DIPLOMARBEIT Stable isotope probing-based ecophysiological analysis of sulphate-reducing microorganisms in an acidic fen angestrebter akademischer Grad Magister der Naturwissenschaften (Mag. rer.nat.) Verfasser: Norbert Bittner Matrikel-Nummer: 0040188 Studienrichtung(lt.Studienblatt): Molekulare Biologie Betreuer: O. Univ. Prof. Dr. Michael Wagner Wien, Jänner 2010 Table of contents 1 Introduction............................................................................................................. 1 1.1 The sulphur cycle .............................................................................................. 1 1.2 Physiology and phylogeny of SRP ................................................................... 2 1.3 Dissimilatory reduction of sulphate .................................................................. 5 1.4 Habitats of SRP................................................................................................. 6 1.5 Wetlands, climate and SRP ............................................................................... 7 1.6 Aim of this study .............................................................................................. 8 2 Material and methods ........................................................................................... 10 2.1 Provided samples ............................................................................................ 10 2.1.1 Sampling site .................................................................................................. 10 2.1.2 Sample incubation .......................................................................................... 10 2.1.3 Soil DNA and RNA extraction ....................................................................... 11 2.2 General material and methods ........................................................................ 11 2.3 Buffers, media and solutions .......................................................................... 15 2.3.1 General solutions ............................................................................................ 15 2.3.2 General solutions for ultracentrifugation ........................................................ 15 2.3.3 Buffers, solutions and standards for gel electrophoresis ............................... 15 2.3.4 Culture media for Escherichia coli strains .................................................... 16 2.3.5 Culture media for Halobacterium salinarum ................................................ 17 2.4 Methods .......................................................................................................... 18 2.4.1 Quantitative analysis of nucleic acids ............................................................ 18 2.4.1.1 Nanodrop analysis........................................................................................... 18 2.4.1.2 Picogreen analysis ........................................................................................... 18 2.4.2 Qualitative nucleic acid analysis by agarose gel electrophoresis ................... 18 2.4.2.1 Qualitative nucleic acid fragment analysis ..................................................... 18 2.4.2.1 Qualitative nucleic acid fragment analysis followed by gel extraction .......... 19 2.4.3 Amplification of specific nucleic acid fragments by PCR ............................. 19 2.4.3.1 Amplification of 16S rRNA genes .................................................................. 19 2.4.3.2 M13 screening PCR ........................................................................................ 21 2.4.3.3 Touchdown hot-start PCR amplification of dsrAB ........................................ 22 2.4.3.4 Amplification of reverse transcribed 16S rRNA fragments by RT-PCR ....... 24 2.4.4 Isopycnic centrifugation of nucleic acids ....................................................... 25 2.4.4.1 Refractrometric density measurements ........................................................... 25 2.4.4.2 Isopycnic centrifugation of DNA ................................................................... 25 2.4.4.3 Isopycnic centrifugation of RNA .................................................................... 26 2.4.5 Nucleic acid precipitation ............................................................................... 26 2.4.5.1 DNA precipitation out of CsCl gradient medium .......................................... 26 2.4.5.2 RNA precipitation out of CsTFA gradient medium ...................................... 26 2.4.6 Terminal restriction fragment length polymorphism analysis ....................... 26 2.4.7 Cloning of PCR products ............................................................................... 28 2.4.7.1 Cloning of 16S rRNA PCR products ............................................................. 28 2.4.7.2 Cloning of dsrAB amplicons .......................................................................... 28 2.4.8 DNA sequencing ............................................................................................ 29 2.4.9 Comparative sequence analysis ..................................................................... 29 2.4.9.1 Sequence alignement ..................................................................................... 29 2.4.9.2 Phylogeny ...................................................................................................... 30 2.4.9.3 Test for chimeric sequences ........................................................................... 31 2.4.9.4 Rarefaction ..................................................................................................... 31 2.4.9.5 Statistical comparison of different habitats ................................................... 31 3 Results ..................................................................................................................... 32 3.1 Gradient centrifugation of nucleic acids ....................................................... 32 3.2 T-RFLP analysis of density resolved nucleic acids ....................................... 34 3.2.1 Bacterial T-RFLP analysis of DNA SIP incubations .................................... 34 4 3.2.2 Archaeal T-RFLP analysis of DNA SIP incubations ..................................... 40 3.2.3 Bacterial T-RFLP analysis of RNA SIP incubations ..................................... 42 3.3 Amplification, cloning and sequencing of 16S rRNA genes and dsrAB ....... 43 3.4 Sequence analysis .......................................................................................... 44 3.4.1 Bacterial 16S rRNA gene analysis ................................................................. 44 3.4.2 DsrAB analysis .............................................................................................. 48 4 Discussion ............................................................................................................... 50 5 Summary ................................................................................................................ 57 6 Literature ................................................................................................................ 59 7 List of abbreviations .............................................................................................. 69 8 Appendix ................................................................................................................. 71 8 Appendix ................................................................................................................. 71 9 Curriculum vitae .................................................................................................... 82 10 Acknowledgements ................................................................................................ 83 5 Introduction 1 Introduction 1.1 The sulphur cycle Besides carbon (C), nitrogen (N) and phosphorus (P), sulphur (S) is one of the main elements found in biomass. It is an essential constituent of many enzymes, vitamins and hormones. Sulphur is present in many different oxidation states, ranging from −2 (H2S) 2− to +6 (SO4 ), being constantly transformed chemically and biologically. Most of earths sulphur is present in rocks and sediments like pyrite (FeS2) or gypsum (CaSO4). As geo- logical turnover times are long and these sulphur-species are biologically inaccessible, most of the biologically used sulphur can be found in the form of sulphate ions and hy- drogen sulphide, playing essential roles in the biological sulphur cycle as illustrated in Fig.1 (Hollemann and Wiberg 1985). Fig.1 Biological sulphur cycle (modified from Madigan 2003). Several plants, fungi and prokaryotes can use sulphate to build up the essential sulphur containing amino acids cystein and methionine through assimilatory sulphate reduction. Many organisms then use these amino acids for incorporation in their biomass. Beside the anabolic utilisation of these amino acids, many microorganisms remineralise them 1 Introduction to sulphide via desulphurylation, which is then converted back to sulphate by sulphur- oxidizing bacteria. Two distinct prokaryotic groups have the ability to oxidize sulphide. One group are anaerobic living, phototrophic sulphur bacteria, the others are aerobic chemolithotrophic bacteria (Madigan et al. 2003). Furthermore, elemental sulphur can be oxidised by some prokaryotes, like Desulfocapsa sulfoexigens, resulting in sulphate and closing the sulphur cycle (Frederiksen and Finster 2004). Alternatively, sulphate, sulphite and thiosulphate