Characterization of a haloarchaeal 16S rRNA gene clone library from Alpine rock salt from Bad Ischl, Austria

Inhaltserzeichnis

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 30
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

8 CITED LITERATURE

9 ABBREVIATIONS

10 PUBLICATIONS FROM THIS THESIS

11 DANKSAGUNGEN/ACKNOWLEDGEMENTS

12 APPENDIX

1 Abstract

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.

2 Zusammenfassung

Mit einer neuen Methode zur Extraktion der Gesamt DNA aus Proben von Alpinem Permo-Triassischem Steinsalz-Sediment, welche Zentrifugation anstelle von Ultrafiltration zur Zellanreicherung und wiederholten Waschschritten zur Ablösung der Zellen von Sedimentpartikeln beinhaltet, wurde die Biodiversität von Haloarchaea untersucht. Für diesen Zweck wurden eine 16S rRNA Gen Klonbank erstellt und die Sequenzen mittels amplifizierter ribosomaler DNA Restriktionsanalyse (ARDRA) mit dem Enzym Dde I und phylogenetischer Analysen der 16S rDNA sowie mittels denaturierender Gradienten Gel Elektrophorese (DGGE) charakterisiert.

Inserts der Klone, die zehn ARDRA Gruppen repräsentierten, wurden mit partiellen Sequenzen analysiert, und Repräsentanten verschiedener Phylotypen wurden doppelsträngig sequenziert.

Die 16S rRNA Gen Klonbank bestand aus 11 unterschiedlichen Phylotypen, die alle zu den Halobacteriaceae gehören. 53 Teilsequenzen sowie 20 totale Insertsequenzen der Klone wurden analysiert.

Vier neue Phylotypen zeigten hohe Ähnlichkeiten mit 4 Sequenz-Clustern aus einer früheren Studie der Biodiversität von Archaea in Alpinem Steinsalz (Radax et al. 2001).

Eine Sequenz (HW50) zeigte 99.0% Identität zu Halococcus morrhuae und eine weitere (HW23) 97% Identität zu Natronomonas pharaonis.

Vier Phylotypen waren sehr eng zu kultivierten Halobacteriaceae (mehr als 97% Sequenzidentität) und zu isolierten Stämmen aus salt mine brines verwandt; einer jener Phylotypen zeigt 94-94.6% Identität zu alkaliphilen Halobakterien.

Sechs Phylotypen waren nur entfernt mit kultivierten Halobacteriaceae verwandt (weniger als 89-92% Sequenzidentität), was vermuten läßt, daß diese neue unkultivierte Taxa in Steinsalz repräsentieren.

Die Neuheit der Sequenzen bestätigt, daß Kontaminationen während der Preparation der DNA und während der PCR Reaktionen vermieden wurden.

Die hier präsentierten Daten zeigen auch, daß unser derzeitger Informationsstand über die Biodiversität in Alpinem Steinsalz noch lange nicht vollständig ist.

3 Introduction

To divide the living world, biology makes a distinction between prokaryotic and eukaryotic cells. The key difference between these two is that eukaryotes contain a nucleus, and they may also contain organelles in addition. Differences in the nature of ribosomes, of the enzymes contributing to the synthesis of DNA, RNA and proteins, and of the cell walls are further distinctive features. Bacteria and cyanobacteria are prokaryotes, while all other organisms known to date are eukaryotes.

In 1977, C.R. Woese (Woese and Fox 1977) postulated the archaebacteria as the third kingdom of life. The classification was based on the comparison of sequence analysis of the 16S ribosomal RNA. It was supported by many features including the unique membrane composition and the cell wall structures of the archaebacteria. So the eukaryote- prokaryote dichotomy was replaced by a division into three new domains, Bacteria, Eucarya and Archaea, based on the comparison of biochemical and molecular features.

illustration not visible in this excerpt

Fig.1: Universal phylogenetic tree, deduced from 16S rRNA sequences. Taken from Brock Biology of Microorganisms (Madigan M. T. et al. 1997).

3.1 General properties of Archaea (Madigan M. T. et al. 1997)

Most of the Archaea live under extreme environmental conditions. They can be divided into three groups, Euryarcheota, Korarcheota and Crenarchaeota.

Closest to the root of the tree is the Korarcheota kingdom, a group of not yet cultured hyperthermophilic Archaea.

Also quite close to the root are the hyperthermophiles, including methanogens and several sulfur-reducing hyperthermophiles (Crenarchaeota).

