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.
Table of Contents
1 ABSTRACT
2 ZUSAMMENFASSUNG
3 INTRODUCTION
3.1 GENERAL PROPERTIES OF ARCHAEA (MADIGAN M. T. ET AL. 1997)
3.1.1 The plasma membrane
3.1.2 Cell walls
3.1.3 Transcription and translation
3.2 HALOPHILIC ARCHAEA (MADIGAN M. T. ET AL. 1997)
3.2.1 Systematics of extremely halophilic Archaea (Boone D. R. et al. 2001)
3.2.2 Isolation of Haloarchaea from rock salt
3.3 MICROBIAL SYSTEMATICS AND EVOLUTION (PAGE R. D. M. AND HOLMES E. C. 1998)
3.3.1 Ribosomal RNAs as phylogenetic chronometers
3.3.2 Producing phylogenetic dendrograms with 16S rRNA gene sequences
3.4 ANALYSIS OF 16S RRNA GENE CLONE LIBRARIES (VON WINTZINGERODE, ET AL. 1997)
3.4.1 General features
3.4.2 Factors influencing the reliability of 16S rDNA clone libraries
3.4.2.1 Formation of chimeric molecules
3.4.2.2 Formation of deletion mutants
3.4.2.3 Formation of point mutants
3.4.3 Analysis of 16S rRNA sequence data from complex communities
3.5 MICROBIAL COMMUNITY-DNA ANALYSIS
3.5.1 ARDRA (amplified rDNA restriction analysis) (Fernandez A. et al. 1999)
3.5.2 DGGE (denaturing gradient gel electrophoresis) (Muyzer 1999)
4 OBJECTIVES OF THIS WORK
5 MATERIAL AND METHODS
5.1 MATERIAL
5.1.1 Bacterial and archaeal strains
5.1.2 Kits
5.1.3 Media
5.1.4 Marker
5.2 EXTRACTION OF COMMUNITY DNA
5.2.1 Dissolved rock salt sample (Radax et al. 2001)
5.2.2 Extraction method with filtration (Radax et al. 2001)
5.2.2.1 Buffers and solutions
5.2.3 Novel extraction method with centrifugation (modified from Martin-Laurent, et al. 2001)
5.2.3.1 Buffers and solutions
5.2.3.2 Designations of DNA samples
5.2.4 PCR amplification of 16S rRNA gene fragments (Radax et al. 2001)
5.2.4.1 Preparation of PCR buffer
5.2.5 DNA isolation from cultivated bacteria
5.3 CREATION OF THE 16S RRNA GENE CLONE LIBRARY
5.3.1 PCR amplification of clones
5.3.2 Plasmid preparation
5.4 CHARACTERIZATION OF CLONES
5.4.1 Restriction analyses (ARDRA)
5.4.1.1 Restriction Endonucleases
5.4.1.2 Sample preparation
5.4.2 DGGE
5.4.2.1 Sample preparation
5.4.2.2 DGGE preparation
5.4.2.2.1 DGGE solutions (BioRad)
5.4.2.3 DGGE run (Muyzer 1993)
5.4.2.4 DNA elution from polyacrylamide gels (Rölleke, et al. 1996)
5.4.3 Partial sequence analysis
5.4.3.1 Cycle-sequence-program
5.5 SEQUENCE ANALYSIS
5.5.1 Primers for 16S rDNA of halophilic Archaea (5´ 3´)
5.5.2 Chimera Detection (Larsen, et al. 1993)
5.6 PHYLOGENETIC ANALYSIS (PAGE R. D. M. ET AL. 1998)
5.6.1 Distance methods
5.6.1.1 Algorithms for finding distance trees
5.6.2 Discrete methods
5.6.3 Estimating sampling error: the bootstrap
5.6.4 Phylogenetic analysis of sequences
5.6.5 Accession numbers
6 RESULTS
6.1 DNA EXTRACTION
6.2 CREATION OF CLONE LIBRARY
6.3 CHARACTERIZATION OF 16S RRNA GENE CLONE LIBRARY
6.3.1 ARDRA (amplified rDNA restriction analysis)
6.3.2 DGGE (denaturing gradient gel electrophoresis)
6.3.3 Partial sequences
6.4 TOTAL SEQUENCES AND PHYLOGENETIC CHARACTERIZATION
7 CONCLUSIONS AND DISCUSSION
7.1 CONCLUSIONS
7.2 DISCUSSION
Research Objectives and Core Themes
The research investigates the microbial diversity within Alpine Permo-Triassic rock salt sediments, aiming to characterize the indigenous haloarchaeal community through culture-independent molecular methods. The primary research question centers on determining the extent of halobacterial diversity and investigating whether alternative DNA extraction and characterization techniques can improve the detection of previously unculturable, novel taxa.
- Extraction of community DNA from rock salt using a novel centrifugation-based method.
- Characterization of the 16S rRNA gene clone library using ARDRA and DGGE.
