molecular phylogeny of archaea in boreal (forest soil and...

80
Molecular phylogeny of Archaea in boreal forest soil, freshwater and temperate estuarine sediment German Jurgens Department of Applied Chemistry and Microbiology Division of Microbiology University of Helsinki Finland ACADEMIC DISSERTATION IN MICROBIOLOGY To be presented with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public examination in Auditorium 2, Viikinkaari 11, Viikki Infocenter, on November 22 th 2002, at 12:00 noon Helsinki 2002

Upload: lamcong

Post on 04-Apr-2019

213 views

Category:

Documents


0 download

TRANSCRIPT

Molecular phylogeny of Archaea in boreal forest soil, freshwater and temperate estuarine sediment

German Jurgens

Department of Applied Chemistry and Microbiology Division of Microbiology

University of Helsinki Finland

ACADEMIC DISSERTATION IN MICROBIOLOGY

To be presented with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public examination in Auditorium 2, Viikinkaari 11, Viikki

Infocenter, on November 22th 2002, at 12:00 noon

Helsinki 2002

Supervisor: PhD Aimo Saano,

Docent, University of Helsinki Metsähallitus, Natural Heritage Services, Vantaa, Finland

Co-supervisors: PhD Uwe Münster,

Docent, University of Helsinki, Lecturer, Institute of Environmental Engineering and Biotechnology, Tampere University of Technology, Finland PhD Kristina Lindström, Docent, University of Helsinki Department of Applied Chemistry and Microbiology, University of Helsinki, Finland

Reviewers: PhD Maarit Niemi,

Docent, University of Helsinki Finnish Environment Institute, Research Programme for Biodiversity, Helsinki, Finland

PhD Hannu Fritze, Docent, University of Helsinki Finnish Forest Research Institute, Vantaa Research Center, Finland

Opponent: Professor Vigdis Torsvik,

University of Bergen, Department of Microbiology, Bergen, Norway

ISSN 1239-9469 ISBN 952-10-0714-1 (paperback)

ISBN 952-10-0715-X (PDF) ISBN 952-10-0716-8 (HTML)

Electronic publication available at http://ethesis.helsinki.fi

Helsinki University Printing House Helsinki, Finland 2002

Front cover: "Archaea's Universe" - collage made by author. Background: combination of same field micrographs made with epifluorescence microscope using filter sets specific for DAPI (blue color) and CY3 (red color, euryarchaeota specific probe EURY499, paper IV), Valkea Kotinen Lake water sample. Center: 16S rRNA secondary structure diagram of the archaeal soil clone FFSB6 - one of the first "non-extreme" crenarchaeota sequences found in forest soil (kindly made by Dr. Robin Gutell in August 1996).

To my mother, Candidate of Biological Sciences Ina Leopoldovna Jurgens

CONTENTS LIST OF ORIGINAL PAPERS THE AUTHOR'S CONTRIBUTION ABBREVIATIONS

1 INTRODUCTION................................................................................................................. 1

1.1 Microorganisms in the biosphere and the recent evolution of their taxonomy......... 1 1.1.1 Microorganisms as part of the biosphere - widespread and important, but

mainly unknown ................................................................................................. 1 1.1.2 History of classification schemes........................................................................ 2 1.1.3 A new era in microorganism studies - introduction of molecular techniques and

novel taxonomy .................................................................................................. 3 1.2 The use of molecular methods to study microbes in natural environments.............. 4

1.2.1 Ribosomal RNA: a key to molecular phylogeny ................................................ 4 1.2.2 Universal phylogenetic tree - the molecular tree of life...................................... 6 1.2.3 Molecular microbial ecology approaches to accessing natural diversity............ 8 1.2.4 Inferring phylogenetic relationships from rRNA gene sequences .................... 11 1.2.5 Limitations of molecular microbial ecology..................................................... 12 1.2.6 Combination of traditional culture-dependent methods and modern molecular

techniques ......................................................................................................... 14 1.3 Archaea .......................................................................................................................... 15

1.3.1 Features distinguishing Archaea from Bacteria and Eucarya ........................... 15 1.3.2 Archaeal phenotypes and phylogenetic division............................................... 16 1.3.3 Phylum Crenarchaeota ...................................................................................... 17 1.3.4 Phylum Euryarchaeota ...................................................................................... 23 1.3.5 Archaea as "non-extremophiles"....................................................................... 28

2 AIMS OF THE STUDY...................................................................................................... 37

3 MATERIALS AND METHODS........................................................................................ 38

3.1 Sampling and nucleic acid extraction ......................................................................... 38 3.1.1 Soil sampling (Finland)..................................................................................... 38 3.1.2 Water sampling for microbial DNA extraction (Finland)................................. 38 3.1.3 Estuarine sediment sampling (Portugal) ........................................................... 39

3.2 PCR amplification, cloning and clones characterization .......................................... 39 3.3 Conditions for in situ hybridization analysis.............................................................. 41

3.3.1 Sampling for in situ hybridization .................................................................... 41 3.3.2 Hybridization conditions and probes description.............................................. 41

3.4 Phylogenetic analysis .................................................................................................... 42

4 RESULTS............................................................................................................................. 43

4.1 ARB database and Archaea trees reconstruction ...................................................... 43 4.2 Phylogeny of Archaea from soil................................................................................... 47 4.3 Phylogeny of Archaea from estuarine sediment ........................................................ 48

v

4.4 Archaea from lake water.............................................................................................. 49 4.4.1 Phylogenetic analysis ........................................................................................ 49 4.4.2 In situ hybridization analysis ............................................................................ 50

4.5 Diagnostic signature and feature analysis of the studied sequences ........................ 51

5 DISCUSSION ...................................................................................................................... 52

6 SUMMARY AND CONCLUSIONS.................................................................................. 54

7 TIIVISTELMÄ.................................................................................................................... 56

8 ACKNOWLEDGEMENTS................................................................................................ 57

9 REFERENCES.................................................................................................................... 59

vi

LIST OF THE ORIGINAL PAPERS

This thesis is based on the following articles, referred to in the text by their Roman

numerals. Additionally, some unpublished results are presented. I. Jurgens, G., Lindström, K. and Saano, A. 1997. Novel group within kingdom

Crenarchaeota from boreal forest soil. Applied and Environmental Microbiology, Vol. 63: 803-805.

II. Jurgens, G. and Saano, A. 1999. Diversity of soil Archaea in boreal forest before, and

after clear-cutting and prescribed burning. FEMS Microbiology Ecology, Vol. 29: 205-213.

III. Abreu, C., Jurgens, G., De Marco, P. , Saano, A. and Bordalo, A.A. 2001.

Crenarchaeota and Euryarchaeota in temperate estuarine sediments. Journal of Applied Microbiology, Vol. 90: 713-718.

IV. Jurgens, G., Glöckner, F.-O., Amann, R., Saano, A. Montonen, L., Likolammi, M. and

Münster, U. 2000. Identification of novel Archaea in bacterioplankton of a boreal forest lake by phylogenetic analysis and fluorescent in situ hybridization. FEMS Microbiology Ecology, Vol. 34: 45-56.

Papers I to IV are reprinted with kind permission of the publishers. THE AUTHOR'S CONTRIBUTION I and II. German Jurgens planned and conducted the experiments, analyzed and interpreted the results as well as wrote the paper, under supervision of project leader Aimo Saano. Kristina Lindström acted as an expert in phylogenetic analysis for paper I. III. The work in this study was done in cooperation with Cristina Abreu (Portugal) while she was on Socrates scholarship in University of Helsinki during spring 1998. Cristina Abreu conducted the experiment and wrote the paper. German Jurgens planned and instructed the research, conducted the phylogenetic analysis and assisted in writing the paper. Aimo Saano, Paolo de Marco and Adriano Bordalo supervised the work. IV. German Jurgens planned and conducted the experiments, analyzed and interpreted the results and wrote the paper. Frank-Oliver Glöckner made the FISH picture and acted as FISH experiments adviser and ARB phylogenetic software expert. Markit Likolammi prepared bacteria biomass and bacteria production measurements and Leone Montonen performed PCR analysis with methyl-coenzyme M reductase primers. Rudolf Amann, Aimo Saano and project leader Uwe Münster supervised the work and assisted in writing.

vii

ABBREVIATIONS ARB from arbor, Latin: tree ATP adenosine triphosphate ATPase adenosine triphosphatase BAC bacterial artificial chromosome DAPI 4',6-diamidino-2-phenylindole DGGE denaturing gradient gel electrophoresis DNA deoxyribonucleic acid EDTA ethylenediaminetetraacetic acid FFS Finnish Forest Soil FFSB Finnish Forest Soil (group) B FISH fluorescence in situ hybridization HCl hydrochloric acid NaCl sodium chloride PCR polymerase chain reaction RDP Ribosomal Database Project RFLP restriction fragment length polymorphism RNA ribonucleic acid rRNA ribosomal ribonucleic acid SDS sodium dodecyl sulphate SSU small subunit TRIS tris (hydroxymethyl) aminomethane tRNA transfer ribonucleic acid

viii

1 INTRODUCTION

1.1 Microorganisms in the biosphere and the recent evolution of their taxonomy 1.1.1 Microorganisms as part of the biosphere - widespread and important, but mainly unknown

Microorganisms occupy a peculiar place in the human view of life. They are almost

unknown to the public except in the context of disease or food production. This is due to the fact that microbes are very small. Consequently, even many biologists consider microorganisms to be only a minor component of the global biomass. In reality, microbial communities represent more than half the biomass on Earth. Plant material accounts for most of the remainder, whereas animals do not contribute significantly (Whitman et al., 1998). Microbial life is present not only in our familiar world, i.e. air, soil, and lakes, but also the depths of the ocean and very deep in the crust of the Earth. Because of the large numbers of microorganisms and their metabolic capabilities, they play a crucial function in the planet's biochemical processes such as the decomposition of organic matter in soil and water, provision of atmospheric components, nitrogen fixation, and photosynthesis. It can be stated that the functioning of the whole biosphere depends absolutely on the activities of the microbial world (Madigan et al., 2003). This is not surprising if we consider how much older microbes are than all other forms of life on our planet (Fig. 1) .

Oxygen atmosphere

forming

Trilobites

Dinosaurs

Horses

Humans

4.0 4.5

Microorganisms

Stabilization of crust

Earth formed

3.5 3.0 2.5 x109 of years ago

2.0 1.5 1.0 0.5 0

Figure 1. Time line for the planet Earth. Various salient events in the planet’s history are shown, including the times at which certain major evolutionary groups are thought to have arisen, as indicated from fossil evidence (modified from Woese, 1994).

