Anaerobic pathways in the Porifera: Strombine dehydrogenase, an opine dehydrogenase, from the sponge Suberites domuncula Dissertation zur Erlangung des Grades Doktor der Naturwissenschaften Am Fachbereich Biologie der Johannes Gutenberg-Universität in Mainz Bruna Pleše geb. in Zagreb (Croatia) Mainz, 2007 Dekan: 1. Berichterstatter: 2. Berichterstatter: Tag der mündlichen Prüfung: Mojoj Baki i Čiki 1 INTRODUCTION .........................................................................................................1 1.1 Sponges (Porifera)................................................................................................1 1.1.1 The origins ...........................................................................................................1 1.1.2 Morphology...........................................................................................................4 1.1.3 Reproduction.........................................................................................................5 1.1.4 Classification.........................................................................................................6 1.1.5 Symbiosis..............................................................................................................7 1.2 Anaerobic pathways............................................................................................10 1.2.1 Evolution of anaerobic pathways........................................................................12 1.2.2 Opines .................................................................................................................14 2 AIM OF THE STUDY...................................................................................................18 3 MATERIALS AND METHODS...................................................................................19 3.1 Materials .............................................................................................................19 3.1.1 Chemicals............................................................................................................19 3.1.2 Equipment...........................................................................................................20 3.1.3 Enzymes and Antibodies.....................................................................................21 3.1.4 Markers ...............................................................................................................21 3.1.5 Kits......................................................................................................................22 3.1.6 Vectors ................................................................................................................22 3.1.7 Primers ................................................................................................................22 3.1.8 Bacterial strains...................................................................................................23 3.1.9 Culture medium ..................................................................................................24 3.1.10 Computer programs ............................................................................................25 3.2 Experimental animals..........................................................................................25 4 Methods ..............................................................................................................26 4.1 DNA extraction from tissue................................................................................26 4.2 Phenol-chloroform DNA extraction ..................................................................27 4.3 DNA precipitation...............................................................................................27 4.4 Restriction digestion of DNA .............................................................................27 4.5 RNA isolation.....................................................................................................28 4.5.1 RNAse inactivation using DEPC (diethylpyrocarbonate) ................................28 4.6 Southern Blotting ...............................................................................................28 4.7 Screening of the S. domuncula genomic library .................................................30 4.7.1 Plating out the library and transferring the lambda phage to nylon membranes 32 4.7.2 Lambda DNA extraction.....................................................................................33 4.8 Isolation of plasmid DNA ..................................................................................34 4.9 Agarose gel electrophoresis ..............................................................................34 4.10 Isolation of DNA from agarose gel.....................................................................35 4.11 Polyacrylamide gel..............................................................................................35 4.12 Photometric measurements .................................................................................36 4.13 Cloning................................................................................................................36 4.13.1 A-Tailing of DNA for TA ligation and cloning..................................................36 4.13.2 TOPO TA cloning...............................................................................................37 4.13.3 pGEM-T cloning.................................................................................................37 4.14 Transformantion of competent cells .................................................................38 4.15 Optic density (OD) of bacterial cultures.............................................................40 4.16 Generation of primers.. .......................................................................................40 4.17 PCR (Polymerase Chain Reaction).....................................................................41 4.17.1 Long and accurate (LA) PCR .............................................................................42 4.17.2 Touch down PCR................................................................................................43 4.17.3 DIG labeling PCR...............................................................................................43 4.17.4 Checking PCR.....................................................................................................45 4.17.5 Semi-quantitative RT-PCR.................................................................................45 4.18. DNA Sequencing................................................................................................46 4.19 Sequence analysis ...............................................................................................47 4.20 Isolation of total protein extract..........................................................................48 4.21 SDS-polyacrylamid gel electrophoresis (SDS-PAGE).......................................48 4.21.1 Native/Seminative PAGE ...................................................................................50 4.21.2 Coomassie staining with GelCode® Blue stain reagent ......................................51 4.21.3 Measuring protein concentration (Bradford) ......................................................51 4.21.3.1 Protein Standards ................................................................................................51 4.22 Western Blot.......................................................................................................52 4.23 Recombinant His-tag fusion protein ...................................................................53 4.23.1 Expression of His-tag fusion protein ..................................................................53 4.23.2 Determination of target protein solubility...........................................................54 4.23.3 Purification of 6xHis-tagged fusion proteins using Ni-NTA spin columns .......55 4.24 Antibody production ...........................................................................................57 4.25 Immunohistochemistry .......................................................................................58 4.26 ELISA (Enzyme Linked Immunosorbent Assay)...............................................59 4.27 Enzyme assays ....................................................................................................61 4.27.1 Substrate Saturation of the Enzyme....................................................................63 4.28 Protein modeling.................................................................................................64 5 RESULTS. .....................................................................................................................65 5.1 Cloning of the cDNA encoding the putative tauropine dehydrogenase (TaDH)65 5.2 Sequence analysis of S. domuncula TaDH-like protein......................................67 5.3 Ornithine dehydrogenases in sponge-associated bacteria...................................68 5.4 Cloning of the gene encoding the S. domuncula TaDH- like protein.................71 5.5 Allelic variations.................................................................................................78 5.6 Semi-quantitative RT-PCR analysis: effect of oxygen on TaDH-like gene expression ...........................................................................................................80 5.7 Recombinant