Pobigaylo, Nataliya: Identification of genes in Sinorhizobium meliloti relevant to symbiosis and competitiveness using signature tagged transposons. 2006
Inhalt
- B.1 NITROGEN FIXATION
- B.2 SYMBIOSIS BETWEEN RHIZOBIA AND THE LEGUMINOUS PLANTS
- B.2.1 Determinate and indeterminate nodules
- B.2.2 Formation of indeterminate nodules
- B.2.3 Structure of mature indeterminate nodules
- B.2.4 Bacterial genes involved in competitiveness, efficient nodule formation and nitrogen fixation
- B.2.4.1 Genes important for survival and competitiveness in the rhizosphere
- B.2.4.2. Genes important for the formation of nodules and infection
- B.2.4.3. Genes important for the efficient nitrogen fixation
- B.3 SYSTEMATIC APPROACHES TO STUDY THE GENES IMPORTANT FOR THE SYMBIOSIS AND COMPETITIVENESS
- B.3.1 Proteomics
- B.3.2 DNA macro- and microarrays in the analysis of gene expression
- B.3.3 Metabolomics
- B.3.4 Promoter-reporter gene fusion mutant libraries and in vivo expression technology
- B.3.5 Signature-tagged mutagenesis
- B.4 OBJECTIVES OF THIS WORK
- B.5 PRESENTATIONS
- C.1 CONSTRUCTION OF THE mTn5-STM TRANSPOSON AND THE CARRIER PLASMID pG18-STM
- C.2 CLONING OF TAGS INTO THE mTn5-STM TRANSPOSON
- C.3 TRANSPOSON MUTAGENESIS OF S. MELILOTI
- C.4 MAPPING OF THE TRANSPOSON INSERTION SITES
- C.5 STATISTICAL ANALYSIS OF THE MUTANT LIBRARY
- C.6 CREATION OF TWO SETS OF MUTANTS FOR THE SUBSEQUENT EXPERIMENTS.
- C.7 PILOT COMPETITION EXPERIMENTS IN FREE-LIVING CONDITIONS
- C.8 TWO-CHANNEL TAG MICROARRAY FOR THE DETECTION OF THE TAGS
- C.9 ANALYSIS OF TAG-MICROARRAY DATA
- C.9.1 Normalization and filtering
- C.9.2 Clustering
- C.9.3 Confirmation of biological significance of the applied statistical analysis
- C.10 STUDY OF SURVIVAL AND COMPETITIVENESS OF MUTANTS IN PLANTA
- C.10.1 Screening procedure
- C.10.2 Determination of quantity of mutants in single nodules
- C.10.3 Phenotype confirmation for attenuated mutants
- C.10.4 Symbiotic phenotypes other than reduced symbiotis competitiveness
- D.1 SIGNATURE TAGGED TRANSPOSON MUTAGENESIS IS AN EFFICIENT APPROACH TO STUDY GENES INVOLVED IN SURVIVAL OF RHIZOBIA IN SYMBIOTIC AND FREE-LIVING CONDITIONS
- D.2 S. MELILOTI MUTANTS, KNOWN TO BE IMPAIRED IN SYMBIOSIS, WERE DETECTED AS ATTENUATED IN STM EXPERIMENT
- D.3 INDIVIDUAL COMPETITIVENESS TEST CONFIRMED THE STM EXPERIMENT RESULTS FOR THE MOST OF CHECKED MUTANTS.
- D.4 FOUR MUTANTS, IMPAIRED IN SYMBIOTIC COMPETITIVENESS, EXHIBIT ALSO OTHER SYMBIOTIC PHENOTYPES
- D.5 CONCLUSIONS AND OUTLOOK
- E.1 MATERIALS
- E.1.1 Sources for chemicals and kits
- E.1.2 Bacterial strains and plasmids
- E.1.3 Plant material
- E.1.4 Primers and other oligonucleotides
- E.1.5 Growth media and supplements
- E.1.6 Buffers and solutions
- E.1.6.1 DNA- and enzyme buffers
- E.1.6.2 DNA electrophoresis buffers
- E.1.6.3 Solutions for microarray-hybridizations
- E.1.6.4 Buffer for the preparation of competent E. coli cells
- E.1.6.5 GUS staining buffer
- E.1.7 Software and databases
- E.1.8 Contents and layout of the tag microarray
- E.2 METHODS
- E.2.1. Cultivation of bacteria
- E.2.1.1 Growth conditions
- E.2.1.2 Determination of bacterial cell number
- E.2.1.3 Storage of bacterial strains
- E.2.2 Cultivation of plants and harvesting of nodules
- E.2.2.1 Sterilization of M. sativa seeds
- E.2.2.2 Cultivation of plants on Petri dishes and inoculation by S. meliloti
- E.2.2.3 Cultivation of plants in aeroponic system
- E.2.2.4 Harvesting and sterilization of nodules from the agar plates
- E.2.2.5 Harvesting and sterilization of nodules from the aeroponic tank
- E.2.2.6 Exogenous climatic factors for plant growth
- E.2.3 DNA transfer
- E.2.4. Isolation and purification of DNA
- E.2.4.1. Isolation of plasmid DNA from bacteria (E. coli)
- E.2.4.2 Isolation of genomic DNA from bacteria (S. meliloti)
- E.2.4.3. Isolation of pure DNA from M. sativa nodules
- E.2.4.4. Isolation of DNA from M. sativa nodules by boiling
- E.2.4.5. Purification of PCR products
- E.2.5 Visualization and quantification of DNA
- E.2.5.1 Agarose gel electrophoresis
- E.2.5.2 Checking fluorescently labeled targets on agarose gels prior to microarray hybridizations
- E.2.5.3 Quantification of DNA
- E.2.6 Polymerase chain reaction (PCR)
- E.2.7 Cloning
- E.2.7.1 DNA digestion using restriction enzymes
- E.2.7.2 DNA annealing
- E.2.7.3 DNA ligation
- E.2.7.4 Blunting of overhangs with Klenow fragment
- E.2.7.5 Dephosphorylation of DNA 5´-termini
- E.2.8 Large scale methods (96-well plate format)
- E.2.8.1 Annealing of tags
- E.2.8.2 DNA digestion with KpnI restriction enzyme
- E.2.8.3 Ligation
- E.2.8.4 Transformation of E. coli
- E.2.8.5 Conjugation
- E.2.9 Growth conditions for bacteria in competition experiments in free-living conditions
- E.2.10 Acetylene reduction assay
- E.2.11 Histochemical GUS staining
- E.2.12 Microarray hybridization
- E.2.13 Tag-microarray data analysis
- TABLE G.1. CLONED SIGNATURE TAGS
- TABLE G.2. CHARACTERISTICS OF ALL MUTANTS IN THE EXPERIMENTAL SET 1 IN FREE-LIVING CONDITIONS
- TABLE G.3. RESULTS OF IN PLANTA STM EXPERIMENT FOR SET 1
- TABLE G.4. RESULTS OF IN PLANTA STM EXPERIMENT FOR SET 2
- G.5 LIST OF ABBREVIATIONS
- G.6 LIST OF FIGURES
- G.7 LIST OF TABLES
- G.8 ACKNOWLEDGEMENTS