The clear deduction from the limited fossil record is that cell-based life arose

comparatively quickly after the planet formed 4.5x109 of years ago, and for two-thirds of the time since then, it was limited to prokaryotic-like life. Indeed, it was the impact and evolution of prokaryotic life that provided a suitable environment for the subsequent evolution of animal and plant species. For example, in geochemical terms, the formation of an oxygenated

1

atmosphere suitable for the evolution of many eukaryotic species was primarily due to bacterial photosynthesis. In biological terms, bacteria provided the chloroplast and mitochondrial organelles found in many current eukaryotic cells through endosymbiotic events (Margulis, 1993).

Our understanding of the importance of microbes coexists with a limited knowledge of what the majority of microorganisms are actually doing in nature. A gram of soil, for example, contains millions of microorganisms and still only around 1% (at most) of them are identifiable (Torsvik et al., 1990). Microbes are individually invisible to the naked eye and even under the microscope, the morphologies of most microbe’s are usually nondescript rods or spheres. It is therefore difficult to determine the relatedness of different microorganisms on the basis of morphology in the same way as that performed for large organisms. A special scientific discipline termed "microbiology" was developed in order to study microorganisms in their environment.

Louis Pasteur (1822-1895), a French chemist, played a significant role in the development of microbiology with his experiments disproving the theory of spontaneous generation. This theory, which became popular during the 18th century, proposed that spoilage organisms arose spontaneously in putrefied food. Pasteur captured airborne microorganisms on guncotton filters to show that these airborne microbes were responsible for food spoilage and did not arise spontaneously.

In 1876 a German country physician named Robert Koch (1843-1910) discovered that the lethal, contagious cattle disease anthrax could be transmitted from animal to animal by injecting blood from an infected cow into a healthy cow. His experiments were formulated into a set of criteria, known as Koch’s postulates, that have become the cornerstone for associating specific microorganisms with specific infectious diseases. Koch’s postulates initiated an era known as the Golden Age of Microbiology (1876-1906), during which the causes of many microbial diseases were discovered.

In the early part of 20th century Winogradsky and Beijerinck developed enrichment culture techniques so that microbes could be studied as individual types, and described and classified by their phenotype. Phenotype is a broad term which encompasses the observable characteristics of a cell such as morphology, physiological activities (e.g. nutrient utilization), cell-wall structure (e.g. types of lipids) and sometimes the ecological niche the cell occupies. Unfortunately,

• such characteristics provide little information about the evolutionary relatedness of

microorganisms, and • many microbes are difficult to cultivate. 1.1.2 History of classification schemes

Naturalists have always tried to build a meaningful classification scheme for living things. Long before Darwin, plants and animals were believed to be the primary divisions of life. In 1866, Haeckel was the first to challenge this dichotomy by suggesting that the Protista should be considered a third kingdom, equal in stature to the Plantae and Animalia. The Bacteria or Monera were designated as the fourth kingdom by Copeland, in 1938. Whittaker added the fungi in 1959, and his five kingdom classification (Plantae, Animalia, Protista, Fungi and Monera) is still taught as part of basic biology curricula.

Over 50 years ago, Chatton, Stanier and van Niel suggested that life could be subdivided into two more fundamental cellular categories, prokaryotes and eukaryotes. The distinction between the two groups was subsequently refined through studies of cellular biology and genetics whereby prokaryotes became universally distinguishable from

2

eukaryotes on the basis of lacking internal membranes (such as the nuclear membrane and endoplasmic recticulum), dividing by binary fission rather than mitosis, and the presence of a cell wall. The definition of eukaryotes was broadened to include Margulis’ endosymbiont hypothesis, which describes how eukaryotes improved their metabolic capacity by engulfing certain prokaryotes which were converted into intracellular organelles, principally mitochondria and chloroplasts.

In the late 1970s the fundamental belief in the prokaryote-eukaryote dichotomy was rejected by the work of Carl Woese and George Fox. By digesting in vivo labeled 16S rRNA using T1 ribonuclease, and accumulating and comparing catalogues of the resultant oligonucleotide “words”, Woese and Fox were able to derive dendrograms showing the relationships between different bacterial species. Analyses involving some unusual methanogenic “bacteria” revealed surprising and unique species clusterings among prokaryotes. The split in the prokaryotes was so deep that in 1977, Woese and Fox proposed that the methanogens and their relatives be termed the “archaebacteria”, a name which reflected their distinctness from the true bacteria or “eubacteria”, and also reflected contemporary preconceptions that these organisms might have thrived in the environmental conditions of a younger Earth (Brown, 1999). 1.1.3 A new era in microorganism studies - introduction of molecular techniques and novel taxonomy

The introduction of modern molecular tools which allowed the study of microbes

without prior cultivation dramatically changed our perception of phylogeny and the diversity of life.

In 1990, Woese, Kandler and Wheelies (Woese et al., 1990) proposed the replacement of the bipartite view of life with a new tripartite scheme based on three urkingdoms or domains; the Bacteria (formerly eubacteria), Archaea (formerly archaebacteria) and Eucarya (formerly eukaryotes although this term is still more often used) (Fig. 2). The rationale behind this revision came from a growing body of biochemical, genomic and phylogenetic evidence which, when viewed collectively, suggested that the archaebacteria were worthy of a taxonomic status equal to that of eukaryotes and eubacteria.

At the centre of the controversy surrounding the concept of the three domains are the Archaea and their degree of uniqueness from the Bacteria. Although discovered much more recently than either the Bacteria or Eucarya, the biochemistry, genetics and evolutionary relationships of the Archaea have been intensively studied. In addition, complete genomic DNA sequences have been obtained for several archaeal species.

Archaea and Bacteria are both groups of prokaryotes, but are phylogenetically distinct from each other. There is evidence to suggest that Archaea and Eucarya are more closely related to each other than either are to Bacteria; the origin of life appears to be on the bacterial line of descent (Fig. 2). Many phenotypic properties of Archaea and Bacteria are consistent with this phylogenetic assessment (Woese, 1987). Archaeal and bacterial metabolic genes share common evolutionary aspects (Brown and Doolittle, 1997). However, transcription and translation machinery in Archaea and Eucarya have common features, distinct from those of Bacteria (Olsen and Woese, 1997). This will be discussed in section 1.3.1 of this thesis.

Novel molecular techniques (Pace et al., 1985; Giovannoni et al., 1988; Amann et al., 1992; Torsvik et al., 1998) have offered new ways of studying microorganisms in diverse environments "in situ". By analyzing relevant genes obtained directly from environmental samples, microorganisms from a particular habitat can be identified (to some extent), characterized and counted. Since 1987, the use of these innovative approaches has resulted in the number of recognized bacterial phyla increasing from the original estimate of 11 (Woese,

3

1987) to 36 (Hugenholtz et al., 1998), more than one-third of which contain no cultured representatives.

Methano−

Pyrodictium

CrenarchaeotaMarine

Thermo−

Thermoproteus

pJP78pJP27

Korarchaeota

Archaea

pyrusMethano−

coccus

coccus

Crenarchaeota

Methanosarcina

halophilesExtreme

Thermoplasma

Euryarchaeota

lobusPyro−

Thermotoga

Cyanobacteria

Mitochondrion

Proteobacteria

bacteria

bacteria

Bacteria

ThermodesulfobacteriumAquifex

Green nonsulfur

Gram−positive

Flavobacteria

Chloroplast

Diplomonads(Giardia)

Flagellates

FungiEntamoebae molds

Slime

EucaryaAnimals

Plants

Ciliates

Microsporidia

Trichomonads

Figure 2. Rooted universal phylogenetic tree showing the three domains based upon

16S (or 18S) rRNA sequences (from Woese et al., 1990). The position of the root was determined by comparing paralogous gene sequences that diverged from each other before the three primary lineages emerged from their common ancestral condition (Iwabe et al., 1989).

One of the most remarkable findings to emerge from the application of new molecular

approaches was the unexpected discovery of high numbers of novel "non-extreme" archaeal phenotypes in open ocean waters (Fuhrman et al., 1992; Delong, 1992; Fuhrman and Ouverney, 1998), the largest of all biotopes on our planet. Until this finding, Archaea were considered to be restricted to specialized environments including those at high temperature, high salinity, extremes of pH, or strict anoxic. After many studies over several years researchers have demonstrated the presence of "non-extreme" Archaea in a wide variety of temperate and cold environments including soils, marine and lake sediments, marine and freshwater picoplankton, etc., that is to say, virtually everywhere. These discoveries mark the beginning of a new era for investigating Archaea and, in particular, their physiological properties and biological roles in complex microbial populations.

In this thesis results on the phylogenetic diversity of Archaea in boreal forest soil

(papers I and II), temperate estuarine sediment (paper III) and boreal freshwater lake (paper IV), obtained using molecular methods, will be reported.

1.2 The use of molecular methods to study microbes in natural environments 1.2.1 Ribosomal RNA: a key to molecular phylogeny

There is no consistent way to classify and relate microorganisms, both prokaryotes and

eukaryotes, other than the use of modern methods of molecular phylogenetic analysis. The use of macromolecular sequence comparisons to define phylogenetic relationships is now well

4

established (Zuckerkandl and Pauling, 1965). Protein sequences were most often used for phylogenetic determinations in the past as techniques for studying nucleic acid sequences were not available. Studies comparing cytochrome c, ribonucleases, globins, etc., have been rewarding, although they have proven most useful with higher eukaryotes (Goodman, 1982). Among microbes, phylogenetic and biochemical diversity is such that even the identification of homologous proteins is not a straightforward task. Because they are required by all cells for protein synthesis, the nucleic acid elements of the translation apparatus - the protein-synthesizing machinery - seem best suited for broad phylogenetic analysis. The translation of mRNAs into proteins using ribosomes and the tRNAs is an ancient mechanism. The similar architecture of the ribosomes and tRNAs in the three primary kingdoms - Archaea, Bacteria and Eucarya - means that the translation apparatus emerged largely in its modern form before the phylogenetic radiation of the three kingdoms. Thus, phylogenetic analysis of the components of the translation apparatus allow, in principle, the relationships among organisms to be traced nearly to the time of the origin of life on the earth.

However, tRNAs are not very useful for phylogenetic characterizations because they are too constrained in structure. The tRNA structure is tightly locked up in a complex tertiary organization. Almost every residue in a tRNA molecule has contact with at least one other residue, and the tight interlocking of the molecule imposes conformational constraints on all residues. Another major problem is the limited number of mutable residues in homologous tRNAs. The small number of changes in compared molecules means that the statistical error in a calculated evolutionary distance is great.

