 44
II.7.4. DNA isolation................................................................................................................................ 44
II.7.4.1. Isolation of plasmids from E. coli ......................................................................................................... 44
II.7.4.2. Streptomyces............................................................................................... 44
II.7.4.3. Isolation of genomic DNA from Streptomyces coelicolor .................................................................... 45
II.7.5. DNA denaturation by alkaline treatment for ssDNA transformation in Streptomyces.................. 46
II.7.6. PCR amplification......................................................................................................................... 46
II.7.6.1. General conditions ................................................................................................................................ 46
II.7.6.2. Conditions for amplification of the apramycin resistance cassette from pUG019 and the apra-tcp830
cassette from pMS80............................................................................................................................. 47
II.7.7. Southern blot analysis.......... 48
II.7.7.1. Probe preparation.................... 49
II.7.7.2. Southern blot preparation...................................................................................................................... 49
II.7.7.3. Prehybridization and hybridization ....................................................................................................... 49
II.7.7.4. Detection............................................................................................