Functional and biochemical analysis of acidic amino acid transport and utilization by Pseudomonas putida KT2440 [Elektronische Ressource] / vorgelegt von Birendra Singh

Birendra

Sujets

• Biologie

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2007
• Nombre de lectures: 21
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Aus dem
Institut für Physiologische Chemie
der
Philipps-Universität Marburg
Geschäftsführender Direktor: Prof. Dr. Jan Koolman

Arbeitsgruppe Molekulare Enzymologie
Leiter: Prof. Dr. Klaus-Heinrich Röhm


Functional and biochemical analysis of acidic amino acid
transport and utilization by Pseudomonas putida KT2440






INAUGURAL-DISSERTATION

Zur Erlangung des Doktorgrades der Humanbiologie

(Dr. rer. physiol.)

dem Fachbereich Humanmedizin
der Philipps-Universität Marburg
vorgelegt


von
Birendra Singh
aus
Chitrakoot, Indien
Marburg, 2007





































Angenommen vom Fachbereich Humanmedizin der Philipps- Universität Marburg am


Dekan: Prof. Dr. Matthias Rothmund

Referent: Prof. Dr. Klaus-Heinrich Röhm
Correferent: Prof. Dr. Alexander Brehm
Prof. Dr. Wolfgang Garten

























This thesis is dedicated to my Parents
as a token of gratitude





















INDEX

1. Introduction.........................................................................................................................................1

1.1 Background of the project.......................................................................................................1
1.2 Choice of organism.................................................................................................................2
1.3 Bacterial amino acid metabolism; an overview......................................................................2
1.4 Ammonia assimilation by enteric bacteria..............................................................................3
1.4.1 GDH pathway............................................................................................................4
1.4.2 GS/GOGAT pathway................................................................................................4
1.5 Enzymes of amino acid utilization..........................................................................................5
1.5.1 Asparagine synthetase...............................................................................................5
1.5.2 Glutaminase/Asparaginase........................................................................................6
1.5.3 Aspartase...................................................................................................................6
1.5.4 Aspartase transaminase.............................................................................................6
1.6 Molecular control of nitrogen metabolism in bacteria............................................................7
1.6.1 Global nitrogen regulatory system (Ntr)...................................................................7
1.7 Two-component systems.........................................................................................................9
1.7.1 Introduction...............................................................................................................9
1.7.2 Mechanism of action..............................................................................................10
1.7.3 Domain structure of two-component systems.........................................................11
1.7.3.1 Histidine kinases........................................................................................11
1.7.3.2 Response regulators...................................................................................14
1.8 Sigma factors in bacterial gene expression...........................................................................16
54
1.8.1 ơ factors and nitrogen metabolism........................................................................17
54
1.8.2 Mechanism of ơ transcriptional regulation...........................................................17
1.9 Transport of nitrogenous compounds in bacteria..................................................................18
1.9.1 Ammonium transport..............................................................................................18
1.9.2 Nitrate transport.......................................................................................................18
1.9.3 Amino acid transporters..........................................................................................19
1.10 ABC transporters...................................................................................................................19
1.10.1 Structure of ABC transporters.................................................................................20
i
1.10.2 Mechanism of transport...........................................................................................21
1.10.3 Amino acid ABC transporters in pseudomonads....................................................22
1.11 Aims and objectives of this study..........................................................................................23

2 Materials....................................................................................................................24
2.1 Microorganisms and plasmids...............................................................................................24
2.2 Antibiotics.............................................................................................................................24
2.3 Oligonucleotide primer..........................................................................................................25
2.3.1 Primers used for protein over-expression................................................................25
2.3.2 Primers used for Knockout of P. putida KT2440 genes.........................................25
2.3.3 Primers used for amplification of promoters...........................................................25
2.3.4 Primers used for site directed mutagenesis in aatJ.................................................26
2.4 DNA and protein ladders.......................................................................................................27
2.5 Enzymes.................................................................................................................................27
2.6 Radioactive labeled chemicals...............................................................................................27
2.7 Kits.........................................................................................................................................28
2.8 Chemicals..............................................................................................................................28
2.9 Instruments............................................................................................................................29
2.10 Other disposable materials....................................................................................................31
2.11 Computer programs and internet links..................................................................................31

3 Methods ........................................................................................................................................32
3.1 Safety.....................................................................................................................................32
3.2 Bacterial culture.....................................................................................................................32
3.2.1 Cultivation...............................................................................................................32
3.2.2 Storage.....................................................................................................................32
3.3 Preparation and transformation of competent cells...............................................................33
3.3.1 Preparation of E. coli competent cells.....................................................................33
3.3.2 Transformation of E. coli competent host cells.......................................................33
3.3.3 Preparation of electro-competent P. putida KT2440 cells......................................33
3.3.4 Electroporation of competent P. putida KT2440 cells.............................................33
ii
3.4 Growth of P. putida KT2440 and mutants............................................................................34
3.4.1 Am