We are therefore left with rRNAs as the most useful tools for phylogenetic explorations. There are several explicit reasons for focusing on the ribosomal RNAs:

• the rRNAs, as key elements of the protein-synthesizing machinery, are of profound

importance to all organisms. • the rRNAs are ancient molecules and extremely conserved in overall structure. Thus,

homologous forms of rRNA are readily identified by their sizes alone. • the conserved nature of rRNA structure extends to the nucleotide sequence level. Some

segments of rRNA sequences do not vary among the biological kingdoms (domains), whereas others vary to greater or lesser extents (Gutell et al., 1994; van de Peer Y. et al., 1996). The conserved sequences and secondary structure allow disparate sequences to be aligned, so that only homologous sequences are used in phylogenetic analysis. The highly conserved regions also provide convenient hybridization targets for cloning rRNA genes and sequencing techniques.

• in general, rRNAs are essential and conserved across all phylogenetic domains, thus "universal" tracts of sequences can be identified (Olsen et al., 1986). In addition, it is possible to identify sequence motifs of increasing phylogenetic resolution and recognize "signature" sequences for the domains Archaea, Bacteria, and Eucarya and their subdivisions (Giovannoni et al., 1988; Woese, 1987).

• rRNA constitutes a significant component of cellular mass, and is generally recovered easily from all types of organisms.

• rRNA sequences are sufficiently long to provide statistically significant comparisons. • rRNA genes seem to be free from artifacts of lateral transfer between phylogenetically

distant organisms. Thus, relationships established by rRNA sequence comparisons represent true evolutionary relationships (Stackebrandt and Woese, 1981).

Taken together, these features indicate that rRNAs may be uniquely suitable for

establishing phylogenetic relationships among very different organisms.

5

Of the three ribosomal RNAs (5S, 16S/18S and 23S/28S), the 5S is too small (∼120 nucleotides) to be used indiscriminately for phylogenetic inferences. One might expect that the 23S/28S rRNA (23S rRNA in most prokaryotes, containing approximately 2900 nucleotides) would provide about twice the phylogenetic information compared with the 16S/18S rRNA (16S rRNA, containing approximately 1500 nucleotides). This is true within limits - the average rate of sequence change (as reflected in frequency of differences between corresponding sequences from a pair of organisms) of 23S rRNA is significantly faster than that of 16S rRNA. Thus, for close relationships, the larger molecule can be quite valuable although it has not proved to be as proportionately useful in the deepest branches of the tree (Olsen and Woese, 1993). Generally, when both sequences are available for a set of organisms, the phylogenies inferred by each rRNA tend to be similar (Ludwig et al., 1995). As 16S and 23S rRNAs are not functionally independent, it is not surprising that they give congruent pictures. We should also take into account the fact that the number of currently available complete 23S rRNA sequences in the databases is rather poor in comparison to the number of 16S rRNA sequences.

Therefore, SSU rRNA has served as the “gold standard” in elucidating bacterial phylogeny in recent years, and the new edition of both Bergey’s Manual of Systematic Bacteriology (Garrity, 2001, second edition) and "Brock Biology of Microorganisms" (Madigan et al., 2003, tenth edition) base their respective phylogenetic relationships among microorganisms upon the SSU rRNA tree.

1.2.2 Universal phylogenetic tree - the molecular tree of life

Figure 3 is a current phylogenetic tree based on small-subunit (SSU, 16S/18S) rRNA sequences of the organisms represented. The construction of such a tree is theoretically simple (Swofford et al., 1996). Firstly, rRNA sequences from different organisms are aligned. Pairwise comparisons are then made between all the sequences and the differences counted are considered to be some measure of the "evolutionary distance" between the organisms.

There is no consideration given to the passage of time but only changes in nucleotide sequence. Pairwise comparisons of different sequences between many organisms can then be used to infer phylogenetic trees, maps that represent the evolutionary paths leading to the

modern-day sequences. The tree in Figure 3 is largely congruent with trees made using any molecule involved in the nucleic acid-based, information-processing system of cells (Pace, 1997). Conversely, phylogenetic trees based on metabolic genes involved in the manipulation

of small molecules and in interaction with the environment, commonly do not agree with the rRNA-based version (Doolittle and Brown, 1994; Palmer, 1997). Differences in the phylogenetic trees made with different molecules may reflect lateral transfers or even the intermixing of genomes during the course of evolution. Some metabolic archaeal genes, for instance, appear much more related to specific bacterial versions than to their eukaryal homologs. Other archaeal genes seem decidedly eukaryotic in nature; still other archaeal genes are unique. Nonetheless, the so far determined sequences of the archaeons Methanococcus jannaschii (Bult et al., 1996), Methanobacterium thermoautotrophicum (Smith et al., 1997), Archaeoglobus fulgidus (Klenk et al., 1997), Pyrococcus horikoshii (Kawarabayasi et al., 1998), Aeropyrum pernix (Kawarabayasi et al., 1999), Halobacterium sp. (Ng et al., 2000), Thermoplasma acidophilum (Ruepp et al., 2000), Thermoplasma volcanium (Kawashima, 2000) and Sulfolobus solfataricus (She et al., 2001) show that the evolutionary lineage of Archaea is independent of both Eucarya and Bacteria.

6

Figure 3. Universal phylogenetic tree based on comparison of SSU rRNA sequences.

Sixty-four rRNA sequences representative of all known phylogenetic domains were aligned, and a tree was produced with fastDNAml (Olsen et al., 1994). That tree was modified, resulting in the composite one shown, by trimming lineages and adjusting branch points to

incorporate results of other analyses. The scale bar corresponds to 0.1 changes per nucleotide (from Pace, 1997).

7

"Evolutionary distance" in this type of phylogenetic tree, the extent of sequence change, is read along line segments. The tree can be considered a rough map of the evolution of the

genetic core of the cellular lineages that led to the modern organisms (sequences) included in the tree. The time of occurrence of evolutionary events cannot be extracted reliably from phylogenetic trees, despite common attempts to do so. Time cannot be accurately correlated

with sequence change because the evolutionary clock is not constant in different lineages (Woese, 1987). This inconsistency is shown in Figure 3 by the fact that lines leading to the different reference organisms are not all of the same length as these different lineages have experienced sequence changes to different extents. Nonetheless, the order of occurrence of branching in the trees can be interpreted as a genealogy, and intriguing insights into the evolution of cells are emerging. 1.2.3 Molecular microbial ecology approaches to accessing natural diversity

Molecular ecology has two general approaches: identification of the species present in an environment, and analysis of their populations in that environment. In the first case, organisms in an environment are “surveyed” using methods similar to analysis of cultivated species. This approach starts with an environmental sample and ends with sequence and phylogenetic data. In the second approach, organisms are studied in terms of their physical arrangement in the microenvironment using rRNA sequences to determine numbers and population dynamics, and distinguish one species or phylogenetic group from the rest. This approach starts with sequence and phylogenetic information and ends with environmental data - the reverse of the previous case. Some of the ways this approach can be used are:

• identification of the predominant microbial groups in a consortium by counting

sequences obtained from various phylogenetic groups to assess their abundance in the ecosystem

• use of fluorescently-labeled rRNA sequences (oligonucleotides) as probes for the identification or enumeration of specific organisms or groups in environmental samples by whole-cell hybridization

• assessment of enrichments aimed at cultivating organisms previously identified by their rRNA sequences

The characterization of an organism in terms of its phylotype requires only a gene

sequence, not a functioning cell. Genes can be obtained by cloning nucleic acids, which can be isolated directly from the environment using standard techniques. Phylogenetic analysis of rRNA genes from the environment provides a survey of the phylotypes present. The retrieved sequence information can then be used to gain further information about, or even retrieve, an organism of particular interest (Head et al., 1998). Figure 4 outlines ”full-cycle” rRNA approach - methods and tools used in the molecular analysis of microbial ecosystems. These techniques avoid the need to cultivate organisms in order to identify them (Pace et al., 1986; Amann et al., 1995). Several specialized methods are available for the extraction and purification of nucleic acids from a wide range of environmental samples, including soil, water, tissue and even rocks. Methods are usually based on chemical and/or physical disruption of cells, combined with treatments to remove contaminating materials, such as humic acids and metals, that "poison" enzymatic steps. A number of strategies can be used to obtain rRNA gene clones from "total community" nucleic acids (Fig. 4).

8

Dot/colony blot

Quantitative dot blot

Whole-cell hybridization

DGGE, RFLP

Comparative analysis

Probe design

g

RT-PCR R

Shotgun cloning or

cloning

Environmental sample

Community nucleic acids DNA RNA

Community rRNA genes

Nucleic acid probes

rDNA gene clones

Community "fingerprints"

rDNA sequand datab

t

Phylogenetic trees

Figure 4. “Full-cycle“ rRNA approach communities without the need for cultivation.

9

Sequencin

g

ences ase

- strategies for characteriz

Screenin

PC

Extraction

Dot/Southern blo

ing microbial

(1) Community DNA can be size-fractionated and shotgun-cloned into bacteriophage lambda (or into bacterial artificial chromosome (BAC) vectors which are able to "hold" larger size fragments), and then screened for the presence of rRNA genes. This is laborious procedure, as rRNA genes will only constitute a small fraction of the total clones. A big advantage of such libraries is that they are also sources of genes other than those encoding for rRNA.

(2) The simplest way to obtain phylotypes from the environment is through the use of the polymerase chain reaction (PCR) (Saiki et al., 1988). rRNA genes can be PCR-amplified directly from community DNA using rRNA-specific primers, and then cloned by standard methods. The resulting "snapshot" of community diversity will depend upon the specificity of the PCR primers and the efficiency with which the rRNA genes are amplified. Taking advantage of the conserved nature of rRNA, "universal" primers which are capable of annealing to rRNA genes from all three phylogenetic domains have been designed. Specific phylogenetic groups of interest in a total community can also be characterized using group-specific primers for rRNA. Although the analysis of a microbial community by PCR and cloning provides a convenient and rapid alternative to shortgun cloning, selective amplification of rRNA genes may bias diversity estimates (Head et al., 1998).

(3) The third alternative for obtaining rRNA gene clones from extracted nucleic acids is to use reverse transcriptase and universal or group-specific primers to make single-stranded DNA that is complementary to rRNA, and then to use PCR to make duplex ribosomal DNA for cloning (RT-PCR). The resulting community profile will offer some reflection of the most metabolically active organisms, because cells that produce more RNA (i.e. those that are metabolically more active) will be represented to a greater extent in the clone library than metabolically inactive cells.

A complete survey of phylotypes in a particular rRNA gene library would require

sequence analysis of all unique clones in the library. Natural microbial communities are highly complex, so several hundreds or thousands of individual clones would require sequencing. Dot- or colony-blotting using specific probes can be useful to sort clones and to identify specific targets for sequencing or other analyses. Screening clones in a library can also be facilitated by restriction fragment length polymorphism (RFLP) and single-nucleotide sequencing. Non-sequencing methods are also available which allows a microbial community to be monitored over space and time without cloning. Denaturing gradient gel electrophoresis (DGGE) (Muyzer et al., 1993; Øvreås et al., 1997), for example, separates different rRNA genes on the basis of their G+C content, and RFLP analysis of community rRNA genes produces rRNA gene fragments that may be specific for different community members. These methods are most informative when used in conjunction with sequence information, allowing individual community members to be qualitatively tracked in environmental samples.

