Analysis of regulatory mechanisms governing aromatic compound degradation in Acinetobacter baylyi [Elektronische Ressource] / by Fenja Sabine Bleichrodt

Sujets

• Biologie

Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2011
• Nombre de lectures: 28
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Analysis of regulatory mechanisms governing
aromatic compound degradation in
Acinetobacter baylyi




Dissertation

Submitted for the fulfillment of the requirements for the doctoral degree Dr. rer. nat
at the Faculty of Natural Sciences, University of Ulm








By Fenja Sabine Bleichrodt from Berlin
2011























The current study was prepared at the Institute of Microbiology and Biotechnology, University of Ulm.




Dekan: Prof. Dr. Thomas Wirth

1. Reviewer: apl. Prof. Dr. Ulrike Gerischer
2. Reviewer: Prof. Dr. Peter Dürre

Tag der Promotion: 31.05.2011
Contents 1
Contents
Abbreviations ............................................................................................................................................... 5
1 Introduction ......... 8
2 Materials and Methods ..................................................................................................................... 16
2.1 Plasmid and strain construction ................................................................................................... 16
2.2 Bacterial strains designed and used in this study ........ 18
2.3 Plasmids constructed and used in this study ................................................................................ 20
2.4 Primers used in this study ............................................ 21
2.5 Growth conditions ....................... 25
2.6 Stock cultures .............................................................................................................................. 26
2.7 Transformation ............................ 27
2.7.1 Transformation of E. coli .................................... 27
2.7.1.1 Competent E. coli DH5  cells ........................................................................................ 27
2.7.1.2 Heat shock transformation ............................... 27
2.7.1.3 Competent E. coli BL 21 cells ......................................................................................... 27
2.7.1.4 Electroporation ................................................ 28
2.7.1.5 Blue white screening ....................................................................... 28
2.7.2 Transformation of A. baylyi strain ADP1 ............ 28
2.8 Working with nucleic acids ......................................... 28
2.8.1 Nucleic acid isolation .......................................................................... 29
2.8.1.1 Minipreparation of plasmid DNA from E. coli - alkaline lysis ....... 29
TM2.8.1.2 Plasmid isolation using the Zyppy Plasmid Miniprep Kit (Zymo research) ............... 29
2.8.1.3 Plasmid isolation using the QIAGEN Plasmid Mini Kit ................................................. 30
®2.8.1.4 Total RNA extraction using TriReagent (Molecular Research Center, Inc.) ................ 30
2.8.2 Nucleic acid enrichment and purification ............................................................................ 30
2.8.2.1 Phenol chloroform extraction .......................... 30
2.8.2.2 Ethanol precipitation ....................................................................................................... 30
®2.8.2.3 Purification of PCR products using the NucleoSpin Extract Kit (Macherey & Nagel) 31 Contents 2
TM2.8.2.4 Purification of radiolabled DNA fragments using MicroSpin G-25 columns (GE
Healthcare) ........................................................................................................................ 31
2.8.3 Enzymatic modification of nucleic acids ............ 31
2.8.3.1 Restriction ....................... 31
2.8.3.2 Ligation ........................................................................................................................... 31
2.8.3.3 End-labeling .................... 32
2.8.4 Polymerase chain reaction (PCR) ........................................................................................ 32
2.8.5 Direct cloning of PCR products .......................... 32
2.8.6 Rapid amplification of cDNA ends (5´RACE).................................................................... 33
2.8.7 DNA sequencing ................................................. 33
2.9 Electrophoresis ............................................................................................ 33
2.9.1 Agarose gel electrophoresis ................................. 33
2.9.2 Polyacrylamide gel electrophoresis (PAGE) ....................................... 34
2.9.3 Denaturing polyacrylamide gel electrophoresis .. 34
2.9.4 SDS polyacrylamide gel electrophoresis (SDS-PAGE) ...................................................... 35
2.9.4.1 Coomassie staining .......................................................................... 35
2.9.4.2 Silver staining .................................................. 36
2.9.5 Size standards ...................................................................................... 37
2.10 Working with proteins ................. 37
2.10.1 Overproduction of recombinant proteins with E. coli BL 21 .............................................. 37
2.10.2 Purification of His-tagged recombinant proteins by high performance liquid
chromatography (HPLC) ..................................................................... 38
2.10.3 Determination of protein concentrations ............................................. 39
2.10.4 Electro mobility shift assays (EMSAs) ............................................... 39
2.10.5 DNase I footprinting assays................................................................. 40
2.10.5.1 Footprint ...................................................................................... 40
2.10.5.2 Sequencing reaction..................................................................................................... 40
2.11 Determination of luciferase activity ............................ 41
2.12 Software tools and data banks ..... 42
2.13 Chemicals and instruments .......................................................................................................... 42
2.13.1 Instruments .......................................................................................................................... 42
2.13.2 Chemicals ............................ 42 Contents 3
3 Results ................................................................................................................................................. 44
3.1 Analysis of carbon catabolite repression ..................... 44
3.2 Expression pattern in the presence of lactate and gluconate ....................................................... 49
3.3 Analysis of cross-regulation ........................................................................................................ 54
3.3.1 Searching the effector .......... 55
3.3.2 Identification of the regulators mediating cross-regulation ................................................. 58
3.3.3 Bioinformatic exploration of regulatory regions ................................................................. 61
3.3.4 Binding of BenM and CatM to intergenic regions .............................. 61
3.3.5 Determination of transcriptional start sites .......................................................................... 65
3.4 Analysis of vertical regulation .................................... 68
3.4.1 Searching the effector mediating vertical regulation ........................................................... 68
3.4.2 Induction of hca and vanA,B expression by PCA alone ...................... 70
3.4.3 Identification of the regulator mediating vertical regulation ............................................... 72
3.4.4 Bioinformatic exploration of regulatory regions ................................. 76
3.4.5 Binding of PcaU to its putative binding sites ...................................... 78
3.4.6 Exact determination of the PcaU binding site upstream of vanK ........ 82
3.5 Analysis of vanK expression ....................................................................... 84
3.5.1 Refining the substrate spectrum of VanK ........................................... 84
3.5.1.1 Overlapping specificity of transport proteins .. 84
3.5.1.2 Expression of vanK in response to several aromatic compounds .................................... 85
3.5.1.3 Expression of vanK in response to several aromatic compounds in strains blocked in
PCA formation ................................................................................................................ 85
4 Discussion ........................................... 87
4.1 The salA and vanK genes undergo CCR by organic acids .......................................................... 87
4.2 CCR by succinate and acetate involves the catabolite repression control protein ...................... 89
4.3 Expression pattern in the presence of lactate and gluconate ....................... 91
4.4 Crc is involved in the expression of operons on pyruvate and lactate, but not on gluconate ...... 92
4.5 Cross-regulation ...............................................................