Using a new method to extract total community DNA of Alpine Permo-Triassic rock salt sediment samples, which involved centrifugation instead of ultrafiltration for enrichment of cells and several washing steps for detaching cells from sediment particles, Archaeal biodiversity was re-examined . For this purpose, a 16S rRNA gene clone library was created and archaeal sequences were characterized via phylogenetic analysis of amplified ribosomal DNA restriction analysis (ARDRA) with DdeI and 16S rDNA sequencing as well as denaturing gradient gel electrophoresis analysis (DGGE). Inserts of clones representing ten ARDRA groups were analysed with partial sequence analysis and representatives of different phylogenetic groups were sequenced double stranded. This possibility of rapid characterization of a total clone library via restriction analysis facilitated the detection of low abundance phylotypes. The 16S rRNA gene library contained 11 different phylotypes, all of which belonged to the Halobacteriaceae. Fifty-three partial haloarchaeal sequences were obtained as well as twenty total insert sequences of clones. Four novel phylotypes clustered tightly with four sequence clusters found in a previous study of Archaeal biodiversity of Alpine rock salt (Radax et al. 2001). One sequence (HW 50) had 99.0% identity to Halococcus morrhuae and another (HW 23) had 97% identity to Natronomonas pharaonis. Four phylotypes were closely related to cultured Halobacteriaceae (more than 97% similarity) and to strains isolated from salt mine brines; one phylotype was 94-94.6% similar to mainly alkaliphilic halobacteria. Six phylotypes were only remotely related to cultured Halobacteriaceae (less than 89-92% similarity), suggesting that they represent uncultured novel haloarchaeal taxa in rock salt. The novelty of the sequences suggested also avoidance of haloarchaeal contaminants during preparation of DNA and PCR reactions. The data presented here also indicate that our present view of halobacterial diversity is far from being complete.
Inhaltsverzeichnis (Table of Contents)
- ABSTRACT
- INTRODUCTION
- GENERAL PROPERTIES OF ARCHAEA (MADIGAN M. T. ET AL. 1997)
- The plasma membrane
- Cell walls
- Transcription and translation
- HALOPHILIC ARCHAEA (MADIGAN M. T. ET AL. 1997)
- Systematics of extremely halophilic Archaea (Boone D. R. et al. 2001)
- Isolation of Haloarchaea from rock salt
- MICROBIAL SYSTEMATICS AND EVOLUTION (PAGE R. D. M. AND HOLMES E. C. 1998)
- Ribosomal RNAs as phylogenetic chronometers
- Producing phylogenetic dendrograms with 16S rRNA gene sequences
- ANALYSIS OF 16S RRNA GENE CLONE LIBRARIES (VON WINTZINGERODE, ET AL. 1997)
- General features
- Factors influencing the reliability of 16S rDNA clone libraries
- Formation of chimeric molecules
- Formation of deletion mutants
- Formation of point mutants
- Analysis of 16S rRNA sequence data from complex communities
- MICROBIAL COMMUNITY-DNA ANALYSIS
- ARDRA (amplified rDNA restriction analysis) (Fernandez A. et al. 1999)
- DGGE (denaturing gradient gel electrophoresis) (Muyzer 1999)
- OBJECTIVES OF THIS WORK
- MATERIAL AND METHODS
- MATERIAL
- Bacterial and archaeal strains
- Kits
- Media
- Marker
- EXTRACTION OF COMMUNITY DNA
- Dissolved rock salt sample (Radax et al. 2001)
- Extraction method with filtration (Radax et al. 2001)
- Novel extraction method with centrifugation (modified from Martin-Laurent, et al. 2001)
- Buffers and solutions
- Plasmid preparation
- CREATION OF THE 16S RRNA GENE CLONE LIBRARY