A joint approach with cloning for the characterization of microbes in the environment is the use of nucleic acid probes - oligonucleotides complementary to rRNA or rRNA gene targets (Amann et al., 1992). Qualitative and quantitative estimates of community structure can be made using oligonucleotide hybridization probes (Fig. 4). Hierarchal probes can be custom-made that target broadly (e.g. at the domain level) or more specifically (e.g. at the strain level). These probes can be visualized directly in the environmental samples using fluorescently labeled oligonucleotide probes and epifluorescence microscopy to establish morphotype and cell numbers (Glöckner et al., 1996). Fluorescently labeled probes can also be combined with confocal scanning laser microscopy to describe spatial arrangements of cells, such as in biofilms and flocks.

Oligonucleotide probes and PCR primers are excellent tools for describing natural communities, but they rely on sequence data for their design. The more data that exist for a

10

particular environment, the more accurate the designed probes and primers will be. At the same time, after adding new data to the database, old probes and primers may not be specific for the new sequences.

1.2.4 Inferring phylogenetic relationships from rRNA gene sequences

The most commonly used form of comparative rRNA gene sequence analysis involves the construction of phylogenetic trees. There are a number of procedures used to achieve this, but the first stage in these analyses is always the careful alignment of the rRNA sequences. This is a relatively straightforward task for regions that have a highly conserved sequence. However, it is considerably more problematic in regions of greater sequence variability. Comparison with the secondary structure model of rRNA can often resolve these difficulties. The importance of careful alignment cannot be overstated. In any phylogenetic analysis, we must compare like with like if we are to be confident that a nucleotide substitution at any particular position in the sequence is, in fact, the result of an evolutionary event. Regions of sequences that cannot be unambiguously aligned are normally not included in phylogenetic analyses.

Once the rRNA sequences have been aligned, taking into account secondary structure interactions, phylogenetic analyses can be undertaken. Three widely used approaches for inferring phylogenetic trees are pairwise distance, parsimony, and maximum likelihood analysis (Swofford et al., 1996). Distance methods perform a modified cluster analysis of matrix of binary distance values. In contrast, the maximum parsimony and maximum likelihood procedures are used for analyzing primary sequence data. These procedures do not only compute the number of changes, but also take into account the character of those changes.

The distance methods (Fitch and Margoliash, 1967) are conceptually most simple. Pairwise comparisons of a set of aligned sequences are used to construct a distance matrix. The distances calculated are generally not simple binary similarities, but include a model of base substitution to account for multiple substitutions at a single site, for example, the Jukes and Cantor model (Jukes and Cantor, 1969). The distance matrix can then be converted into a phylogenetic tree by grouping the most closely related pairs of sequences.

Maximum parsimony procedures search for tree topologies which require a minimum number of base changes to correlate with the sequence data. The maximum likelihood procedure is considered the most sophisticated method for developing a phylogenetic tree (Felsenstein, 1981). It also searches tree topologies in ways that reflect how current sequences were most likely to have been generated, according to the criteria specified by whichever model is being applied. Not surprisingly, applying different treeing methods upon the same data often results in locally different tree topologies.

All these methods may assign organisms incorrectly to positions along a phylogenetic tree as a result of "false identity" in sequence positions. However, the extent of this problem varies from one method to another. Identical residues at particular alignment positions are typically treated as evolutionarily identical. This practice holds also for plesiomorphies which may have resulted from multiple base changes during the course of evolution. Depending on the number of "false'' identities in a sequence data set, misleading branch attractions may occur in a particular tree, which are difficult to detect as such. Branch attraction as a result of "false identities" may also prevent a stable positioning of long "naked'' branches represented by only one or a few sequences.

Because of such difficulties, the topology of every tree needs to be carefully evaluated. Maximum likelihood and, to a lesser extent, maximum parsimony methods handle these assignment problems more efficiently than distance methods. Therefore, different methods

11

should be applied to estimate the robustness of any tree's topology. The use of filters that remove, include, or give weight to particular sequence positions according to overall or positional variability also helps to detect and reduce the impact of "false identities" on tree topologies (Ludwig and Schleifer, 1999). The choice of tree-building method is somewhat arbitrary and often depends on time requirements, or the philosophical predisposition of the researcher. This is due to two reasons. Firstly, no method is uniformly better in reconstructing the true tree when the sequence length is small and secondly, all methods tend to perform well given enough data (Nei and Kumar, 2000).

The next step in constructing a sequence phylogeny is to assess the reliability of the inferred branching pattern. This is often accomplished by a bootstrap analysis (Felsenstein, 1985). Bootstrap procedures involve construction of new sequence sets by resampling with replacement sites (columns) of the original set, building a tree for each new set, and calculating the percentage of times a cluster reappears in the bootstrap replications. This percentage is called the bootstrap value and clusters with a bootstrap value >95% are widely considered to reflect correct relationships, although some authors have suggested that 70% may be a more realistic cutoff point.

1.2.5 Limitations of molecular microbial ecology

While we have undoubtedly gained much new and valuable knowledge using the

techniques described, as with all methods, there are important limitations that must be minimized, eradicated, or, at the very least, recognized. The limitations relate to the extraction of nucleic acids from natural samples, biases, and artifacts associated with enzymatic amplification of the nucleic acids, cloning of PCR products, and sensitivity and target site accessibility in whole-cell hybridization techniques (Head et al., 1998).

♦ Nucleic Acid Extraction

A major limitation of all the methods described, with the exception of whole-cell

hybridization techniques, is the quantitative recovery of nucleic acids from environmental samples. There is always the philosophical argument that if you do not know the total amount of nucleic acids present in a sample, then it is difficult to assess the efficiency of recovery by any extraction technique. This is compounded by the fact that Gram-positive cells are more resistant to cell lysis than Gram-negative cells. While this is irrefutable, a reasonable indication of the efficiency of cell lysis in an environmental sample can be obtained by microscopic enumeration of the cells in a sample before and after lysis treatments. There are many published methods for extracting DNA from natural samples (Fuhrman et al., 1988; Tsai and Olson, 1991) but there have been few systematic studies that have addressed this issue. It is possible that the same lysis technique may give different results with different types of sample such as water, sediment, or soil, and the degree of cell lysis should be determined independently. It has been demonstrated that a combination of physical and chemical treatments, such as freezing and thawing, lysis with detergents, and bead beating can lyse approximately 96% of cells in soil and also lyse bacterial endospores with high efficiency (More et al., 1994). It was noted however, that smaller cells (0.3-1.2 µm) were more resistant to lysis. This clearly has implications for the recovery of sequences from environmental samples where many cells may be in a state of starvation and, hence are likely to be small. Other workers have found, however, that, even without harsh physical treatments such as bead beating, up to 99.8% lysis can be obtained (Rochelle et al., 1992), although this did require long incubations with lysozyme and up to six freeze-thaw cycles.

12

PCR and Cloning Selectivity in PCR amplification of rRNA genes is another source of bias that can affect

the results of molecular measures of diversity. Small differences in the sequence of universally conserved regions may result in selective amplification of some sequences, particularly when primer annealing is at high stringency. The frequency of different sequence types in PCR-derived rRNA gene clone libraries has sometimes been assumed to accurately represent the relative abundance of different components of a microbial community. This cannot be claimed with any confidence, as the copy number of rRNA genes present within the genomes of different organisms can range from 1 to 14 (Cole and Saint, I, 1994). Thus, assuming unbiased amplification, a mixture of equal cell numbers of Bacillus subtilis (10 rRNA operons) and Thermus thermophilus (2 rRNA operons) would produce a library that indicated a 5 to 1 greater number of B. subtilis in the original mixture. In this example, the copy number of the genes in each genome and the size of the genome of both bacteria are known, and this can be accounted for in our estimation of species abundance. In natural samples, we have no such information about the constituent microbial types. There is also concern that more abundant sequences are preferentially amplified, and low-abundance sequences are discriminated against (Ward et al., 1992). It has been further suggested that high percent G+C templates are discriminated against due to lower efficiency of strand separation during the denaturation step of the PCR reaction (Reysenbach et al., 1992). PCR amplification using artificial mixes of genomic DNA from organisms with different genome sizes and numbers of rRNA operons has demonstrated that, in general, the ratio of rRNA genes in the PCR product mix do, in fact, reflect the ratio in the starting mixture of DNA (Farrelly et al., 1995). However, when rRNA operons are clustered together rather than evenly distributed throughout a genome, the clustered genes dominate in PCR products (Farrelly et al., 1995). The implication of these results is that we can never confidently extrapolate sequence composition of a clone library to a quantitative population composition of an environmental sample.

The formation of chimeric PCR products has also been observed where fragments from two different sequences become fused during the amplification process (Liesack et al., 1991). One study demonstrated that up to 30% of the products generated during coamplification of similar templates were chimeric (Wang and Wang, 1996). The experimental conditions used may well have promoted chimera formation to some extent. Nonetheless, the results demonstrated the considerable potential for chimera formation during PCR amplification.

A number of computer programs have been developed to help identify chimeric sequences (Kopczynski et al., 1994), but these have difficulty in identifying chimeras when the two sequences from which the chimera is formed show greater than 85% homology. The programs may also indicate the presence of chimeric sequences even when none exist (Kopczynski et al., 1994). These programs are best used as a guide to the presence of chimeric sequences. The authenticity of a sequence should be confirmed by independent sequence analyses, using the putative chimeric fragments. Discrepancies in secondary structure can also aid in the identification of genuine chimeric molecules.

Whole Cell In Situ Hybridization While the complex problems of enumeration associated with quantitative analyses

involving PCR do not hold for whole cell hybridization, a number of other methodological constraints do exist. These can be divided into four main categories: cell permeability problems, target site accessibility, target site specificity and sensitivity.

13

The first obstacle that must be overcome for successful in situ whole-cell hybridization is entry of the probe into the cell. This is normally achieved by fixation with denaturants such as alcohols, or cross-linking reagents such as formaldehyde or paraformaldehyde. These fixation procedures not only aid in cell permeability, but also help maintain the cells morphological integrity during hybridization.

Even when cell permeabilization has been achieved, there is no guarantee that probe hybridization to rRNA will occur within the cell. This is believed to be the result of the target sequence in the rRNA being inaccessible due to strong interactions with ribosomal proteins or highly stable secondary structure elements of the rRNA itself. This problem can normally be detected by a strong hybridization signal being obtained with a universal probe that is known to target an accessible site on the rRNA molecule. If another probe does not give a hybridization signal in the same cell(s), this generally indicates poor accessibility of the target site (Amann et al., 1995).