Going up the tree, several phylogenetically distinct groups of methanogenes branch off, including the Methanococcus group, the Methanobacterium group and the Methanosarcina-Methanospirillum group. The extreme halophilic Archaea are a group unto themselves, as are the acidophilic, thermophilic cell-wall-less prokaryote Thermoplasma. Collectively, these groups make up the kingdom Euryarcheota.

3.1.1 The plasma membrane

Polar membrane lipids of Archaea are chemically unique, because instead of fatty acids, as in eubacterial and eukaryotic ester lipids, phytanes or biphytanes are bound to glycerol by a sn -2,3 ether linkage.

Two phytanyl chains can be bound head-to-head, thus forming dibiphytanyldiglycerol tetraethers which form lipid monolayers instead of bilayer structures. The length of the biphytanes can be altered by the production of pentacyclic rings without changing the number of C atoms. These tetraether structures can only be found in thermoacidophilic Archaea and some methanogenes.

The ether lipids account for 80-95% of the membrane lipids. The remaining 5-20% are neutral squalenes and other isoprenoids. C40 and C50 carotinoides are also components of the membranes of halophilic archaea.

3.1.2 Cell walls

Muramic acid and D -amino acids, two of the major compounds of eubacterial peptidoglycan, are not present in archaeal cell walls. Instead some Archaea contain pseudopeptidoglycan, a polymer of alternating repeats of N -acetylglucosamine and N - acetyltalosamin uronic acid linked by a β1-3 bond, as found in Methanobacterium sp. The latter amino sugars are cross-linked by several L -amino acids.

Halococcus and Methanosarcina cell walls consist of heteropolysaccharide only. Most of the other Archaea produce surface layers made of glycoprotein or protein subunits, which are often hexagonally structured. Methanospirillum and Methanotrix possess sheath-like protein cell walls. Thermoplasma does not have a cell wall.

3.1.3 Transcription and translation

Eubacterial DNA-dependent RNA polymerases consist of four different subunits. The structure of archaeal RNA polymerases indicates a closer relationship to eukaryotic RNA polymerases. They are composed of about 10-11 subunits and only the latter enzymes are inhibited by rifampicin and streptolydigin, and only archaeal and eukaryotic RNA polymerases are stimulated by silybin.

The archaeal translatory apparatus shares some common features with both eubacterial and eukaryotic ones. Like the eubacterial ribosomes, archaeal ribosomes also sediment at 70S. Inhibitory patterns indicate a closer relationship to eukaryotic 80S ribosomes. In contrast to elongation factors present in mitochondria and eubacteria, the archaeal elongation factor EF-II and the eukaryotic EF-G can be ADP-ribosylated by fragment A of the diphteria toxin.

3.2 Halophilic Archaea (Madigan M. T. et al. 1997)

Extremely halophilic Archaea are a group of prokaryotes that inhabit highly saline environments such as solar salt evaporation ponds and natural salt lakes, or artificial saline habitats such as the surfaces of heavily salted foods. Such habitats are often called hypersaline. The term extreme halophile is used to indicate not only that these organisms are halophilic, but also that their requirement for salt is very high, in some cases near that of saturation. A generally accepted definition of an extreme halophile is that the organism requires at least 1.5 M (about 9%) NaCl for growth, but most species require 2-4 M (12- 23%) for optimal growth. Virtually all extreme halophiles can grow at 5.5 M NaCl (32%, the limit of saturation for NaCl), although some species grow only very slowly at this salinity.

To compensate for the high salt concentration in the environment, halobacteria accumulate K+ ions (up to 4.6 M). Osmotic stability can be supported by production of organic compatible solutes. With respect to their requirements for magnesium ions, halobacteria can differ widely. Alkaliphilic halobacteria need only traces of these ions, H. volcanii and H. sodomense cells have a high requirement for magnesium ions.

The often reddish colour of habitats of extreme halophiles is due to carotinoid pigments (bacterioruberins and β-carotene) also present in halobacterial membranes.

In some halobacteria additional pigments can be found. Bacteriorhodopsin is a light-driven proton pump that produces a membrane potential which can drive ATP synthesis. Halorhodopsin (a chloride pump) and a sensory rhodopsin which is involved in phototaxis are also present in some halobacteria.

All known halophilic Archaea stain gram negatively, reproduce by binary fission, and are not known to form resting stages or spores.

