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Biofilm Formation and EPS Analysis of the Thermoacidophilic Archaeon Biofilm formation and EPS analysis of the thermoacidophilic Archaeon Sulfolobus acidocaldarius Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften – Dr. rer. nat. – vorgelegt von Silke Jachlewski geboren in Willich Biofilm Centre – Molekulare Enzymtechnologie und Biochemie der Universität Duisburg-Essen 2013 Die vorliegende Arbeit wurde im Zeitraum von Oktober 2010 bis September 2013 im Arbeitskreis von Prof. Dr. Bettina Siebers am Biofilm Centre der Universität Duisburg-Essen durchgeführt. Tag der Disputation: 20.11.2013 Gutachter: Prof. Dr. Bettina Siebers Prof. Dr. Hans-Curt Flemming Vorsitzender: Prof. Dr. Matthias Epple Table of Contents TABLE OF CONTENTS TABLE OF CONTENTS ................................................................................................................ I LIST OF TABLES...................................................................................................................... V LIST OF FIGURES .................................................................................................................... VI ABSTRACT.......................................................................................................................... VIII 1. INTRODUCTION ............................................................................................................ 1 1.1 Archaea – The third domain of life ................................................................................... 1 1.1.1 The Crenarchaeon Sulfolobus acidocaldarius ........................................................ 2 1.2 Bacterial and archaeal biofilms ........................................................................................ 3 1.2.1 Role of archaeal surface structures in attachment ................................................ 6 1.3 Extracellular polymeric substances .................................................................................. 7 1.3.1 Isolation of EPS ....................................................................................................... 7 1.3.2 Function of EPS ....................................................................................................... 8 1.3.3 Secretion of EPS ................................................................................................... 10 1.3.4 Role of glycosyltransferases in synthesis of exopolysaccharides ........................ 12 1.4 Aims of this study ........................................................................................................... 19 2. MATERIALS & METHODS .............................................................................................. 21 2.1 Chemicals and plasmids ................................................................................................. 21 2.2 Commercial kits .............................................................................................................. 21 2.3 Instruments .................................................................................................................... 22 2.4 Software and databases ................................................................................................. 24 2.5 Strains and precultivation .............................................................................................. 25 2.5.1 Escherichia coli ..................................................................................................... 25 2.5.2 Sulfolobus acidocaldarius and Sulfolobus solfataricus......................................... 25 2.6 General characterization of S. acidocaldarius biofilms .................................................. 27 2.6.1 Optimization of growth conditions ...................................................................... 27 2.6.2 Determination of biofilm mass ............................................................................ 28 2.6.3 Determination of total cell counts ....................................................................... 29 2.6.4 Determination of colony counts .......................................................................... 29 i Table of Contents 2.6.5 Microscopy of biofilms and EPS components ...................................................... 29 2.6.6 Determination of cell surface hydrophobicity ..................................................... 30 2.7 EPS isolation ................................................................................................................... 31 2.8 Quantitative biochemical analysis .................................................................................. 32 2.8.1 Multi-element analysis of biofilms and EPS ......................................................... 32 2.8.2 Quantification of carbohydrates .......................................................................... 32 2.8.3 Quantification of proteins .................................................................................... 33 2.8.4 Quantification of DNA .......................................................................................... 34 2.9 Identification of monosaccharides ................................................................................. 34 2.9.1 Hydrolysis of polysaccharides .............................................................................. 34 2.9.2 Thin layer chromatography (TLC) ......................................................................... 34 2.10 Sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) .................... 35 2.10.1 1D gel electrophoresis.......................................................................................... 35 2.10.2 2D gel electrophoresis.......................................................................................... 36 2.11 Gel staining and image acquisition................................................................................. 38 2.11.1 Silver staining ....................................................................................................... 38 2.11.2 Enhanced Coomassie Brilliant Blue staining ........................................................ 38 2.11.3 Rapid Coomassie Blue staining ............................................................................ 39 2.11.4 Staining of glycoproteins ...................................................................................... 39 2.12 Determination of enzyme activities ............................................................................... 39 2.12.1 Fluorimetric screening of enzyme activity ........................................................... 39 2.12.2 Zymographic analysis of protease activity ........................................................... 40 2.12.3 Zymographic analysis of esterase activity ............................................................ 41 2.13 Identification of EPS proteins via MALDI-TOF/MS ......................................................... 41 2.14 Molecular biology methods ........................................................................................... 42 2.14.1 Isolation of chromosomal DNA from S. acidocaldarius ....................................... 42 2.14.2 Isolation of plasmid DNA from E. coli DH5α ........................................................ 42 2.14.3 Agarose gel electrophoresis ................................................................................. 42 2.14.4 Purification of DNA fragments ............................................................................. 42 2.14.5 Amplification of S. acidocaldarius genomic DNA via polymerase chain reaction (PCR) ..................................................................................................................... 43 2.14.6 Restriction and ligation of DNA ............................................................................ 44 ii Table of Contents 2.14.7 Preparation of chemically competent E. coli cells ............................................... 45 2.14.8 Preparation of competent S. acidocaldarius MW001 cells for electroporation .. 45 2.14.9 Transformation of competent E. coli cells ........................................................... 45 2.14.10 Electroporation of competent S. acidocaldarius MW001 cells ........................... 46 2.14.11 Identification of positive E. coli clones ................................................................. 46 2.14.12 DNA Sequencing ................................................................................................... 46 2.14.13 Deletion of glycosyltransferase genes in S. acidocaldarius MW001 ................... 46 2.14.14 Construction of thermopsin insertion mutant S. acidocaldarius ∆1714 ............. 47 2.14.15 Screening for positive S. acidocaldarius mutants ................................................ 47 2.14.16 Heterologous expression of S. acidocaldarius glycosyltransferases .................... 47 2.14.17 Cell disruption and heat precipitation ................................................................. 48 2.14.18 Ni-TED affinity chromatography .......................................................................... 48 2.14.19 Western blotting and immunodetection ............................................................. 49 3. RESULTS ................................................................................................................... 50 3.1 Optimization of growth and general characterization of S. acidocaldarius biofilms ..... 50 3.1.1 Cell surface hydrophobicity.................................................................................
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