The sensitivity of in situ hybridization is also an issue. In general, probes containing a single labeled molecule give a strong signal only if cells are metabolically active and, hence, contain large numbers of ribosomes and target rRNA (Hahn et al., 1992; Manz et al., 1993). A number of approaches have been used to improve sensitivity by using multiple singly labeled probes (Amann et al., 1990; Lee et al., 1993), multiple labeled probes (Wallner et al., 1993), and enzyme-linked probes or detection systems (Zarda et al., 1991; Amann et al., 1992) that allow signal amplification. In addition, the development of highly sensitive cameras has improved the sensitivity of in situ hybridization assays.

As more rRNA sequences become available in sequence databases, the problem of probe specificity has been highlighted, and the design of diagnostic probes is becoming more difficult. While this problem has always existed, it is only with the rapidly expanding database of sequences that the problem has become more apparent. These problems are not exclusive to whole-cell hybridization but are equally relevant to PCR and other oligonucleotide-dependent techniques. It has been stated that for an 18mer probe targeting a variable region of an rRNA molecule, there is a 1:418 chance of an unrelated target cell being detected. However, because there may be only a few positions that vary between taxa even in variable regions, the probability of detecting an unrelated cell is increased considerably (1:45, if only 5 positions are variable). It has been suggested that this problem can be overcome by using multiple specific oligonucleotide probes that target different sites on the rRNA molecule and are labeled with different fluorochromes (Amann, 1995). An elegant solution which takes advantage of additive color mixing is the use of differently labeled probes. This method has been demonstrated to work well and considerably reduces the detection of false positives (Amann, 1995).

Analogous to this approach is the use of specific PCR primers and confirmation of the identity of the amplified sequence(s) by the use of a specific oligonucleotide probe. While a single oligonucleotide target sequence may be found in a number of related taxa, the probability that target sites for three specifically designed oligonucleotides are found in a non-target organism is much reduced.

1.2.6 Combination of traditional culture-dependent methods and modern molecular techniques

Due to the limitations of traditional culture-dependent methods the use of molecular

techniques has become of growing importance for the study of microbial communities in various ecosystems. Above all, the rRNA approach has been successfully applied to reveal the existence of several novel lineages of hitherto unknown prokaryotes leading to a broadening of our view on microbial diversity. It must be stressed, however, that cultivation-independent,

14

PCR-based methods can also have inherent biases preventing a reliable assessment of the structure of bacterial populations which may lead to a misinterpretation of the abundance of certain phylogenetic groups. Such pitfalls may be avoided by hybridizing whole cells or extracted rRNA from the studied habitat with specific oligonucleotide probes in order to verify the initial results. Furthermore, the retrieval of a novel 16S rRNA sequence reveals very little about the phenotypic traits of the respective organism and its metabolic activity. It is only when the retrieved sequence can be clearly affiliated to a monophyletic lineage which is characterized by a common phenotypic trait that some conclusions may be drawn about the function of the corresponding microorganism. In most cases, however, the simple knowledge of the phylogenetic diversity in an environment is not very helpful in understanding the interacting metabolic processes and factors which control them. Nevertheless, a molecular approach can help in the identification of microorganisms which are ecologically relevant because of their abundance or activity. These microorganisms can then be the subject of detailed studies or a target of directed cultivation experiments.

The majority of prokaryotes living in natural environments are rather inconspicuous. Therefore, several molecular techniques were developed in order to overcome the lack of information about the function of bacteria identified by cultivation-independent methods. Despite the progress which has been made in linking the identification of distinct microorganisms with their functions in situ, it may still be necessary to isolate or enrich novel bacteria to reveal their metabolic potential under various environmental conditions.

The results of molecular ecology research has established that experimental strategies based on the combination of molecular techniques with traditional cultivation-dependent methods have great potential in revealing some of the hidden complexity of natural microbial ecosystems.

1.3 Archaea 1.3.1 Features distinguishing Archaea from Bacteria and Eucarya

In order to study and be able to predict properties of the uncultivated Archaea, one must

be familiar with the main features, properties and ecology of this kingdom's known cultivated members (Huber and Stetter, 1999a; Huber and Stetter, 1999b; Madigan et al., 2003).

Microbiologists have perceived Archaea as exotic, highly atypical microorganisms. Prior to their recognition as a phylogenetically coherent group (Woese and Fox, 1977) however, their individual idiosyncrasies were interpreted simply as adaptations: the lipids of Thermoplasma were unusual because the organism evolved to live at high temperatures or in highly acidic environments or both (Brock, 1978); the wall of Halococcus was an adaptation to an extremely saline environment (Larsen, 1973); the uniqueness of their coenzymes merely reflected the capacity of methanogens to produce methane from carbon dioxide (Zeikus, 1977). The fact that different Archaea had the same unusual lipids was even interpreted as convergent adaptation (Brock, 1978).

♦ General aspects

The Archaea share a number of features with both the Bacteria and the Eucarya, but

they also possess some unique characteristics (Beveridge, 2001; König, 2001). Typical bacterial features are the small cell size, the lack of a nucleus, a small genome size and non-mitotic cell division. The circular chromosome is not membrane bound and exhibits a large range of DNA base composition (Thomas et al., 2001). Restriction enzymes are present. The

15

sedimentation coefficient of the ribosomes is 70S. The cells can possess polyphosphate and polyhydroxybutyrate inclusions. Membrane-surrounded organelles are absent, which is also true, however, for some eukaryotic Archaezoa. Like Bacteria, the flagella are single filamentous protein helices. Some Archaea are able to fix dinitrogen. The organization of the rRNA cistrons (except Thermoplasma) is also bacteria-like.

A number of features are Eucarya-like (eukaryal). The elongation factor EF-2 contains the amino acid diphthamide and is therefore ADP-ribosylated by the diphtheria toxin. Amino acid sequences of the ribosomal A proteins exhibit sequence homologies with the corresponding eukaryotic proteins. The methionyl initiator tRNA is not formylated and some tRNA genes contain introns. The aminoacyl stem of the initiator tRNA terminates with the base pair AU. Like the a-DNA polymerases of Eucarya, the replicating archaeal DNA polymerases are not inhibited by aphidicolin and butylphenyl-dGTP. The inhibition of peptide synthesis by anisomycin, but not by chloramphenicol, is also a eukaryotic feature. The pigment retinal is a compound found formerly in Eucarya only.

A unique archaeal feature is the membrane structure. (1) The membrane lipids are glycerol isopranyl ethers; (2) the Archaea possess unique cell envelopes; and (3) interestingly, there is an absence of ribothymidine in the “common” arm of the tRNAs. In the Archaea, it is replaced by 1-methyl-pseudouridine or pseudouridine. In addition, especially in methanogens, a number of unusual cofactors have been found.

Cell envelopes The Archaea possess no common cell wall polymer and all Archaea lack murein

(Kandler and König, 1993). The cell walls of Gram-positive Archaea consist of pseudomurein (König et al., 1982), methanochondroitin, heterosaccharide or a glutaminylglycan. The Gram-negative Archaea possess surface layers of protein or glycoprotein subunits forming two-dimensional crystalline arrays which are directly located on the outside of the plasma membrane. The Thermoplasmas are cell wall-less and Methanospirilli possess additional proteinaceous sheaths. Due to their distinct chemical composition, the Archaea exhibit high resistance against cell wall antibiotics and lytic agents. The biosynthetic pathways follow different modes compared to the well-known pathways of cell wall polymers in bacteria.

Plasma memranes Typical fatty acid glycerolipids are absent in archaeal membranes. Instead, glycerol

phytanyl diether and biphytanyl tetraether lipids form the lipids of the plasma membrane (Langworthy, 1985). The isopranyl glycerol ethers are a convenient molecular marker to distinquish Archaea from Bacteria and Eucarya. The phytanyl residues are linked to atoms C2 and C3 of glycerol, while in the other domains the atoms C1 and C2 are substituted with fatty acids. The tetraethers form monolayered and not bilayered membranes.

1.3.2 Archaeal phenotypes and phylogenetic division

The cultivated Archaea, as recognized from a phenotypic perspective, comprise of three

different phenotypes: the methanogens (that produce methane), the extreme halophiles (that live at very high concentrations of salt (NaCl)) and the extreme thermophiles (that live at very high temperatures) (Woese, 1987).

On the basis of SSU rRNA analysis, the domain Archaea consists of two phylogenetically distinct phyla, the Euryarchaeota and the Crenarchaeota. Pure culture of a third kingdom, the Korarchaeota (Fig. 3, clones pJP78 and pJP27) are not yet available,

16

although stable mixed cultures, containing representatives of this group, can be grown in the laboratory (Burggraf et al., 1997). From the media and incubation conditions supporting these cultures, we can deduce that the Korarchaeota are hyperthermophiles and may have metabolic properties similar to those of the hyperthermophilic Crenarchaeota.

A cultured nanosized hypertermophylic symbiont (Huber et al., 2002a) and several novel SSU rRNA clones, which constitute new sister clades, were reported recently (Kim et al., 2000, Takai and Horikoshi, 1999, Fig. 5A).

Cultivated crenarchaeotes are phenotypically monotonous in that they all possess a thermoacidophilic phenotype. The term "cren" means spring or fount and should express "the ostensible resemblance of this phenotype to the ancestor (source) of the domain Archaea". In contrast, the Euryarchaeota phylum contains organisms that are highly diverse in their physiology, morphology and natural habitats. The term "eury" takes this into account with its meaning of "broad" or "wide".

The following descriptions of the most studied members of the cultivated Archaea are based on Bergey’s Manual of Systematic Bacteriology (Table 1, Garrity, 2001) as a leading reference source in prokaryotic taxonomy.

1.3.3 Phylum Crenarchaeota

The Crenarchaeota are a well-defined branch of the archaeal domain, which is obvious

from sequence data and biochemical investigations. All cultured representatives of the Crenarchaeota are extreme thermophiles or

hyperthermophiles. A broad variety of metabolic pathways is evident (Stetter, 1998). Aerobically growing chemolithotrophs gain energy by the oxidation of various sulfur compounds, molecular hydrogen or ferrous iron. Anaerobic chemolithotrophs reduce sulfur, thiosulfate or produce nitrate, hydrogen sulfide or ammonia. Organotrophic growth occurs on complex organic substrates, sugars, amino acids or polymers such as starch (Robb and Place, 1995).

One should remember that even though we think of thermophilic environments as being unusual, they are actually quite common. Hot springs, after all, are just the "tip of the iceberg" of the thermophilic world below us. All the subterranean environments and the mid-oceanic spreading zones form a continuous ecosystem several kilometers wide and deep. All indications are that the water that saturates the crushed rock and sinter in the spreading zone is full of thermophilic microbial growth. Water that flows out from hot springs contains typically 107 to 108 cells per milliliter, and these are just the cells that grow in suspension.