Most Halobacteria are nonmotile, but a few strains are weakly motile by lophotrichous flagella.

The genomic organisation of Halobacterium and Halococcus is highly unusual in that large plasmids containing up to 25-30% of the total cellular DNA are frequently present and the GC base ratio of these plasmids (57-60%GC) is significantly different from that of chromosomal DNA (66-68%GC).

3.2.1 Systematics of extremely halophilic Archaea (Boone D. R. et al. 2001)

16S ribosomal ribonucleic acid gene sequencing and other studies have defined eight genera of extreme halophiles: Halobacterium, Halococcus, Haloferax, Haloarcula, Halorubrum, Halobaculum, Halogeometricum, Natronorubrum, Haloterrigena, Natrinema, Natrialba, Natronomonas, Natronobacterium and Natronococcus. The extremely halophilic Archaea are frequently referred to collectively as “halobacteria”, because the genus Halobacterium was the first in this group to be described and is still the best-studied representative of the group. Natronobacterium and Natronococcus differ from other extreme halophiles in being alkaliphilic as well as halophilic. Natronobacteria contain unusual diether lipids not found in other extreme halophiles and cluster tightly as a phylogenetic group.

3.2.2 Isolation of Haloarchaea from rock salt

Growing interest is emerging in the exploration of microbial life in subterranean environments, such as deep sub-sea floor sediments, crustal rocks, sedimentary rocks, and also ancient salt deposits (Pedersen 2000). It was estimated that the total amount of carbon in the “intraterrestrial” procaryotic mass on Earth may be as large or even exceed that of plants and prokaryotes growing on the surface of the Earth. As reviewed by McGenity, (McGenity , et al. 2000) Dombrowski and Reiser & Tasch were the first to describe viable microorganisms which were isolated from ancient rock salt.

In 1994 an archaeon which was designated H. salifodinae BIp was isolated from Permian rock salt collected in a salt mine in Bad Ischl, Austria (Denner et al. 1994). Since H. salifodinae BIp included initially only one strain, additional salt samples from the same site were obtained and several halococci which proved to be identical to H. salifodinae were recovered (Stan-Lotter et al. 1999). These taxonomic data demonstrated clearly that the coccoid salt mine isolates were representatives of the species Halococcus salifodinae. Thus it was demonstrated that in geographically separated halite deposits of similar geological age, identical species of halococci are present.

In general, haloarchaeal colonies grew rather slowly on agar plates. Sometimes it took several month of incubation before they became visible, possibly because haloarchaea in the rock salt were in a state of starvation, from which they had to recover. The colonies analyzed varied in shape, size and surface structure, suggesting a pronounced microbial diversity in this unique habitat.

3.3 Microbial systematics and evolution (Page R. D. M. and Holmes E. C. 1998)

Certain cellular macromolecules are evolutionary chronometers - actual measures of evolutionary change. From studies on the sequence of monomers on certain informational macromolecules, it has been shown that the evolutionary distance between two species can be measured by differences in the nucleotide or amino acid sequence of homologous macromolecules from the two species. This is so because the number of sequence differences in a molecule is believed to be proportional to the number of stable mutational changes fixed in the DNA encoding that molecule in species that diverged from a common ancestor. As different mutations become fixed in different populations, biological evolution results.

In order to determine true evolutionary relationships between two species, it is essential to choose the correct molecules for sequence studies. The molecule should be universally distributed across the group chosen for study and it must be functionally homologous in each organism; phylogenetic comparison must start with molecules of identical function. Thirdly, it is crucial in sequence comparisons to be able to properly align the two molecules in order to identify regions of sequence homology and sequence heterogeneity. Finally, the sequence of the molecule chosen should change at a rate commensurate with the evolutionary distance measured.

3.3.1 Ribosomal RNAs as phylogenetic chronometers

Ribosomal RNA genes are used for identification and phylogenetic characterisation because they are ubiquitous, their functions are conserved (because of the likely antiquity of the protein-synthesising process), they are moderately well conserved across broad phylogenetic distances, and a large database (Ribosomal Database Project RDP) is available for sequence alignment and identification. Also because the number of different possible sequences of large molecules as ribosomal RNAs is so extensive, similarity in two sequences indicates some phylogenetic relationship. The degree of similarity in rRNA sequences between two organisms yields their phylogenetic relationship. From comparative sequence analysis molecular trees can be constructed, leading to phylogenetic dendrograms and showing the true evolutionary positions of organisms to one another.