- PCR amplification of 16S rRNA gene fragments (Radax et al. 2001)
- Preparation of PCR buffer
- DNA isolation from cultivated bacteria
- PCR amplification of clones
- Designations of DNA samples
- CHARACTERIZATION OF CLONES
- Restriction analyses (ARDRA)
- Restriction Endonucleases
- Sample preparation
- DGGE
- Sample preparation
- DGGE preparation
- DGGE solutions (BioRad)
- DGGE run (Muyzer 1993)
- DNA elution from polyacrylamide gels (Rölleke, et al. 1996)
- Partial sequence analysis
- Cycle-sequence-program
- SEQUENCE ANALYSIS
- Primers for 16S rDNA of halophilic Archaea (5'⇒3′)
- Chimera Detection (Larsen, et al. 1993)
- PHYLOGENETIC ANALYSIS (PAGE R. D. M. ET AL. 1998)
- Distance methods
- Algorithms for finding distance trees
- Discrete methods
- Estimating sampling error: the bootstrap
- Phylogenetic analysis of sequences
- Accession numbers
- RESULTS
- DNA EXTRACTION
- CREATION OF CLONE LIBRARY
- CHARACTERIZATION OF 16S RRNA GENE CLONE LIBRARY
- ARDRA (amplified rDNA restriction analysis)
- DGGE (denaturing gradient gel electrophoresis)
- Partial sequences
- TOTAL SEQUENCES AND PHYLOGENETIC CHARACTERIZATION
- CONCLUSIONS AND DISCUSSION
- CONCLUSIONS
- DISCUSSION
- CITED LITERATURE
- ABBREVIATIONS
- PUBLICATIONS FROM THIS THESIS
- DANKSAGUNGEN/ACKNOWLEDGEMENTS
- APPENDIX
Zielsetzung und Themenschwerpunkte (Objectives and Key Themes)
This thesis aims to re-examine the archaeal biodiversity in Alpine Permo-Triassic rock salt sediment samples using a new method for extracting total community DNA. This method involves centrifugation instead of ultrafiltration for cell enrichment and multiple washing steps to separate cells from sediment particles.
- Novel DNA extraction method for archaeal community analysis
- Characterization of archaeal biodiversity in Alpine rock salt
- Phylogenetic analysis of 16S rRNA gene sequences
- Comparison of archaeal communities with previous studies
- Identification of potential novel haloarchaeal taxa
Zusammenfassung der Kapitel (Chapter Summaries)
- INTRODUCTION provides a general overview of archaea, including their properties, the systematics of halophilic archaea, and the use of 16S rRNA gene sequences for phylogenetic analysis.
- MICROBIAL SYSTEMATICS AND EVOLUTION discusses the importance of ribosomal RNAs as phylogenetic chronometers and methods for constructing phylogenetic dendrograms.
- ANALYSIS OF 16S RRNA GENE CLONE LIBRARIES outlines the general features of clone libraries and factors that can influence their reliability.
- MICROBIAL COMMUNITY-DNA ANALYSIS introduces two techniques for analyzing microbial communities: amplified rDNA restriction analysis (ARDRA) and denaturing gradient gel electrophoresis (DGGE).
- OBJECTIVES OF THIS WORK clearly states the goals of the study.
- MATERIAL AND METHODS details the materials, methods, and procedures used in the research, including DNA extraction, creation of the 16S rRNA gene clone library, characterization of clones, sequence analysis, and phylogenetic analysis.
Schlüsselwörter (Keywords)
The primary keywords and focus topics of this work include archaeal biodiversity, halophilic archaea, 16S rRNA gene, clone library, phylogenetic analysis, ARDRA, DGGE, Alpine rock salt, novel taxa, and DNA extraction methods.
- Arbeit zitieren
- Dr. Heidemarie Kloninger (Autor:in), 2002, Characterization of a haloarchaeal 16S rRNA gene clone library from Alpine rock salt from Bad Ischl, Austria, München, GRIN Verlag, https://www.grin.com/document/25869
