Phenotypic variation and molecular signaling in the interaction of the rhizosphere bacteria Acidovorax sp. N35 and Rhizobium radiobacter F4 with roots Dan Li Helmholtz Zentrum München Deutsches Forschuungszentrum für Gesundheit und Umwelt Abteilung Mikroben-Pflanzen Interaktionen Dissertation Zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften der Fakultät für Biologie der Ludwig-Maximilians-Universität München September 2010 1. Gutachter : Prof. Dr. Anton Hartmann 2. Gutachter: Prof. Dr. Kirsten Jung Eingereicht am: 23. September 2010 Tag der mündlichen Prüfung: 04. February 2011 To my family and to my Xu Contents Contents Abbreviations ........................................................................................................................ 4 1 Introduction ........................................................................................................................ 5 1.1 Plant-microorganisms interactions in the rhizosphere ................................................ 5 1.1.1 The rhizosphere .................................................................................................... 5 1.1.2 Effects of microorganisms on plants .................................................................... 6 1.1.3 Molecular microbial techniques to study microbe-plant interactions .................. 7 1.2 Cell-cell communication in bacteria............................................................................ 7 1.2.1 Quorum sensing in Gram-negative bacteria ......................................................... 7 1.2.2 Quorum sensing in plant-associated bacteria ....................................................... 9 1.2.3 Cross-kingdom signaling...................................................................................... 9 1.3 Phase variation in bacteria......................................................................................... 10 1.3.1 Phase variation in bacteria mediated by genome modification .......................... 10 1.3.2 Phase variation in human pathogens .................................................................. 10 1.3.3 Phase variation in rhizosphere bacteria .............................................................. 10 1.4 The Genus Acidovorax, a phenotypically diverse Gram-negative bacterium ........... 11 1.5 Rhizobium radiobacter, associated closely with the plant growth promoting fungus Piriformospora indica ..................................................................................................... 12 1.5.1 Fungus associated bacteria ................................................................................. 12 1.5.2 Piriformospora indica - a plant growth promoting fungus ................................ 13 1.5.3 Rhizobium radiobacter - an endofungal bacterium ............................................ 13 1.6 Aim of the work......................................................................................................... 14 2 Material and methods ....................................................................................................... 16 2.1 Cultivation of microorganisms .................................................................................. 16 2.1.1 Bacterial strains and plasmids ............................................................................ 16 2.1.2 Media and buffers............................................................................................... 19 2.1.3 Selective agents .................................................................................................. 23 2.1.4 Cultivation of microorganisms ........................................................................... 25 2.2 Cultivation of barley.................................................................................................. 25 2.2.1 Barley in monoxenic system .............................................................................. 25 2.2.1.1 Monoxenic system....................................................................................... 25 2.2.1.2 Seed sterilization ......................................................................................... 26 2.2.2 Barley in soil system .......................................................................................... 26 2.3 Localization of bacteria on barley roots .................................................................... 26 2.3.1 Inoculation of barley roots with bacteria............................................................ 26 2.3.2 Root harvesting................................................................................................... 27 2.3.3 Preparation of roots for microscopic detection of bacteria................................. 27 2.4 Microscopy ................................................................................................................ 28 2.4.1 Epifluorescence microscopy............................................................................... 28 2.4.2 Confocal laser scanning microscopy (CLSM).................................................... 28 2.4.3 Scanning electron microscopy............................................................................ 29 2.5 Rhizobacteria-fungi interactions................................................................................ 29 2.6 Metabolic analyses of bacteria .................................................................................. 30 2.6.1 Detection and characterization of N-acyl-homoserine lactones (AHLs)............ 30 2.6.1.1 Detection of AHLs via biosensor ................................................................ 30 2.6.1.2 Detection and characterization of AHLs via Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and ultra performance liquid chromatography (UPLC) ......................................................................................... 31 2.6.2 Phenotypic characterization of bacterial strains ................................................. 32 2.7 Molecular genetic methods........................................................................................ 33 1 Contents 2.7.1 Fluorescence in situ hybridization (FISH).......................................................... 33 2.7.1.1 Oligonucleotide probes................................................................................ 34 2.7.1.2 Cell fixation with paraformaldehyde........................................................... 35 2.7.1.3 Hybridization with oligonucleotide probes ................................................. 36 2.7.2 DNA isolation..................................................................................................... 38 2.7.2.1 Plasmid DNA isolation................................................................................ 38 2.7.2.2 Chromosomal DNA isolation ...................................................................... 39 2.7.3 DNA purification................................................................................................ 39 2.7.4 Quantification of DNA concentration ................................................................ 39 2.7.5 Enzymatic DNA modifications .......................................................................... 40 2.7.5.1 Digestion of DNA with restriction enzymes ............................................... 40 2.7.5.2 Dephosphorylation of linear DNA .............................................................. 40 2.7.5.3 Ligation of DNA.......................................................................................... 40 2.7.6 Gel electrophoresis ............................................................................................. 41 2.7.7 Amplification of specific DNA fragments via PCR........................................... 41 2.7.7.1 PCR primer.................................................................................................. 41 2.7.7.2 Standard PCR .............................................................................................. 42 2.7.7.3 Cloning of PCR amplicons.......................................................................... 43 2.7.8 DNA sequence analysis...................................................................................... 43 2.7.8.1 DNA sequencing with ABI 3730 sequencer................................................ 43 2.7.8.2 Genomic DNA sequencing with a 454 pyrosequencer................................ 43 2.7.8.3 Sequencing data analysis............................................................................. 44 2.7.9 DNA-DNA hybridization ................................................................................... 45 2.7.10 DNA transfer into Gram-negative bacteria ...................................................... 45 2.7.10.1 Electrocompetent cells after Hanahan (1983, modified)........................... 45 2.7.10.2 Electroporation after Dower et al. (1988) ................................................. 45 2.7.10.3 DNA transfer into E. coli via heat shock................................................... 46 2.7.10.4 DNA transfer via conjugation after Kristensen et al. (1995, modified).... 46 2.7.11 Plasmid constructions....................................................................................... 47 2.7.12 GFP- and YFP-labeling of isolate N35 ............................................................ 48 2.7.12.1 GFP labeling of N35 and N35v by transposon tagging............................. 48 2.7.12.2 YFP labeling of N35 and N35v