Molecular evidence for the antiquity of group I introns interrupt- ing transfer RNA genes in cyanobacteria Dissertation zur Erlangung des Doktogrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg – August – Universität zu Göttingen vorgelegt von David Fewer M.Sc. aus Waterford/Irland Göttingen 2001 D7 Referent: Prof. Dr. Thomas Friedl Korreferent: Prof. Dr. Burkhard Büdel Tag der mündlichen Prüfung: Contents ii Contents Abstract ...............................................................................................................................................1 1. Introduction ....................................................................................................................................2 1.1 Cyanobacteria fossil record..........................................................................................................2 1.2 Cyanobacteria systematics ..........................................................................................................3 1.3 Molecular evolution of cyanobacteria .........................................................................................4 1.4 Group I introns interrupting tRNA genes in cyanobacteria ........................................................5 1.5 Aims of thesis ..............................................................................................................................7 2. Materials and Methods ................................................................................................................8 2.1 General Materials and Methods ..............................................................................................8 2.1.1 Origin of the strains of cyanobacteria and eukaryotes used in this study .............................8 2.1.2 DNA extraction .....................................................................................................................8 2.1.3 PCR conditions ......................................................................................................................8 2.1.4 Cloning of PCR products ......................................................................................................10 2.1.5 Automated sequencing ..........................................................................................................10 2.1.6 Phylogenetic analyses ............................................................................................................10 2.1.7 Statistical tests .......................................................................................................................11 2.2 Materials and Methods for section 3.1. ...................................................................................11 2.2.1 PCR Amplification of the 16S rRNA, rpoC1 and tufA genes ...............................................11 2.2.2 Large 16S rRNA datasets ......................................................................................................12 2.2.3 Reduced Chroococcidiopsis 16S rRNA dataset ....................................................................13 2.2.4 Individual analyses of the genes comprising the combined dataset ......................................14 2.2.5 Analyses of the combined dataset .........................................................................................16 2.2.6 Statistical tests .......................................................................................................................16 2.3 Materials and Methods for section 3.2. ...................................................................................16 2.3.1 Systematic survey of cyanobacteria and chloroplasts ............................................................16 2.3.2 Large 16S rRNA dataset.........................................................................................................17 2.3.3 The tRNA-fMet intron dataset ..............................................................................................18 2.3.4 Reduced 16S rRNA dataset for congruency tests .................................................................18 2.3.5 Host and intron divergences ..................................................................................................18 2.3.6 Statistical tests .......................................................................................................................19 2.4 Materials and Methods for section 3.3. ...................................................................................19 2.4.1 PCR amplification and sequencing of the divergent intron ...................................................19 2.4.2 Culturing experiments ...........................................................................................................19 2.4.3 DNA extraction .....................................................................................................................20 2.4.4 Alignment of introns interrupting tRNA genes .....................................................................20 2.4.5 Distribution of the tRNA-Arg intron and the phylogenetic position of contaminant.............21 2.4.6 Congruency between 16S rRNA and tRNA-Arg intron ........................................................21 4.4.7 Statistical tests ........................................................................................................................22 2.5. Materials and Methods for section 3.4. ..................................................................................22 2.5.1 Systematic survey of cyanobacteria and chloroplasts ...........................................................22 2.5.2 Large 16S rRNA dataset ........................................................................................................22 2.5.3 The tRNA-Leu (UAA) intron dataset.....................................................................................23 2.5.4 Reduced 16S rRNA dataset for congruency tests ..................................................................23 2.5.5 Statistical tests .......................................................................................................................24 2.6. Materials and Methods for section 3.5. ..................................................................................24 2.6.1. Systematic survey of plastid containing eukaryotes .............................................................24 2.6.2. Intron alignment ...................................................................................................................24 2.6.3. rbcL and intron alignment ....................................................................................................25 2.6.4. Nuclear encoded 18S rRNA alignment ................................................................................25 2.6.5. Plastid encoded 16S rRNA and intron alignment .................................................................26 2.6.6. In vitro transcription and intron splicing ..............................................................................26 2.6.7. Northern analysis ..................................................................................................................27 3. Results and discussion .................................................................................................................28 3.1. Chroococcidiopsis and the heterocysts differentiating cyanobacteria are each others 28 closest living relatives ...................................................................................................................... 3.1.1 Abstract .................................................................................................................................28 3.1.2 Introduction ...........................................................................................................................28 3.1.3 Results and discussion ...........................................................................................................30 3.1.3.1 Phylogenetic analyses of the 16S rRNA dataset ..............................................................30 3.1.3.1 Phylogenetic analyses of the combined dataset ...............................................................39 3.2. Phylogenetic evidence for the antiquity of the intron interrupting the initiator tRNA 41 gene in cyanobacteria ...................................................................................................................... 3.2.1 Abstract ..................................................................................................................................41 3.2.2 Introduction ............................................................................................................................41 3.2.3 Results and discussion............................................................................................................44 3.2.3.1 Scattered and sporadic distribution of the intron..............................................................44 3.2.3.2 The open reading frames are recent acquisitions .............................................................49 3.2.3.3 Instances of congruence between the host and intron ......................................................54 3.2.3.4 The antiquity of the tRNA-fMet intron ............................................................................56 3.3 Phylogenetic analyses does not support the horizontal transfer of a group I intron from 57 α-proteobacteria to cyanobacteria.................................................................................................