♦ Morphology of the Crenarchaeota

Within the Crenarchaeota, a broad variety of morphologies exist including rods of

different width and length, regular or highly irregular cocci, and very unusual disc-shaped cells integrated in a network of hollow cannulae (Huber and Stetter, 1999a). As in all Archaea, no murein (peptidoglycan) is present in the cell walls of the Crenarchaeota. All Crenarchaeota species stain Gram-negative.

17

Table 1. Taxonomic outline of the domain “Archaea”. Bergey’s manual of Systematic Bacteriology, 2nd Edition (Garrity, 2001). Phylum Euryarchaeota

Class I. Methanobacteria Order I. Methanobacteriales Family I. Methanobacteriaceae

Genus I. Methanobacerium Genus II. Methanobrevibacter Genus III. Methanosphaera Genus IV. Methanothermobacter

Family II. Methanothermaceae Genus I. Methanothermus

Class II. Methanococci Order I. Methanocaccales Order II. Methanomicrobiales Order III. Methanosarcinales Family I.Methanococcaceae Family I. Methanomicrobiaceae Family I. Methanosarcinaceae

Genus I. Methanomicrobium Genus II. Methanoculleus Genus III. Methanofollis Genus IV. Methanogenium Genus V. Methanolacinia Genus VI. Methanoplanus

Family II. Methnocorpusculaceae

Genus I. Methanosarcina Genus II. Methanococcoides Genus III. Methnohalobium Genus IV. Methanohalophylus Genus V. Methanolobus GenusVI. Methanomicrococcus Genus VII. Methanosalsum

Genus I. Methnocorpusculum Family II. Methanosaetaceae Family III. Methnospirillaceae

Genus I. Methanospirillum Genera insertae sedus

Genus I. Methanococcus Genus II. Methenothermococcus

Genus I. Methanocalculus

Genus I. Methanosaeta

18

Table 1, continued Phylum Euryarchaeota (continued)

Class III. Halobaceria Class IV. Thermoplasmata Order I. Halobaceriales Order I. Thermoplasmatales Family I. Halobaceriaceae Family I. Thermoplasmataceae

Genus I. Thermoplasma Family II. Picrophilaceae

Genus I. Picrophilus Family III. Ferroplasmataceae

Genus I. Halobacterum Genus II. Haloarcula Genus III. Halobaculum Genus IV. Halococcus Genus V. Haloferax Genus VI. Halogeometricum Genus VII. Halorhabdus Genus VIII. Halorubrum Genus IX. Haloterrigena Genus X. Natrialba Genus XI. Natrinema Genus XII. Natronobacterium Genus XIII. Natrococcus Genus XIV. Natronomonas Genus XV. Natronorubrum

Genus I. Ferroplasma

Class V. Thermococci Class VI. Archaeaoglobi Class VII. Methonopyri Order I. Thermococcales Order I. Archaeaoglobales Order I. Methanopyrales Family I. Thermococaceae Family I. Archaeaoglobaceae Family I. Methanopyraceae

Genus I. Thermococcus Genus II. Palaeococcus Genus III. Pyrococcus

Genus I. Archaeaoglobus Genus II. Ferroglobus

Genus I. Methanopyrus

19

Table 1, continued Phylum Crenarchaeaota

Class I. Thermoprotei Order I. Thermoproteales Order II. Desufurococcales Order III. Sulfolobales Family I. Thermoproteaceae Family I. Desulfurococcaceae Family I. Sulfolobaceae

Genus I. Thermoproteus Genus II. Caldivirga Genus III. Pyrobaculum Genus IV. Thermocladium

Family II. Thermofilaceae

Genus I. Desufurococcus Genus II. Acidolobus Genus III. Aeropyrum Genus IV. Ignicoccus Genus V. Staphylothermus Genus VI. Stetteria Genus VII. Sulfophobococcus Genus VIII. Thermodiscus Genus IX. Thermosphaera

Family II. Pyrodictiaceae

Genus I. Thermofilum

Genus I. Pyrodictium Genus II. Hyperthermus Genus III. Pyrolobus

Genus I. Sulfolobus Genus II. Acidianus Genus III. Metallosphaera Genus IV. Stygiolobus Genus V. Sulfurisphaera Genus VI. Sulfurococcus

20

The rod-shaped representatives of the Crenarchaeota are found within the order Thermoproteales. The separation into the two families Thermoproteaceae and Thermofilaceae is in agreement with morphological characteristics. The members of the Thermoproteaceae exhibit cell diameters of 0.4 to 0.5 µm, while the Thermofilaceae are thin rods (filaments), with cell diameters of 0.15 to 0.35 µm. The length of all these rod-shaped organisms is usually between 1 and 8 µm, although cells of up to 100 µm occur within the genus Thermofilum and Thermoproteus.

As indicated by the name ”lobus”, cells representative of the order Sulfolobales are usually lobed cocci with cell diameters between 0.5 and 2 µm occurring singly or in pairs. As an exception, both Metallosphaera species exhibit a regular coccoid shape.

The representatives of the Desulfurococcales are cocci or discs with diameters between 0.3 and 5 µm, which are often highly irregular. The cells occur usually singly or in pairs. In addition, the cells of Thermosphaera aggregans and Staphylothermus marinus form short chains or grape-like aggregates. In culture media inoculated with Pyrodictium cells, greyish flakes 1 to 10 µm in diameter, can be seen macroscopically. These flakes are composed of cells interconnected by a network of hollow cannulae. This unique interconnecting structure is formed only by Pyrodictium species (Rieger et al., 1995). Single cannulae have a diameter of 25 nm, and can aggregate in bundles of 10 or more. The cells of Pyrodictium display a further unusual feature, the so-called ultraflat areas, with a width of only 80 to 100 nm. The two cytoplasmic sites of the membrane are often in direct contact, with hardly any cytoplasm left in between.

The phylum Crenarchaeota comprises of one Class - Thermoprotei which consists of

three orders: Thermoproteales (Zillig et al., 1981), Sulfolobales (Stetter, 1989) and Desulfurococcales (Zillig et al., 1982) (Table 1).

♦ The order Thermoproteales

All members of the Thermoproteales live at a neutral or slightly acidic pH. With the

exception of Pyrobaculum aerophilum, they are strict anaerobes, which gain energy by respiration of elemental sulfur using organic compounds as substrates, yielding carbon dioxide and hydrogen sulfide. In addition, Thermoproteus tenax, Thermoproteus neutrophilus and Pyrobaculum islandicum can grow chemolithotrophically by H2/S autotrophy. In contrast, growth of Pyrobaculum aerophilum is inhibited by elemental sulfur. This organism grows autotrophically by oxidation of molecular hydrogen or thiosulfate, using oxygen, nitrate or nitrite as electron acceptors. Complex organic compounds are used during heterotrophic growth in the presence of nitrate. Pyrobaculum aerophilum is the only aerobic representative of the Thermoproteales. The two Thermofilum species can be distinguished by the requirement for a polar lipid of Thermoproteus tenax or cell extracts of other archaea by Thermofilum pendens. Thermofilum librum grows without these supplements in the culture medium.

The Thermoproteales are hyperthermophiles with temperature optima between 85 and 100°C and temperature maxima from 95 to 104°C. With the exception of Pyrobaculum aerophilum, the only marine species within the order, they are found in soils, mud holes or surface waters of solfataric fields which exhibit low ionic strength. Therefore, growth occurs at sodium chloride concentrations between 0 and 2% in the culture medium. Pyrobaculum aerophilum grows between 0 and 3.6% NaCl with an optimum at 1.5% (Huber and Stetter, 1992).

21

The order Desulfurococcales All members of the order Desulfurococcales are neutrophiles with pH optima between 5

and 8. The representatives of the two families are diverse in their temperature maxima: while the Desulfurococcaceae can grow up to around 100°C, the Pyrodictiaceae reach temperature maxima between 108 and 113°C. Below 80°C, no growth can be obtained with any representatives of the Pyrodictiaceae. Pyrolobus fumarii (Blöchl et al., 1997) is unable to grow even below 90°C and exhibits the highest growth temperature of all known organisms, 113°C. It gains energy anaerobically by reduction of nitrate (forming ammonia) or thiosulfate (forming hydrogen sulfide). Under microaerobic conditions it can grow by oxidation of molecular hydrogen. The closely related members of the genera Pyrodictium and Hyperthermus (Zillig et al., 1991) exhibit different metabolisms. The Pyrodictium species grow chemolithoautotrophically by reduction of elemental sulfur using molecular hydrogen. Sulfur can be replaced by sulfite or thiosulfate in some strains. In addition, P. abyssi (Pley et al., 1991) gains energy by fermentation of peptides. The obligate fermentative Hyperthermus produces organic acids and butanol from peptides. All members of the Pyrodictiaceae have been exclusively isolated from shallow submarine hydrothermal systems and from deep-sea hot vents.

With the exception of Igneococcus, all representatives of the family Desulfurococcaceae are obligate organotrophs. Members of the genera Thermosphaera, Sulfophobococcus and Staphylothermus gain energy by fermentation of sugars or complex organic substrates, such as yeast extract or peptides. Furthermore, sulfur respiration, yielding hydrogen sulfide in addition to organic acids or alcohols, is found within the genera Desulfurococcus, Thermodiscus and Stetteria. In addition, Desulfurococcus amylolyticus can use starch as an organic substrate. The only obligate aerobic member of this family, Aeropyrum pernix (Sako et al., 1996), grows by respiration of complex organic material in the presence of oxygen. Igneococcus gains energy by reduction of elemental sulfur using molecular hydrogen as an electron donor (Huber et al., 2000). Members of the Desulfurococcaceae have been isolated from continental and marine high temperature biotopes all over the world.

Due to their hyperthermophily, some species have been examined for heat-shock proteins. In Pyrodictium a thermosome (a chaperonin-like protein complex with ATPase activity) is expressed which is accumulated after heat shock. Chaperone-like heat-shock protein can be detected in cells of Pyrolobus after immunoblotting crude cell extracts.

Formerly, representatives of the Crenarchaeota were often termed the "sulfur-metabolizing" hyperthermophiles. However, several representatives of this kingdom are not only unable to metabolize sulfur (e.g. growing obligately by fermentation), but are even inhibited by elemental sulfur. This includes Pyrolobus fumarii, Sulfophobococcus zilligii and Thermosphaera aggregans.

The order Sulfolobales As a rule, all members of the order Sulfolobales are thermoacidophiles, exhibiting

temperature optima between 65 and 90°C and pH optima between 1.5 and 4 (Brock et al., 1972). The six genera within this group are well defined by physiological and biochemical properties, although this chemotaxonomic classification does not always correlate with their phylogeny, based on 16S rRNA sequence data.

