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Publié par | philipps-universitat_marburg |
Publié le | 01 janvier 2004 |
Nombre de lectures | 15 |
Langue | English |
Poids de l'ouvrage | 3 Mo |
Extrait
Aus dem
Institut für Physiologische Chemie
der
Philipps-Universität Marburg
Geschäftsführender Direktor: Prof. Dr. Andrej Hasilik
Arbeitsgruppe Molekulare Enzymologie
Leiter: Prof. Dr. Klaus-Heinrich Röhm
Regulation of Pseudomonas putida genes involved in the
metabolism of acidic amino acids
INAUGURAL-DISSERTATION
Zur Erlangung des Doktorgrades der Humanbiologie
(Dr. rer. physiol.)
dem Fachbereich Humanmedizin
der Philipps-Universität Marburg
vorgelegt
von
Avinash Sonawane
aus
Yesgaon
Marburg 2003
Dedicated to my parents
as a token of gratitude
I
Angenommen vom Fachbereich Humanmedizin der Philipps- Universität Marburg
am
Dekan: Prof. Dr. B. Maisch
Referent: Prof. Dr. K.- H. Röhm
Correferent: Prof. Dr. G. Suske
II
INDEX
1. Introduction ................................................................................................1
1.1 Amino acid metabolism: general overview ................................................................. 1
1.2 Pathways for ammonia assimilation in enteric bacteria ............................................... 2
1.2.1 Glutamate dehydrogenase pathway .............................................................. 2
1.2.2 GS/GOGAT pathway..................................................................................... 2
1.3 Enzymes of amino acids utilization.............................................................................. 3
1.3.1 Asparagine synthetase ................................................................................... 4
1.3.2 Glutaminase/asparaginase 4
1.3.3 Aspartase........................................................................................................ 5
1.3.4 Aspartate transaminase .................................................................................. 5
1.4 Nitrogen control by the Ntr system 5
1.4.1 Components ................................................................................................... 5
1.4.2 Uridylyltransferase-uridylyl removing enzyme (UT/UR)............................ 7
1.4.3 Control of the NtrB/NtrC system by P ........................................................ 7 II
1.4.4 Genes regulated by the Ntr system ................................................................ 8
1.5 Two Component Systems............................................................................................. 8
1.6 Sigma factors in bacterial gene expression 10
541.6.1 σ -dependent promoters ............................................................................. 11
541.6.2 σ -dependent genes of nitrogen metabolism.............................................. 11
1.7 Transport of Nitrogenous Compounds ....................................................................... 12
1.7.1 Ammonium transport................................................................................... 12
1.7.2 Nitrate transport........................................................................................... 12
1.7.3 Amino acid transport 12
1.8 Plant growth-promoting rhizobacteria........................................................................ 13
1.9 Root colonization by bacteria..................................................................................... 14
1.10 The role of root exudates in plant-bacterial interactions.......................................... 15
1.11 Components of root exudates 16
1.12 Choice of organism................................................................................................... 17
1.13 Aims and objectives of this study............................................................................. 18
2. Materials.........................................................................................................19
2.1 Microorganisms.......................................................................................................... 19
III
2.2 Antibiotics .................................................................................................................. 20
2.3 Plasmids ..................................................................................................................... 20
2.4 Oligonucleotides......................................................................................................... 20
2.4.1 Oligonucleotide primers for gene expression.............................................. 20
2.4.2 Oligonucleotide primers for transposon mutant sequencing ....................... 21
2.4.3 Oligonucleotide primers for gene replacement............................................ 21
2.4.4 Oligonucleotide primers for protein overexpression. .................................. 22
2.5 DNA and RNA Markers ............................................................................................ 22
2.6 Kits ............................................................................................................................ 22
2.7 Enzymes and Chemicals 23
2.7.1 Enzymes....................................................................................................... 23
2.7.2 Chemicals .................................................................................................... 24
2.8 Instruments ................................................................................................................. 25
2.8.1 Bacterial growth........................................................................................... 25
2.8.2 Centrifuges................................................................................................... 25
2.8.3 Photometers ................................................................................................. 25
2.8.4 Electrophoresis ............................................................................................ 26
2.9 Membranes and special materials .............................................................................. 26
2.10 HPLC Analysis......................................................................................................... 26
2.11 Other apparatus 27
2.12 Computer programs and Internet-Links ................................................................... 27
3. Methods ..........................................................................................................................29
3.1 Safety.......................................................................................................................... 29
3.2 Bacterial growth ......................................................................................................... 29
3.2.1 Storage and revival of bacterial cultures ..................................................... 29
3.2.2 Cultivation ................................................................................................... 29
3.3 Preparation and transformation of competent cells .................................................... 31
3.3.1 Preparation of competent E. coli cells ......................................................... 31
3.3.2 Transformation of competent host cells ..................................................... 31
3.3.3 Preparation of electro-competent Pseudomonas cells ................................. 31
3.3.4 Electroporation of competent cells ...................................... 32
3.4 Motility and chemotaxis assays.................................................................................. 33
IV
3.5 Survival in nitrogen limiting conditions..................................................................... 33
3.6 Isolation of bacterial DNA ......................................................................................... 33
3.6.1 Isolation of genomic DNA by the DNA Mini Kit ....................................... 33
3.6.2 Phenol-chloroform extraction 34
3.6.3 DNA precipitation ....................................................