Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München A non-canonical pathway for aromatic amino acid biosynthesis in haloarchaea Identification and function of essential genes Miriam (Mirit) Kolog Gulko Tel-Aviv 2010 Erklärung Diese Dissertation wurde im Sinne von §13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 von Herrn Prof. Dr. Dieter Oesterhelt betreut. Ehrenwörtliche Versicherung Diese Dissertationn wurde selbständig, ohne unerlaubte Hilfe erarbeitet. München, am ........................... .......................................... Miriam (Mirit) Kolog Gulko Dissertation eingereicht am: 25/03/2010 1. Gutacher: Prof. Dr. Dieter Oesterhelt 2. Guttacher: Prof. Dr. Nediljko Budisa Mündliche Prüfung am: 07/06/2010 Ricardo, as always, for all things- past, present and yet to come "The outcome of any serious research can only be to make two questions grow where only one grew before" Thorstein Veblen, (1857-1929) U.S. economist and sociologist Table of contents 1 Abbreviations ....................................................................................................... 1 2 Acknowledgments ................................................................................................ 3 3 Summary ............................................................................................................... 5 4 Introduction .......................................................................................................... 7 4.1 The three domains of life ............................................................................... 7 4.2 Diversity and unity ......................................................................................... 8 4.3 Metabolism of halophilic archaea .................................................................. 9 4.3.1 Shikimate pathway of aromatic amino acids (AroAAs) biosynthesis ................. 11 4.3.2 Pathway reconstruction from genomic data ...................................................... 13 4.3.2.1 Shikimate pathway in Methanocaldococcus and Methanococcus ............ 15 4.3.2.2 The Shikimate pathway in Haloarchaea.................................................... 16 4.3.2.3 Postulating de novo pathway in Haloarchaea ........................................... 21 4.4 Enzyme classification .................................................................................. 22 4.4.1 Aldolases EC 4.1.2.13 ........................................................................................ 22 4.4.2 Dehydroquinate synthase EC 4.6.1.3 ................................................................. 24 4.4.3 dehydroquinate dehydratase EC 4.2.1.10 .......................................................... 24 4.5 Microarray analysis ...................................................................................... 25 4.6 Objectives .................................................................................................... 27 5 Materials and Methods ...................................................................................... 28 5.1 Materials ...................................................................................................... 28 5.1.1 Chemicals .......................................................................................................... 28 5.1.2 Kits and Enzymes ............................................................................................... 28 5.1.3 Media, Buffers and Stock solutions .................................................................... 29 5.1.3.1 Media ........................................................................................................ 29 5.1.3.2 Buffers ...................................................................................................... 33 5.1.3.3 Antibiotics ................................................................................................. 34 5.1.4 Strains, Vectors and Oligonucleotides ............................................................... 35 5.1.4.1 Strains ....................................................................................................... 35 5.1.4.2 Plasmids .................................................................................................... 36 5.1.4.3 Oligonucleotides ....................................................................................... 37 5.2 Microbiological methods ............................................................................. 37 5.2.1 Cultivation of E. coli .......................................................................................... 37 5.2.2 Cultivation of H. salinarum and N. pharaonis .................................................. 37 5.3 Molecular biological methods ..................................................................... 38 Table of contents 5.3.1 Transformation of E. coli ................................................................................... 38 5.3.1.1 Preparation of chemically-competent cells ............................................... 38 5.3.1.2 Transformation of chemically-competent cells ......................................... 40 5.3.1.3 Transformation of electro-competent cells ............................................... 40 5.3.2 Transformation of H. salinarum ........................................................................ 41 5.3.3 Transformation of N. pharaonis ........................................................................ 42 5.3.3.1 PEG method .............................................................................................. 42 5.3.3.2 Electroporation .......................................................................................... 42 5.3.3.3 Gun particle ............................................................................................... 42 5.3.4 Preparation of genomic DNA from H. salinarum .............................................. 43 5.3.5 Isolation of plasmid DNA from E. coli .............................................................. 43 5.3.6 Agarose gel electrophoresis ............................................................................... 43 5.3.7 Isolation of DNA from preparative agarose gels ............................................... 44 5.3.8 Determination of DNA concentration ................................................................ 45 5.3.9 Sequencing of DNA ............................................................................................ 45 5.3.10 Polymerase chain reaction (PCR) ................................................................. 45 5.3.10.1 Ligation of DNA fragments ...................................................................... 47 5.3.10.2 Digestion of DNA by restriction endonucleases ....................................... 48 5.3.10.3 ORFs OE1472F, OE1475F, OE1477R, and OE2019F from R1 ............... 48 5.3.10.4 Plasmids for gene deletion ........................................................................ 48 5.3.10.4.1 Blue/red screening of transformant colonies ..................................... 51 5.3.10.5 Southern blot analysis ............................................................................... 52 5.3.11 Microarrays ................................................................................................... 53 5.3.11.1 Microarray design ..................................................................................... 53 5.3.11.2 Isolation of total RNA ............................................................................... 53 5.3.11.3 Microarray analysis ................................................................................... 54 5.3.12 RT-PCR ......................................................................................................... 55 5.4 Protein Methods ........................................................................................... 56 5.4.1 SDS-PAGE of proteins ....................................................................................... 56 5.4.2 Western blot analysis ......................................................................................... 57 5.4.3 Determination of protein concentration ............................................................ 58 5.4.4 EasyXpress protein Synthesis Kit (Qiagen) ....................................................... 58 5.4.5 Expression and purification of OE1472F, OE1475F and OE1477R from R1 ... 58 5.4.6 N-terminal sequencing ....................................................................................... 59 5.4.7 Amino acid analysis ........................................................................................... 59 5.4.8 Activity assays .................................................................................................... 59 5.4.8.1 Aldolase activity using a coupled enzyme assay ...................................... 59 II Table of contents 5.4.8.2 Colorimetric assay of Aldolase ................................................................. 60 5.4.8.3 3-Dehydroquinate dehydratase ................................................................. 60 5.4.8.4 LC-MS ...................................................................................................... 61 5.4.8.5 GC-MS ...................................................................................................... 61 5.5 Synthesis .....................................................................................................