There are three ribosomal RNA molecules, which in prokaryotes have size of 5S, 16S, and 23S. The large rRNAs 16S and 23S contain several regions of highly conserved sequence for proper sequence alignment and sufficient sequence variability in other regions of the molecule to serve as excellent phylogenetic chronometers.

3.3.2 Producing phylogenetic dendrograms with 16S rRNA gene sequences

For comparative sequence analysis of amplified 16S rRNA gene sequences and for construction of phylogenetic trees there are different algorithms available.

First of all sequences are aligned with a sequence editor, where novel data are aligned to known sequences. Then comparative analysis is carried out with distance or discrete methods.

With distance methods phylogenetic or evolutionary distances are revealed with computer analysis by counting every position in which there is a difference in the sequence. From these data a matrix of the phylogenetic differences between two sequences can be constructed. Next the data set is correlated with a proof factor considering statistical probabilities. At last a phylogenetic tree is calculated, in which the length of the lines is proportional to the phylogenetic distance.

In contrast to distance methods, discrete methods operate directly on the sequences, or on functions derived from sequences, rather than on pairwise distances. Hence they endeavour to avoid the loss of information that occurs when sequences are converted into distances. The two major discrete methods are maximum parsimony and maximum likelihood. Maximum parsimony chooses the tree that requires the fewest evolutionary changes. Maximum likelihood chooses the tree that from all possible trees is the one that is most likely to have produced the observed data.

The data given from maximum parsimony comprise individual nucleotide sites. For each site the goal is to reconstruct the evolution of that site on a tree subject to the constraint of invoking the fewest possible evolutionary changes.

The principle of maximum likelihood suggests that the explanation that makes the observed outcome the most likely, so the most probable occurrence is one to be preferred. The tree that makes the data the most probable evolutionary outcome is the maximum likelihood estimate of the phylogeny.

3.4 Analysis of 16S rRNA gene clone libraries (von Wintzingerode et al. 1997)

3.4.1 General features

Analysis of 16S rRNA (as well as 18S rRNA) from clone libraries was introduced in molecular microbial studies. C. Woese initiated the study of rRNA in 1970´s because they are among the most evolutionarily conserved macromolecules in all living systems and large portions of their sequences are well conserved.

16S rRNA genes are large enough to provide enough information (nucleotides) for comparison and small enough to be conveniently analysed (1500 nt). Large amounts of these macromolecules are produced in growing cells. Sequences led to the development of oligonucleotide probes and PCR primers used in the determination of community complexity.

The amplified 16S rRNA genes (16S rDNA) approach combined with other molecular techniques can rapidly evaluate gross similarities and differences within complex microbial communities. It provides a rapid means of identifying bacterial isolates and it can detect and identify microorganisms that are no longer viable or culturable.

illustration not visible in this excerpt

Fig.2: Secondary structure of 16S subunit ribosomal RNA (rRNA) of E.coli. Taken from Brock Biology of Microorganisms (Madigan M. T. et al. 1997).

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.

3.4.2.1 Formation of chimeric molecules

In vitro recombination of homologous DNA leading to chimeric molecules composed of parts of two different sequences has been widely observed and is not restricted to 16S rDNA amplification from complex microbiota. Chimeras between two different DNA molecules with high sequence similarity can be generated during the PCR process as DNA strands compete with specific primers during the annealing step.

In addition to incomplete strand synthesis during the PCR process DNA damage has been suggested to promote the formation of chimeric molecules in PCR co-amplification of templates with high sequence similarities (Paabo et al. 1990).

DNA damage during DNA preparation and cell lysis from environmental samples were shown to support production of recombinant PCR products.

3.4.2.2 Formation of deletion mutants

PCR templates containing stable secondary structures often yield very low amplification efficiency or deletion mutagenesis in PCR products. As ribosomal RNA´s usually exhibit some secondary structures, RT (reverse transcriptase)-PCR could lead to deletion mutants, which could be excluded from subsequent analysis as amplified 16S rRNA genes are often size selected to avoid cloning or gel electrophoresis of non-specific amplicons.

3.4.2.3 Formation of point mutants

Since the first application in PCR reactions it is known that some polymerases, which lack in proofreading activity are known to have an intrinsic misincorporation rate during strand synthesis, which can lead to base substitutions. But the small error rate seems to have little impact on the phylogenetic evaluation of PCR-amplified 16S rRNA genes since the maximum misincorporation rate would lead to 0.3% sequence divergence (in about 1500bp).