Representatives of the genera Sulfolobus, Metallosphaera and Sulfurococcus are obligate aerobes. Acidianus and the recently described Sulfurisphaera are facultative anaerobes, while Stygiolobus is an obligate anaerobe. For Sulfolobus, Metallosphaera,

22

Sulfurococcus and Acidianus, the typical energy-yielding reaction is the oxidation of elemental sulfur to sulfuric acid. Alternatively, sulfide, tetrathionate or ferrous iron can serve as electron donors for several species. However, it should be mentioned that the type strains of Sulfolobus acidocaldarius and S. solfataricus, deposited in culture collections, have lost their capability of oxidizing elemental sulfur. Due to their ability to oxidize sulfides, some of these strains are highly efficient in mobilizing metals from sulfidic ores producing soluble sulfates (Huber et al., 1989). Oxygen can be replaced by MoO4

2- or ferric iron as electron acceptors. Furthermore, most strains can gain energy by the oxidation of molecular hydrogen (the "Knallgas" reaction). However, significant differences have been determined for the optimal concentration of oxygen in different strains. It varies from 0.5% (Metallosphaera sedula) to 12% (Metallosphaera prunae) oxygen in the atmosphere. In addition to chemolithoautotrophic growth, many strains are able to grow on complex organic substrates, like yeast extract peptone, tryptone, meat extract or casamino acids. Some Sulfolobus and Sulfurococcus strains can use various sugars and amino acids as carbon and energy sources. In the absence of oxygen, Acidianus is able to grow by reduction of elemental sulfur. Anaerobic growth occurs via S/H2 autotrophy and large amounts of H2S are produced. This reaction is also characteristic for the strictly anaerobic Stygiolobus. Sulfurisphaera is a facultative aerobe, which grows on complex organic substrates, or under anaerobic conditions on elemental sulfur.

The Sulfolobales are usually found in biotopes of low ionic strength (and low pH), like sulfur rich solfataric fields. Therefore, optimal growth occurs at NaCl concentrations between 0 and 1% NaCl in the culture medium. An exception, however, is Acidianus, which can grow at concentrations of up to 4% NaCl.

1.3.4 Phylum Euryarchaeota

Five major groups are known within this kingdom; the obligate anaerobic methanogens,

the extreme halophiles, the hyperthermophilic sulfate reducers, the Thermoplasma group, and finally the Thermococcus-Pyrococcus group. During the last decade, numerous reclassifications within the Euryarchaeota have been carried out, mainly based on results of 16S rRNA sequence comparisons. This is especially true for the orders Halobacteriaceae, the Methanobacteriaceae, and the Methanomicrobiales.

♦ Morphology of the Euryarchaeota

Within the Euryarchaeota a broad variety of morphologies is evident. Besides straight to

crooked rods (sometimes embedded in a sheath) of different width and length, regular or highly irregular cocci, or pleiomorphic forms occur; with some even lacking a cell wall (Garrity, 2001). Although no murein (or peptidoglycan) is present in the cell walls of the Euryarchaeota, a quite similar structure called pseudomurein is found within the Methanobacteriales and the Methanopyrales. Therefore, in addition to the halococci and some strains of the genus Methanosarcina, these organisms stain Gram-positive. All other Euryarchaeota exhibit a Gram-negative reaction.

Within the Methanopyrales, all cells are exclusively rod-shaped, while all members of the Methanococcales are regular cocci. With the exception of the coccoid Methanosphaera species, the Methanobacteriales harbour only rod-shaped strains and species, which, however, differ significantly in width and length. Several genera of the Methanomicrobiales and Methanosarcinales exhibit quite unusual cell morphologies, like the plate- or disc-shaped Methanoplanus, the pleiomorphic Methanolacina, the aggregate-forming Methanosarcina, or the spiral-shaped Methanospirillum. Methanospirillum and Methanosaeta have unique

23

ultrastructures. The individual cells are covered by a sheath with a striated surface appearance and are spaced apart by multiple lamellae or a plug composed of concentric rings. The filaments of Methanosaeta form large bundles or mats and are up to several 100 µm long. The sheaths exhibit remarkable resistance to chemical reagents. Different types of cell walls have been observed for methanogens. Many methanogens (especially coccoid organisms) are motile by one or more flagella, which are sometimes arranged in a polar tuft. Due to the possession of F420 all methanogens show a blue-green fluorescence under an ultraviolet microscope at 436 nm. In a few species, cylindrically shaped gas vesicles are found (e.g. Methanosarcina vacuolata). The Thermococcales are characterized by a coccoid shape. With the exception of Thermococcus litoralis, all Thermococcus and Pyrococcus species are motile and possess one or more flagella (in Pyrococcus they are always arranged in at least one polar tuft). Archaeoglobus and Ferroglobus cells are highly irregular cocci, occurring singly or in pairs. Very often they are triangular-shaped and appear to be flat at the broader base. Cell diameters vary from 0.7 to 1.3 µm. Like the methanogens, all members of the Archaeoglobales show a blue-green fluorescence under the UV microscope. Most of the strains are flagellated.

The lack of a cell wall is characteristic for members of the genus Thermoplasma. Consequently, their cell shape and diameters are highly variable. During early exponential growth phase, filamentous, disc-shaped and coccoid-shaped cells occur with diameters between 0.2 and 5 µm. Later, spherical forms predominate. All strains are motile by possession of flagella. Members of the Halobacteriaceae are rods, cocci, sarcinas, or flat triangles and squares. These organisms stain Gram positive or negative, depending on the species.

Due to their extreme diversity, the Euryarchaeota cover the whole spectrum of

physiological properties: from psychrophiles to hyperthermophiles, from strict aerobes to obligate anaerobes, from freshwater strains to extreme halophiles, and from extreme acidophiles to alkalophiles (Zinder, 1993). Furthermore, a broad variety of metabolic pathways is evident, represented by strict chemolithoautotrophy to organotrophy.

♦ The methanogens

Within the methanogens five orders have been described (Balch et al., 1979; Kurr et al.,

1991), the Methanopyrales, the Methanococcales, the Methanobacteriales, the Methanomicrobiales, and the Methanosarcinales (Table 1).

In contrast to their enormous phylogenetic diversity, methanogens can only use a few simple substrates, most of them being C1 compounds, like H2/CO2, formate, methanol or methylamines. With the exception of Methanosarcina and Methanolacina, many strains are restricted to only one or two such energy sources. The most common energy-yielding reaction within the methanogens is the reduction of carbon dioxide by molecular hydrogen producing methane, followed by the utilization of formate. Only the representatives of the Methanosarcinaceae and Methanosphaera are unable to grow on these substrates. Acetate can be used by Methanosarcina and Methanosaeta, while the methylotrophic genera (e.g. most members of the Methanosarcinaceae) utilize methanol, several methylamines or methylsulfide. Furthermore, some species grow on primary and secondary short-chain alcohols. Many species are dependent on special growth factors like vitamins, amino acids or acetate. All methanogens can use ammonium as a nitrogen source. In addition, a few species are able to fix molecular nitrogen (e.g. Methanosarcina barkeri (Scherer, 1989) and Methanococcus thermolithotrophicus).

24

All methanogens are strict anaerobes, although some strains tolerate oxygen for a short time (especially Methanosarcina). They usually grow at neutral pH, but a few strains of Methanobacterium still grow at pH 5 and Methanohalobium zhilinae exhibits a pH optimum of 9.2. Most of the methanogens live in environments of low ionic strength (e.g. Methanobacteriales) or in marine biotopes (e.g. Methanopyrales, Methanococcales). However, some methylotrophic methanogens, such as Methanohalobium evestigatum (Zhilina and Zavarzin, 1987), Methanohalophilus mahii and M. halophilus, grow in salt concentrations of up to 3 or 4 mol/l NaCl. Although most methanogens are mesophiles, numerous thermophilic and hyperthermophilic strains and species have been isolated. While Methanothermobacter thermoautotrophicum, the first thermophilic methanogenic organism to be described, grows at temperatures up to 75°C, the hyperthermophilic Methanothermus fervidus (Stetter et al., 1981) and Methanopyrus kandleri (Kurr et al., 1991) have extended the maximum growth temperature of methanogens, growing at 97°C and 110°C, respectively. Within the Methanococcales two hyperthermophiles are known, M. jannaschii and M. igneus, the latter growing at temperatures up to 91°C. Recently, one psychrophilic species, Methanogenium frigidum, has been described (Franzmann et al., 1997). This marine organism grows from the freezing point of the medium to 17°C with an optimum at 15°C.

Methanogens are common organisms, found in all types of anaerobic environments, and are certainly the most prevalent cultivated Archaea in the "moderate" world:

• anoxic sediments and soils - "swamp gas" is methane, which, because of its low ignition

temperature and low threshold concentration, is readily ignited, resulting in the faint white glow of "will-o-the-wisps" visible at night in swamps.

• animal digestive tracts - o rumen of ruminant animals such as cattle, sheep, elk, deer and camels. A cow

belches about 50 liters of methane a day during the process of eructation (chewing the cud).

o cecum of cecal animals such as horses and rabbits. o large intestine of monogastric animals such as humans, swine, and dogs. o hindgut of cellulolytic insects (termites). African termite mounds are thoroughly

aerated by the insects not for oxygen, but to keep methane concentrations low. • wastewater and landfills - the whole anaerobic wastewater process works because

organics in the wastewater are converted first to biomass (in the early stages of treatment), then digested anaerobically to H2, CO2, and acetate which in turn is converted by methanogenesis into methane, which diffuses into the atmosphere.

• oil deposits - natural gas is methane, produced by methanogens living in the subterranean oil deposits, or geochemically in the deeper layers of the bedrock.

• geothermal sources of H2+CO2: hydrothermal vents.

Methanogens form a variety of symbioses with plants, animals and protists, but despite these close associations there are no known pathogenic methanogens. Methanogens also form close syntrophic associations with heterotrophic Bacteria that generate hydrogen (i.e. use protons as the terminal electron acceptor). Hydrogen-generating heterotrophism is only energetically-favorable where the ambient concentration of hydrogen is extremely low. Methanogens associate with these organisms, utilizing the hydrogen they generate for methanogenesis maintain a low hydrogen concentration favourable to the heterotrophs. Neither of these organisms could persist in the environment alone, but together they are successful.

25

The Class Arhaeoglobi The Class consists of one order - Archaeoglobales, contains two genera, Archaeoglobus

(Stetter, 1988) and Ferroglobus (Hafenbradl et al., 1996) (Table 1). All members of the Archaeoglobales are strict anaerobic hyperthermophiles, growing

between 60 and 95°C with an optimum at 80-85°C. They are neutrophilic with a pH optimum around pH 7 (range pH 4.5 to 8.5). Due to their natural habitat (abyssal hot sediments, submarine hydrothermal systems), sodium chloride concentrations up to 4.5% are tolerated, although all species grow optimally at around 2%.