- Phylogenetic analysis of the obtained 16S rRNA gene sequences.
- Evaluation of the reliability and efficiency of different molecular techniques in assessing environmental microbial diversity.
Excerpt from the Book
3.4.2 Factors influencing the reliability of 16S rDNA clone libraries
Wintzingerode et al. (von Wintzingerode et al. 1997) have given a review about specific aspects and pitfalls concerning PCR-based analysis of prokaryotic small-subunit ribosomal RNAs for ecological studies.
Factors identified to change the relative proportion of naturally occurring taxa during the generation of rDNA clone libraries include methodological shortcomings, problems intrinsic to the molecule of choice and problems due to the interpretation of data.
Methodological problems include obtaining and handling of samples. A severe bias is introduced by cell lysis and extraction of DNA since the release of nucleic acids depends markedly on the structure of membranes. On the other hand, rigorous lysis procedures may be detrimental to the intactness of genes, leading to the increased formation of chimeric structures or non-amplification of DNA targets. Bias introduced by PCR amplification includes differences in differential PCR amplification and PCR artefacts (e.g. chimeric structures, formation of deletion mutants and point mutants).
Differences in the clonability may be due to differences in commercially available cloning kits and in the relative ratio of PCR amplicons, such that products representing minor fractions of populations may be suppressed.
Interpretation problems are connected with the analysis of sequences. To avoid these problems, sequences have to be analysed with diverse computer programs (Chimera_Check, structural analysis (Felsenstein J 1993)) and have to be compared to those of cultured strains and clones from other libraries.
Summary of Chapters
1 ABSTRACT: Provides a summary of the methodology and key findings regarding the characterization of haloarchaeal biodiversity in Alpine rock salt.
2 ZUSAMMENFASSUNG: A German language version of the abstract summarizing the research methodology and findings.
3 INTRODUCTION: Explores the classification of the three domains of life and provides background information on haloarchaeal characteristics and molecular methods for identifying microbes.
4 OBJECTIVES OF THIS WORK: Describes the motivation for conducting this research and the specific goals, including testing new DNA extraction and characterization methods.
5 MATERIAL AND METHODS: Details the biological samples used, chemical reagents, extraction protocols, PCR parameters, and statistical/computational tools for phylogenetic analysis.
6 RESULTS: Presents the findings of the study, including successful DNA extraction from specific samples and the resulting 16S rRNA clone library characterization.
7 CONCLUSIONS AND DISCUSSION: Evaluates the implications of the findings, confirming higher-than-expected biodiversity and the utility of the implemented molecular methods.
Keywords
Archaea, Halobacteriaceae, 16S rRNA gene, clone library, DNA extraction, ARDRA, DGGE, rock salt, Bad Ischl, Alpine, molecular biology, phylogenetic analysis, microbial diversity, PCR amplification, uncultured taxa.
Frequently Asked Questions
What is the core focus of this research?
The work primarily focuses on examining and characterizing the microbial diversity—specifically haloarchaea—contained within ancient Alpine Permo-Triassic rock salt.
What are the primary themes addressed?
The central themes include the development of improved DNA extraction methods, the use of culture-independent molecular techniques (ARDRA, DGGE, and 16S rRNA sequencing) to overcome limitations in assessing environmental microbial populations, and phylogenetic characterization of novel, uncultured taxa.
What is the primary objective of this work?
The main goal is to prove that the biodiversity in Permo-Triassic rock salt is much higher than previously thought and to refine methods for identifying microorganisms that are difficult to cultivate through traditional laboratory techniques.
Which scientific methods were employed?
The study utilizes a novel centrifugation-based DNA extraction method, followed by PCR amplification of 16S rRNA genes, clone library construction, amplified rDNA restriction analysis (ARDRA), denaturing gradient gel electrophoresis (DGGE), and comprehensive phylogenetic sequence analysis.
What is covered in the main body of the work?
The main body details the experimental procedures used to extract DNA from rock salt, the steps taken to build and analyze a 16S rRNA gene clone library, and the subsequent efforts to classify the resulting phylotypes using restriction analysis and phylogenetic tree construction.
Which keywords define this work?
Key terms include Archaea, Halobacteriaceae, 16S rRNA gene, clone library, DNA extraction, ARDRA, DGGE, Alpine rock salt, phylogenetic analysis, and uncultured microbial taxa.
Why was the novel centrifugation method used for DNA extraction?
It was implemented to improve the enrichment of cells and overcome the limitations and potential biases introduced by the previously used ultrafiltration method, particularly to better detach cells from sediment particles.
What was the significance of detecting Halococcus in the sample?
This study provided the first molecular evidence for the presence of the genus Halococcus in environmental rock salt samples, after previous investigations had failed to detect it.
- Quote paper
- Dr. Heidemarie Kloninger (Author), 2002, Characterization of a haloarchaeal 16S rRNA gene clone library from Alpine rock salt from Bad Ischl, Austria, Munich, GRIN Verlag, https://www.grin.com/document/25869