3.4.3 Analysis of 16S rRNA sequence data from complex communities

The goal of a PCR-mediated analysis of 16S rRNA molecules from complex microbiota is the retrieval of sequence information, which allows determination of microbial diversity, by comparative 16S rRNA sequence analysis.

The quality of results obtained by comparative sequence analysis depends on the available data set. The number of sequences reflects only a minor part of the expected microbial diversity. 16S rRNA genes retrieved from environmental samples often exhibit a low sequence similarity to known sequences making their phylogenetic affiliation difficult. This leads to the question if environmental sequence represent unculturable, novel microorganisms.

As explained above, amplification of a mixture of 16S rRNA genes may lead to the formation of chimeric molecules. These chimeras have to be recognized and excluded from further phylogenetic analysis. Several methods have been developed for the recognition of chimeric 16S rRNA genes, which rely on checking the secondary structures and performing comparative sequence analysis of different fragments of the sequence (pairwise similarity analysis).

3.5 Microbial community-DNA analysis

The application of culture-independent techniques based on molecular biological methods, especially on the PCR amplification of 16S rRNA genes, attempts to overcome some shortcomings of conventional cultivation methods and reveals far more complex bacterial and archaeal communities than can be shown by cultivation methods, because cultivation dependent methods intrinsically favour the growth of specific community members (Amann et al. 1995) One of the major challenges of investigating microbial growth by molecular means is the extraction of DNA.

Thus, molecular nucleic acid-based characterisation techniques are a promising approach to increase our knowledge about microbial communities. Directly extracted DNA can be used as a template for PCR amplification and detection of specific microorganisms without the need of cultivation. Target sequences can include 16S rRNA genes using universal primers for bacteria or archaea.

Determination of microbial diversity of environmental ecosystems can be optimised comparing results of different nucleic acid extractions. Specific oligonucleotides derived from environmental 16S rRNA sequences could be used as dye-labeled probes for 16S rRNA targeting in situ hybridization of fixed sample material with fluorescent in situ hybridization (FISH) (Fernandez-Lago et al. 2000).

From cloned PCR products which were generated from a community DNA, patterns can be generated by electrophoretic techniques. Such genetic „fingerprints” should reflect the structure of the microbial community or a specific fraction of it. A variety of techniques has recently been developed for community analysis: denaturing gradient gel electrophoresis (DGGE) (Muyzer 1999), terminal restriction fragment length polymorphism (tRFLP) (Kerkhof et al. 2000) and DNA single-strand-conformation polymorphism (SSCP) (Stach et al. 2001). The use of PCR amplification of 16S rRNA genes of cultivated isolates followed by restriction enzyme analysis of their amplified products (ARDRA) (Fernandez A. et al. 1999) is an efficient way to differentiate bacterial or archaeal isolates from diverse locations and obtain information about their phylogenetic position.

3.5.1 ARDRA (amplified rDNA restriction analysis) (Fernandez A. et al.1999)

ARDRA is a technique based on RFLP (restriction fragment length polymorphism) in which organisms can be differentiated by analysis of patterns derived from cleavage of their DNA. If two organisms differ in the distance between sites of cleavage of a particular restriction endonuclease, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme. The similarity of the patterns generated can be used to differentiate species from one another.

3.5.2 DGGE (denaturing gradient gel electrophoresis) (Muyzer 1999)

In denaturing gradient gel electrophoresis (DGGE), the double stranded DNA is subjected to electrophoresis in gel that has an increasing concentration of denaturant along the length of the gel. The fragment melts while travelling through the gel. The melting proceeds in segments, called melting domains, because of the cooperative nature of the denaturation of the double-stranded DNA. When a domain melts, the fragment assumes a branched structure that causes significant retardation of movement. Thus, the position of the fragment in the gel after a certain time of electrophoresis is determined by the history of melting of the fragment that is altered if the sequence is different. The principle of separation in DGGE is such that sequence changes in the melting domain of highest stability cannot be detected, because the fragment no longer has a branched structure when the last domain melts. If, however, a stretch of sequence that serves as an extremely stable domain is attached to one side of the fragment, then mutations at any sites within certain types of sequence context can be detected by DGGE. This extra sequence of extremely high stability can be conveniently attached to the target sequence of PCR by using one primer that has 40 nucleotides of an artificial GC-rich sequence (GC-clamp) extending at its 5'-end. With the use of this clamp, DGGE may be able to detect nearly all possible sequence- differences in any given sequence.

