Materials and Methods
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Regulation of glutamate dehydrogenase in Corynebacterium glutamicum Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades vorgelegt von Eva Hänßler aus Aachen Als Dissertation genehmigt von der Naturwissen- schaftlichen Fakultät der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 15.02.2008 Vorsitzender der Promotionskommission: Prof. Dr. Eberhard Bänsch Erstberichterstatter: Prof. Dr. Andreas Burkovski Zweitberichterstatter: Prof. Dr. Reinhard Krämer Content Content 1 Zusammenfassung/Summary........................................................1 2 Introduction.....................................................................................3 2.1 Corynebacterium glutamicum.............................................................................3 2.2 Uptake and assimilation of nitrogen sources....................................................4 2.3 Nitrogen-dependent regulation...........................................................................6 2.4 GDH at the interface between nitrogen and carbon metabolism...................11 2.4.1 Glutamate dehydrogenase of E. coli ................................................................12 2.4.2 Glutamate dehydrogenase of B. subtilis...........................................................13 2.4.3 Glutamate dehydrogenase of C. glutamicum ...................................................15 2.5 Objectives ...........................................................................................................17 3 Materials and methods .................................................................19 3.1 Bacterial strains and plasmids .........................................................................19 3.2 Cultivation of bacteria........................................................................................22 3.2.1 Culture medium for E. coli ................................................................................22 3.2.2 Culture media for corynebacteria .....................................................................22 3.2.3 Antibiotics .........................................................................................................23 3.2.4 Growth conditions.............................................................................................24 3.3 Genetic manipulation of bacteria......................................................................25 3.3.1 Preparation of competent E. coli cells and transformation ...............................25 3.3.2 Preparation of competent C. glutamicum cells and transformation..................26 3.4 Working with DNA ..............................................................................................26 3.4.1 Isolation of plasmid DNA from E. coli ...............................................................26 3.4.2 Gel electrophoresis and extraction of DNA from agarose gels.........................27 3.4.3 Preparation of chromosomal DNA from C. glutamicum....................................27 3.4.4 Purification and enrichment of DNA .................................................................27 3.4.5 Polymerase chain reaction (PCR) ....................................................................28 3.4.6 Two-step PCR ..................................................................................................28 3.4.7 Restriction of DNA............................................................................................29 3.4.8 Ligation of DNA fragments ...............................................................................29 3.4.9 Sequencing of DNA..........................................................................................29 3.5 Working with RNA ..............................................................................................30 3.5.1 Isolation of total RNA and RNA gel electrophoresis.........................................30 Content 3.5.2 Synthesis of digoxigenin-labeled RNA probes .................................................31 3.5.3 Northern blot analysis.......................................................................................31 3.5.4 Dot blot analysis ...............................................................................................33 3.5.5 Reverse transcriptase (RT) PCR......................................................................34 3.5.6 Quantitative real time RT PCR .........................................................................34 3.5.7 Primer extension analysis ................................................................................35 3.6 Working with proteins........................................................................................37 3.6.1 Protein purification............................................................................................37 3.6.2 Quantification of proteins..................................................................................38 3.6.3 SDS polyacrylamide gel electrophoresis (PAGE).............................................38 3.6.4 Staining with Coomassie Brilliant Blue .............................................................39 3.6.5 Western blotting ...............................................................................................39 3.6.6 Determination of enzyme activity .....................................................................41 3.6.6.1 GDH activity measurements.....................................................................41 3.6.6.2 Glutamyltransferase test...........................................................................42 3.6.7 Determination of promoter activity....................................................................43 3.6.8 Gel shift assays and competition assays .........................................................43 4 Results...........................................................................................45 4.1 Purification and characterization of glutamate dehydrogenase....................45 4.1.1 Purification of GDH...........................................................................................45 4.1.2 Characterization of GDH in lysine-producing strains........................................47 4.1.3 Gradual expression of gdh ...............................................................................49 4.2 Transcriptional regulation of gdh .....................................................................51 4.2.1 Mutational analyses of the gdh promoter region ..............................................51 4.2.2 Determination of the transcription start.............................................................57 4.2.3 Nitrogen-dependent transcription .....................................................................60 4.2.3.1 Function of AmtR......................................................................................61 4.2.3.2 Influence of putative regulators on gdh transcription................................66 4.2.4 Studies on sigma factor-dependent gdh expression ........................................71 4.2.5 Investigation of the putative orf Cg2281...........................................................73 4.3 Identification of AmtR and FarR target genes .................................................75 4.3.1 Identification of FarR target genes ...................................................................75 4.3.1.1 Characterization of arginine biosynthesis genes ......................................76 4.3.1.2 Determination of FarR and ArgR binding sites .........................................79 4.3.1.3 Transcriptional regulation of arginine biosynthesis genes........................81 Content 4.3.2 Identification of AmtR target genes ..................................................................83 5 Discussion.....................................................................................89 5.1 Characterization of GDH in the context of systems biology..........................89 5.2 Transcriptional regulation of gdh .....................................................................93 5.3 Identification of FarR and AmtR target genes ...............................................100 5.4 The interface between nitrogen and carbon metabolism.............................106 6 Appendix......................................................................................109 6.1 Regulation of glutamine synthetase in corynebacteria................................109 6.2 Plasmid constructions.....................................................................................112 References ........................................................................................117 Publications ......................................................................................131 Abbreviations and units...................................................................132 Zusammenfassung 1 1 Zusammenfassung Die Glutamatdehydrogenase (GDH) aus Corynebacterium glutamicum, einem Actinomyceten mit herausragender biotechnologischer Bedeutung, befindet sich an einer wichtigen Position innerhalb des Stoffwechsels, da sie Stickstoffassimilation und den Zentralstoffwechsel verbindet. Unter Überschussbedingungen ist die GDH an der Ammoniumassimilation beteiligt und über das Substrat α-Ketoglutarat besteht eine direkte Verknüpfung zum Citrat Zyklus. Aufgrund der NADPH-Abhängigkeit kann weiterhin der pool an Reduktionsäquivalenten beeinflusst werden. Trotz dieser scheinbar bedeutsamen Stellung