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Characterisation of Metal-Resistant and Chloroaromatics-Degrading Bacteria - - - - Faculteit Wetenschappen Vakgroep Biochemie, Fysiologie & Microbiologie Laboratorium voor Microbiologie Academiejaar 2002 - 2003 CHARACTERISATION OF METAL-RESISTANT AND CHLOROAROMATICS-DEGRADING BACTERIA - - - - - KARAKTERISERING VAN METAAL-RESISTENTE EN CHLOORAROMAAT-DEGRADERENDE BACTERIËN JOHAN GORIS Proefschrift ingediend tot het verkrijgen van de graad van Doctor in de Wetenschappen, Biotechnologie Promotor: Prof. Dr. P. De Vos CHAPTER 1 Literature overview................................................................................. 1 1.1 Introduction....................................................................................................... 1 1.2 Bacterial systematics......................................................................................... 2 1.2.1 General definitions.................................................................................... 2 1.2.2 Current tools in bacterial systematics ....................................................... 3 1.2.2.1 “Classical” phenotypic characterisation ............................................... 4 1.2.2.2 Chemotaxonomic methods ................................................................... 5 1.2.2.3 Phenotypic typing methods................................................................... 5 1.2.2.4 Whole-genome DNA hybridisation and nucleotide composition......... 6 1.2.2.5 DNA-based typing methods.................................................................. 6 1.2.2.6 Sequencing of selected genes ............................................................... 7 1.2.2.7 Whole-genome sequencing................................................................... 8 1.2.2.8 Uncultivable organisms ........................................................................ 9 1.2.3 General conclusions and future perspectives.......................................... 10 1.3 Organisms of interest in this study ................................................................. 12 1.3.1 Taxonomy and ecology of the genus Burkholderia................................ 12 1.3.1.1 Soil inhabitants................................................................................... 13 1.3.1.2 Nitrogen-fixing and nodulating strains............................................... 13 1.3.1.3 Plant endosymbionts........................................................................... 14 1.3.1.4 Plant pathogens................................................................................... 14 1.3.1.5 The Burkholderia cepacia complex .................................................... 14 1.3.1.6 Biotechnological interest.................................................................... 15 1.3.1.7 Human pathogens............................................................................... 16 1.3.1.8 B. gladioli and B. fungorum ............................................................... 16 1.3.1.9 Genome flexibility.............................................................................. 16 1.3.2 Taxonomy and ecology of the genus Ralstonia...................................... 18 1.3.2.1 Plant pathogens................................................................................... 19 1.3.2.2 Clinical importance............................................................................. 19 1.3.2.3 Xenobiotics degraders......................................................................... 20 1.3.2.4 Metal resistance.................................................................................. 21 1.3.2.5 Symbiotic nitrogen fixation ................................................................ 21 1.3.2.6 Diversity of the genus Ralstonia......................................................... 21 1.4 Microbiological aspects of bioremediation..................................................... 22 1.4.1 Toxic metals: resistance mechanisms and bioremediation approaches.. 22 1.4.1.1 General bacterial resistance mechanisms against metals.................... 22 1.4.1.2 Metal resistance determinants of R. metallidurans CH34 .................. 22 1.4.1.3 Bioremediation strategies for metal pollution .................................... 24 1.4.2 The degradation of chlorinated aromatics .............................................. 25 1.4.2.1 Introduction......................................................................................... 25 1.4.2.2 General degradation pathways............................................................ 26 1.4.2.3 Degradation of 2,4-dichlorophenoxyacetic acid................................. 28 1.4.2.4 Degradation of chloroanilines............................................................. 30 1.4.3 Bioremediation strategies....................................................................... 32 1.4.3.1 Natural attenuation, biostimulation and bioaugmentation.................. 32 1.4.3.2 Bioaugmentation through horizontal transfer of catabolic genes ....... 34 1.5 Cited literature................................................................................................ 37 INDEX i CHAPTER 2 Goals of the thesis .................................................................................51 CHAPTER 3 Microplate DNA-DNA hybridisations ..................................................53 3.1 Introduction .....................................................................................................53 3.2 Experimental procedure..................................................................................56 3.2.1 Immobilization of DNA in microplate wells...........................................56 3.2.2 Preparation of probe DNA ......................................................................56 3.2.3 Prehybridisation ......................................................................................57 3.2.4 Actual hybridisation................................................................................57 3.2.5 Enzymatic development of the hybridisation reaction............................57 3.2.6 Calculation of reassociation values.........................................................58 3.3 Evaluation of a microplate DNA-DNA hybridisation method compared to the initial renaturation method ..........................................................................................59 3.3.1 Summary .................................................................................................59 3.3.2 Introduction .............................................................................................59 3.3.3 Materials and methods ............................................................................60 3.3.3.1 Strains..................................................................................................60 3.3.3.2 DNA extraction...................................................................................60 3.3.3.3 Determination of the G+C content......................................................60 3.3.3.4 DNA-DNA hybridisation....................................................................61 3.3.3.5 Numerical interpretation.....................................................................61 3.3.4 Results .....................................................................................................61 3.3.4.1 Bacillus / Virgibacillus hybridisations ................................................61 3.3.4.2 Myroides hybridisations......................................................................61 3.3.4.3 Vibrio hybridisations...........................................................................64 3.3.4.4 Xanthomonas / Stenotrophomonas hybridisations..............................64 3.3.4.5 Global comparison..............................................................................64 3.3.5 Discussion ...............................................................................................68 3.4 Further experience with microplate DNA-DNA hybridisations .....................69 3.4.1 Possible sources of error .........................................................................69 3.4.1.1 Effect of DNA quality.........................................................................69 3.4.1.2 Enzymatic development of the hybridisation reaction........................69 3.4.2 Influence of hybridisation temperature ...................................................70 3.4.3 Applications of the microplate DNA-DNA hybridisation technique......71 3.5 Literature cited................................................................................................72 CHAPTER 4 Determination of nucleotide composition via HPLC ............................75 4.1 Introduction .....................................................................................................75 4.2 Experimental procedure..................................................................................77 4.3 Evaluation of the method ................................................................................79 4.3.1 HPLC separation of nucleosides. ............................................................79 4.3.2 Enzymatic degradation of DNA..............................................................80 4.3.3 Quantification of nucleosides and calculation of %G+C........................82 4.4 Conclusions .....................................................................................................84 4.5 Literature cited................................................................................................85 CHAPTER 5 Metal-resistant
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