4 Objectives of this work

Discovery of bacteria that remain viable in a dormant state for lengthy periods is significant for understanding patterns of microbial diversity and evolution on Earth.

It remains to be proven how Halobacteriaceae can survive for extended times (Grant et al. 1998). To approach this question it is necessary to conduct a survey of the indigenous haloarchaeal community in rock salt.

A faster evaluation of microbial diversity in an environment should be obtained by PCR amplification of diagnostic molecules, such as the 16S rRNA genes, and subsequent sequencing of cloned products. This technique obviates culturing of microorganisms and has permitted the detection of novel and unexpected phylogenetic groups, e.g. in ocean samples (DeLong 1992).

Here an analysis of the microbial community in several Alpine Permo-Triassic rock salt samples by PCR amplification of 16S rRNA genes is reported.

To obtain more information on the true halobacterial biodiversity in Alpine rock salt sediments, two extraction methods were carried out in order to examine, whether different DNA extraction methods reveal different species.

Another intention was to discover if Halococcus -DNA can be extracted with an alternative extraction method, because up to this time no Halococcus -DNA could was found in rock salt with any DNA extraction method. Nevertheless Halococcus could be cultivated from rock salt samples on M2-Agar.

It was also tried to establish new methods to characterize the total 16S rRNA gene clone library more rapidly and completely, such as amplified ribosomal DNA restriction analysis (ARDRA) (Fernandez A. et al. 1999) as well as phylogenetic analysis of 16S rDNA sequences and denaturing gradient gel electrophoresis analysis (DGGE) (Pinar et al. 2001).

5 Material and Methods

5.1 Material

5.1.1 Bacterial and archaeal strains

illustration not visible in this excerpt

5.1.2 Kits

- pGEMR-T easy vector system (Promega)
- NucleoTrap extraction kit (Macherey and Nagel, Düren, Germany)
- GFXTM micro plasmid preparation kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA)

5.1.3 Media

illustration not visible in this excerpt

- LB plates with ampicillin: add ampicillin to a final concentration of 100µg/ml.

- LB plates with ampicillin/IPTG/ X-Gal: supplement with 0.5mM IPTG and 80µg/ml X-Gal and pour the plates. Alternatively, 100µl of 100mM IPTG and 20µl of 50mg/ml X-Gal may be spread over the surface of an LB-ampicillin plate and allowed to absorb for 30 minutes at 37°C prior to use.

illustration not visible in this excerpt

5.1.4 Marker

- PCR Marker (Sigma): fragment sizes: 2000, 1500, 1000, 750, 500, 300, 150, 50 base pairs
- 100bp marker ladder (Sigma)
- 20bp marker ladder (Sigma)

5.2 Extraction of community DNA

5.2.1 Dissolved rock salt sample (Radaxet al. 2001)

The samples were taken from salt mines, which are still in operation (Bad Ischl-Perneck in Austria). The following sample was used: Pieces of visibly stratified rock salt from a depth of about 650 m, taken in a newly created tunnel in Bad Ischl-Perneck 3 days after blasting operations. Approximately 500g of rock salt was surface sterilized by soaking in ethanol and flaming, placed in an Erlenmeyer flask equipped with a cotton stopper, slowly dissolved in sterile water, and incubated at 37°C for ten days after the addition of 0.05% (w/v) yeast extract (Difco, Augsburg, Germany) and 0.05% HyCase (Sigma, St.Louis, MO, USA) with gently shaking.The DNA analyzed in Radax et al. (2001) was prepared from the resulting solution that was designated Brine 2nd Hor; The DNA examined in this study was also prepared from Brine 2nd Hor. that had been kept at ambient temperature for approximately 12 month and the DNA extracted from this sample was designated BI 2nd Hor.

The second sample, a cylindrical bore core, was taken from Berchtesgaden. The bore core was 80mm in diameter, weight was at least 180g and was desiganted BG core 80.

5.2.2 Extraction method with filtration (Radax et al. 2001)

- Filtration of 100ml dissolved rock salt Brine 2nd Hor . through a 0.22µm pore size autoclaved membrane filter using a Sartorius filtration unit with a low pressure vacuum.
- The filter was incubated over night at -70°C.
- The thawed filter was cut in small pieces and vortexed in 5ml TE Puffer (+1%SDS; BioRad; filter-sterilized).

[...]