Analysis of potential host-colonization factors in Bifidobacterium bifidum S17 Dissertation zur Erlangung des Doktorgrades Dr. rer. nat. der Fakultät für Naturwissenschaften der Universität Ulm vorgelegt von Christina Westermann aus Ulm 2015 Amtierender Dekan: Prof. Dr. Joachim Ankerhold Erstgutachter: PD Dr. Christian Riedel Zweitgutachter: Prof. Dr. Bernhard Eikmanns Tag der mündlichen Prüfung: 15.10.2015 Die vorliegende Arbeit wurde im Institut für Mikrobiologie und Biotechnologie an der Universität Ulm angefertigt. Die Arbeit wurde durch ein Stipendium nach dem Landesgraduiertenförderungsgesetz Baden-Württemberg finanziert. TABLE OF CONTENTS 1. Introduction ....................................................................................................................... 8 1.1. General features of the genus Bifidobacterium ................................................................. 8 1.2. Bifidobacteria as a part of the human microbiota ............................................................. 9 1.3. Bifidobacteria as probiotics .............................................................................................. 10 1.3.1. Definition of probiotics ........................................................................................ 10 1.3.2. Probiotic bifidobacteria and their health-promoting effect on human host ....... 11 1.4. Potential adhesive structures of the human gut.............................................................. 12 1.4.1. Mucus ................................................................................................................... 12 1.4.2. Intestinal epithelial cells and the glycocalyx ........................................................ 13 1.4.3. Extracellular matrix proteins ................................................................................ 14 1.5. Bacterial cell surface architecture – potential adhesion mediating structures ............... 15 1.5.1. Exopolysaccharides .............................................................................................. 17 1.5.2. Cell envelope proteases ....................................................................................... 18 1.5.3. Pili ......................................................................................................................... 19 1.5.4. Identified single adhesins ..................................................................................... 20 1.5.4.1. Translocation and cell wall anchoring of cell surface proteins ........................ 21 1.6. The aim of this study ........................................................................................................ 23 2. Materials and Methods .................................................................................................... 24 2.1. Bacterial strains ................................................................................................................ 24 2.1.1. Bifidobacteria ....................................................................................................... 24 2.1.2. Escherichia coli ..................................................................................................... 25 2.2. Plasmids ............................................................................................................................ 26 2.3. Eukaryotic Caco-2 cell line ................................................................................................ 27 2.3.1. Subcultivation of Caco-2 cells............................................................................... 27 2.3.2. Cryoconservation of Caco-2 cells ......................................................................... 28 2.3.3. Thawing of cryo-stocks ......................................................................................... 28 2.3.4. Determination of the cell number ....................................................................... 28 2.4. Adhesion assays ............................................................................................................... 29 2.4.1. Adhesion assay with Caco-2 cells ......................................................................... 29 2.4.2. Competitive adhesion assay ................................................................................. 29 2.4.3. Adhesion to extracellular matrix proteins............................................................ 30 2.5. Working with nucleic acids ............................................................................................... 31 2.5.1. Chromosomal DNA extraction ............................................................................. 31 2.5.1.1. Preparation and cell lysis .................................................................................. 31 2.5.1.2. Phenol/chloroform/isoamylalcohol extraction ................................................ 32 2.5.1.3. Ethanol precipitation ........................................................................................ 32 2.5.2. Isolation of plasmid DNA ...................................................................................... 32 2.5.3. DNA purification from agarose gels or PCR mixtures .......................................... 33 2.5.4. Agarose gel electrophoresis ................................................................................. 33 2.5.5. Quantification of dsDNA ...................................................................................... 33 2.5.6. Oligonucleotides ................................................................................................... 34 2.5.7. Amplification of DNA by polymerase chain reaction ........................................... 34 2.5.7.1. Temperature gradient PCR ............................................................................... 35 2.5.8. Screening colonies by PCR .................................................................................... 36 2.5.9. DNA digestion with restriction enzymes .............................................................. 37 2.5.10. Klenow fill-in of DNA ends with 5`-overhang ....................................................... 37 2.5.11. Ligation ................................................................................................................. 37 2.5.12. Sequencing of plasmid constructs ........................................................................ 38 2.5.13. RNA extraction ..................................................................................................... 38 2.5.14. Reverse Transcription PCR ................................................................................... 39 2.6. Transformation on E. coli ................................................................................................. 40 2.6.1. Preparation of electrocompetent E. coli cells ...................................................... 40 2.6.2. Transformation of electrocompetent E. coli ........................................................ 41 2.6.3. Plasmid artificial modification .............................................................................. 41 2.7. Transformation of Bifidobacterium sp. strains................................................................. 42 2.7.1. Preparation of electrocompetent bifidobacteria ................................................. 42 2.7.2. Transformation of electrocompetent bifidobacteria ........................................... 42 2.8. Working with proteins...................................................................................................... 43 2.8.1. Preparation of bacterial cell fractions .................................................................. 43 2.8.2. Quantification of proteins .................................................................................... 44 2.8.3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis ............................... 44 2.8.4. Staining of SDS gels .............................................................................................. 45 2.8.5. Western Blot ........................................................................................................ 46 2.9. Microscopy ....................................................................................................................... 47 2.9.1. Ruthenium red staining ........................................................................................ 47 2.9.2. Sample embedding and ultrathin sectioning ....................................................... 47 2.9.3. Transmission electron microscopy ....................................................................... 48 2.9.4. Scanning electron microscopy ............................................................................. 48 2.9.5. Fixing and staining of cells for fluorescence microscopy ..................................... 48 2.9.6. Fluorescence microscopy ..................................................................................... 48 2.10. Bioinformatic analysis .............................................................................................. 49 2.11. Statistical analysis ..................................................................................................... 49 3. Results .............................................................................................................................. 50 3.1. Bioinformatic analysis .....................................................................................................