The branched Entner-Doudoroff pathway in hyperthermophilic archaea [Elektronische Ressource] / vorgelegt von Hatim Ahmed

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• Biologie

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• Publié par: universitat_duisburg-essen
• Publié le: 01 janvier 2006
• Nombre de lectures: 61
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

THE BRANCHED ENTNER-DOUDOROFF PATHWAY IN
HYPERTHERMOPHILIC ARCHAEA


Inaugural-Dissertation


zur
Erlangung des Doktorgrades
Dr. rer. nat.
des Fachbereichs
Biologie und Geographie
an der
Universität Duisburg-Essen


vorgelegt von
HATIM AHMED
aus Hannover


Juni 2006














Die dieser Arbeit zugrundeliegenden Experimente wurden am Institut für Biologie
in der Abteilung Mikrobiologie I der Universität Duisburg-Essen, Campus Essen,
durchgeführt.

1. Gutachter: HD. Dr. Bettina Siebers

2. Gutachter: Prof. Dr. Peter Bayer

Vorsitzender des Prüfungsausschusses: Prof. Dr. Reinhard Hensel

Tag der mündlichen Prüfung: 25.10.2006































He knows what lies before them and what is
after them, and they comprehend not anything

of His knowledge save such as He wills.

Surat al-Baqara: 255




TABLE OF CONTENTS I
TABLE OF CONTENTS

1. INTRODUCTION........................................................................................................1
2. MATERIALS AND METHODS ................................................................................10
2.1 CHEMICALS AND PLASMIDS..........................................................................................10
2.2 INSTRUMENTS ..............................................................................................................10
2.3 STRAINS AND GROWTH CONDITIONS............................................................................12
2.4 MOLECULAR BIOLOGICAL METHODS WITH DNA .........................................................16
2.4.1 Genomic DNA preparation............................................................................... 16
2.4.2 Isolation of plasmid DNA from E. coli............................................................. 17
2.4.3 DNA precipitation.............................................................................................18
2.4.4 Quantification of DNA......................................................................................
2.4.5 Agarose gel electrophoresis for DNA............................................................... 18
2.4.6 Purification of DNA fragments ........................................................................ 19
2.4.7 Polymerase chain reaction (PCR) .................................................................... 19
2.4.8 Amplification of genomic DNA and plasmid DNA by PCR........................... 20
2.4.9 Enzymatic manipulation of DNA..................................................................... 20
2.4.9.1 Restriction of DNA.......................................................................................... 20
2.4.9.2 5`-Dephosphorylation of the linearized vector DNA................................... 20
2.4.9.3 Ligation of vector DNA and insert................................................................. 21
2.4.10 Transformation..................................................................................................21
2.4.10.1 Preparation of competent E. coli cells.......................................................... 21
2.4.10.2 Transformation of the competent E. coli cells............................................. 22
2.4.11 Sequencing.........................................................................................................22
2.4.11.1 Automated DNA sequencing........................................................................22
2.4.11.2 Computer assisted analysis of the nucleotide sequence .............................. 22
2.5 MOLECULAR BIOLOGICAL METHODS WITH RNA .........................................................23
2.5.1 Handling of solutions, glassware and equipments .......................................... 23
2.5.2 Isolation of total RNA from T. tenax and S. solfataricus................................. 23
2.5.3 Quantification of RNA......................................................................................24
2.5.4 Agarose/Formaldehyde gel electrophoresis of RNA .......................................
2.5.5 Capillary transfer of RNA to a nylon membrane (Northern Blot)................. 25
2.5.6 Hybridization of RNA with Digoxigenin-labelled RNA probes .................... 26
2.5.6.1 Synthesis of DIG-Labelled specific antisense mRNA probes by in vitro
transcription .................................................................................................... 26
2.5.6.2 Hybridization of immobilized RNA with DIG-labelled specific antisense
mRNA probes .................................................................................................. 27
2.5.6.3 Detection of RNA-RNA hybrid by immunological detection...................... 27
2.5.7 Primer extension analysis ................................................................................. 28
2.6 BIOCHEMICAL METHODS .............................................................................................29
2.6.1 Heterologous expression of the T. tenax and the S. solfataricus ED proteins in
E. coli ................................................................................................................. 29 TABLE OF CONTENTS II
2.6.2 Preparative protein purification.......................................................................29
2.6.3 Enzyme assays...................................................................................................30
2.6.3.1 2-keto-3-deoxy-6-(phospho)gluconate (KD(P)G) aldolase...........................
2.6.3.2 Gluconate dehydratase (GAD) 31
2.6.3.3 2-keto-3-deoxygluconate (KDG) kinase ........................................................ 31
2.6.3.4 Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase
GAPN...............................................................................................................32
2.6.3.5 Kinetic parameters..........................................................................................33
2.6.4 Biocatalytic synthesis of KDG .......................................................................... 34
2.6.5 In vitro assays with crude extracts................................................................... 34
142.6.6 C-Labelling experiments and Thin Layer Chromatography (TLC) ............. 34
2.6.7 In vitro reconstruction of the ED pathway ..................................................... 35
2.6.8 Analytical protein methods .............................................................................. 35
2.6.8.1 Protein quantification.....................................................................................35
2.6.8.2 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) ................................. 35
2.6.8.3 Molecular mass determination.......................................................................37
3. RESULTS....................................................................................................................38
3.1 SEQUENCE ANALYSIS AND GENE CONTEXT ...................................................................39
3.2 PRIMER EXTENSION ANALYSIS43
3.2.1 Northern Blot analysis ...................................................................................... 43
3.3 HETEROLOGOUS EXPRESSION OF THE T. TENAX AND S. SOLFATARICUS ED
PROTEINS IN E. COLI ....................................................................................................46
3.4 ENZYMES ENRICHMENT AND PURIFICATION.................................................................47
3.5 BIOCHEMICAL CHARACTERIZATION .............................................................................49
3.5.1 Catalytic and kinetic parameters...................................................................... 49
3.5.1.1 Glucose dehydrogenase...................................................................................49
3.5.1.2 KD(P)G aldolase..............................................................................................49
3.5.1.3 Gluconate dehydratase....................................................................................53
3.5.1.4 KDG kinase......................................................................................................54
3.5.1.5 Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase
(GAPN) from S. solfataricus ........................................................................... 57
3.6 CRUDE EXTRACTS STUDIES...........................................................................................61
3.7 IN VITRO RECONSTRUCTION OF THE ED PATHWAY IN T. TENAX...................................62
4. DISCUSSION .............................................................................................................64
4.1 THE ED GENE CLUSTER-COMPARATIVE GENOMICS .....................................................64
4.2 ED GENE ORGANIZATION AND TRANSCRIPT ANALYSIS OF T. TENAX AND
S.SOLFATARICUS .......................................................................