Representatives of the Archaeoglobi show the same blue-green fluorescence that is characteristic of methanogens. Moreover, in Archaeoglobus fulgidus, most of the enzymes and coenzymes typical of methanogenesis are present (with the exception of coenzyme M and F430). Lactate is oxidized to carbon dioxide via a modified acetyl-CoA/CO dehydrogenase pathway, where the methyl group is successively converted to CO2 by the reversion of the methane formation pathway from CO2 in methanogens. Members of the genus Archaeoglobus are resistant to ampicillin, vancomycin, rifampicin and streptolydigin.

The Class Thermococci The Class consists of one order - Thermococcales. Three genera in one family,

represented by numerous species, have been described: Thermococcus, Pyrococcus and very recently Palaeococcus (Takai et al., 2000) (Table 1). Due to its fast growth and easy cultivation, Pyrococcus furiosus (Fiala and Stetter, 1986) has become a model organism for molecular and biochemical studies of (hyperthermophilic) archaea.

All members of the order Thermococcales are strict anaerobic hyperthermophiles with temperature maxima between 85 and 105°C. Optimal growth temperatures are between 75 and 88°C for Thermococcus species and 96-100°C for representatives of the genus Pyrococcus. With the exception of T. alcaliphilus (pH optimum 9.0, maximum 10.5) all organisms are neutrophiles with pH optima between 6 and 8. They grow optimally at NaCl concentrations of between 2 and 3%. This is in agreement with the natural habitats of the Thermococcales, which are marine hydrothermal systems, shallow solfataric marine water holes or hot oil reservoirs. The Thermococcales grow heterotrophically by fermentation or sulfur respiration on a variety of organic compounds such as peptone, yeast extract, meat extract, casein, peptides, casamino acids, and starch. Some strains can use maltose or pyruvate as substrates, and growth on chitin was reported for T. chitonophagus (Huber et al., 1995). The main fermentation products are carbon dioxide, hydrogen, organic acids (isovaleriate, isobutyrate, acetate, formate, lactate), alcohols (e.g. butanol) and amino acids (e.g. alanine). Elemental sulfur significantly stimulates growth of many strains and hydrogen sulfide is produced. For some species (e.g. P. furiosus, P. abyssi, P. horikoshii) molecular hydrogen inhibits growth and the production of hydrogen sulfide in the presence of elemental sulfur is a kind of a detoxification reaction. Growth by sulfur respiration was reported for P. woesei (Zillig et al., 1987) and some Thermococcus strains.

Due to their ability to use a great variety of substrates, members of the Thermococcales contain many extracellular hydrolases. Especially in Pyrococcus furiosus, the pathways of sugar or pyruvate degradation have been intensively studied. Although not all strains and species have been investigated, the Thermococcales exhibit resistance to the following antibiotics: vancomycin, penicillin, kanamycin, streptomycin and chloramphenicol. Some species are sensitive to rifampicin (e.g. T. profundus, T. stetteri, T. litoralis), while T. celer is resistant.

26

The Class Thermoplasmata The Class consists of one order - Thermoplasmatales, which contains three families; the

Thermoplasmaceae, the Picrophilaceae, and the new mesophilic Ferroplasmataceae (Golyshina et al., 2000) (Table 1).

Representatives of this Class are extreme acidophiles, exhibiting pH optima between 0.7 and 2. Picrophilus (Schleper et al., 1995) surpasses all other prokaryotes in this ability by growing well even at around pH 0. All species (exept Ferroplasmataceae family) exhibit temperature optima of around 60°C and a maxima of between 65 and 67°C. Due to their natural habitat (solfataric springs or smouldering coal refuse piles), they need culture media with low ionic strength.

In contrast to the obligate aerobe Picrophilus, Thermoplasma species are facultative anaerobes. They are able to grow in the absence of oxygen by respiration on elemental sulfur with production of hydrogen sulfide. Under aerobic conditions, sulfur is not metabolized (e.g. to sulfuric acid). All Thermoplasmatales are obligate heterotrophs growing on yeast extract, meat extract or bacterial extracts from, for example, Bacillus acidocaldarius, Sulfolobus solfataricus or Desulfurococcus species. It is probably oligopeptides present in these substrates which are metabolized. Therefore, they are scavengers utilizing decomposition products of organisms present in their natural habitat. The addition of sugars results in higher final cell densities, although no growth is obtained on sugars alone. Carbon dioxide, acetate and formate are mainly detected as metabolic products.

Members of the Thermoplasmatales are resistant to vancomycin, bacitracin and streptomycin.

Extreme halophiles (The Class Halobacteria) The extremely halophilic Archaea require at least 2M NaCl or equivalent ionic strength

for growth - most grow in saturated or near-saturated brines. They are the primary inhabitants of salt lakes. Red pigments make it obvious when large numbers of these organisms are present - blooms often occur after a rain carries organic material into a salt lake, and the Red Sea gets its name from such blooms. They are common in hypersaline seas, salt evaporation pools, salted meats, dry soil, salt marshes, etc. They are also found in subterranean salt deposits, where micropockets of saturated water "diffuse" around the otherwise solid salt. Other halophilic organisms (e.g. fungi, brine shrimp) have normal cytoplasmic salt concentrations, expending energy to continuously pumping salt out of the cell and water into the cell, and contain organic osmolytes like glycerol or sugars. Halophilic Archaea grow at much higher salt concentrations, and the internal salt concentrations are as high as they are outside. For this reason, there is little or no osmotic pressure on the cell wall, and some organisms take advantage of this by adopting high surface-area shapes that are not possible for organisms in "normal" ionic strength. One example is Haloarcula (Oren et al., 1999), which occurs in squares and triangles with straight edges, sharp corners, and is very flat. Other halophiles are rods or cocci.

The Class Halobacteria (Grant and Larsen, 1989) consists of one order - Halobacteriales, with one family Halobacteriaceae including 15 genera (Table 1).

Halophiles are mesophilic facultative aerobes. Aerobically, they grow heterotrophically, via respiration using oxygen as the terminal electron acceptor. Anaerobically, they grow photochemotrophically - obtaining energy (ATP) from light, but still require organic compounds as a carbon source.

They do not contain the usual photosystems or electron transport chain for gathering energy from light. Phototrophy is driven by a single protein, bacteriorhodopsin, a light-driven

27

proton pump. This proton pump generates a proton gradient used to make ATP via ATPase, just like in other organisms. It is not nearly as efficient as the bacterial photosystems, but light is rarely limits growth in the desert salt lakes where they predominate.

Some halophiles grow at high pH (up to pH 10-10.5) i.e. Natronobacterium (Xin et al., 2001) in soda lakes. At that pH, any protons pumped to the outside, by electron transport or rhodopsin, are gone forever. Even though the resulting electric potential is still there, it cannot be harvested by an ATPase unless it can obtain protons from the outside. How they get around this is not known, and it is probably this issue that limits the upper pH range of life.

1.3.5 Archaea as "non-extremophiles" ♦

First discovery of new archaeal types The known phenotypic patterns of cultivated Archaea (see the previous sections 1.3.1-

1.3.4) are still largely represented by extreme halophiles, thermoacidophiles and methanogens. Judging solely from cultivated strains, archaeal phenotypic diversity appears limited, in comparison to the wide variety of phenotypes in the Bacteria (Woese, 1987). Consequently it was assumed that Archaea were of ecological significance only in few highly specialized (and predominantly anaerobic) „extreme“ habitats, and were therefore described as "extremophiles". This picture has altered considerably as new molecular biological methods have been applied to the study of naturally occurring microorganisms (Pace, 1997).

The presence of novel uncultivated types of Archaea was first suggested during molecular phylogenetic surveys of marine planktonic microorganisms. This survey of PCR-amplified SSU rRNA genes revealed archaeal-like rRNA gene sequences in seawater samples from 100 m and 500 m depth in the Pacific Ocean (Fuhrman et al., 1992). These oceanic archaeal rRNAs were most closely related to those of Crenarchaeota, a branch of Archaea previously thought to consist of hypertermophiles exclusively. At the same time, microorganisms collected in surface water off the North American coast showed the presence of two new archaeal groups; one crenarchaeotal, one euryarchaeotal (Delong, 1992). Initially, it had to be considered that the planktonic archaea might be allochthonous thermophiles, transported far from a putative hydrothermal vent habitat. However, due to the widespread distribution and the relatively high abundance of the planktonic Archaea, it was thought unlikely. The discovery of high numbers of Archaea in anaerobic, Antarctic waters at temperatures of minus 1.8°C (Delong et al., 1994) and the association of one crenarchaeotal species, Cenarchaeum symbiosum, with a marine sponge living at 10°C (Preston et al., 1996) provided further evidence that the new Archaea were native to cold seawater biotopes.

Diversity and distribution of "non-extreme" Archaea Since their initial detection, evidence for a widespread distribution of new, uncultivated

Archaea has been further extended to marine plankton, shallow and deep-sea sediments, freshwater lakes and sediments, various soils and many other "extreme" and "non-extreme" environments. Table 2 lists the key published research work (up to May 2002) performed on the subject of uncultivated environmental archaeal SSU rRNA. Three major new uncultured archaeal groups (Delong, 1998) were (until recently) encountered in culture-independent ecological surveys (Table 2, Fig. 5 B, C). Group I includes Archaea living in a variety of soil, sediment, marine and freshwater habitats and is related to Crenarchaeota. The other two archaeal clades (Group II and III) fall within the Euryarchaeota with little less variation in habitat occupation.

28

Table 2. Summary of published uncultivated environmental archaeal SSU rRNA clones by their environments. ………………………………………………………………………………………………………………………………………………………… * - as given by the authors in the corresponding publications. ** - Group I is related to Crenarchaeota; Group II and III belong to the Euryarchaeota (section 1.3.5 and Delong, 1998). Sign "+" in column "other" indicates that reported sequences do not affiliated with Groups I, II or III. Sign "-" indicates that no sequences of that group were found.

Reported Group** Clones name Environment description* I II III other

Reference

„Hot“ environments

pJP hot spring, Yellowstone National Park, USA - - - + Barns et al., 1994; Barns et al., 1996

pSL hot spring, Yellowstone National Park, USA + - - + Barns et al., 1996 PVA_OTU hydrothermal vent microbial mat, Pacific Ocean, Hawaii + + - - Moyer et al., 1998

pOSA, pOWA, pUWA

marine and terrestrial hot water environments, Japan + - - + Takai and Sako, 1999

pMC, pIVWA, pISA

deep-sea hydrothermal vent, Japan + - + + Takai and Horikoshi, 1999

O23, O14, O18 high temperature petroleum reservoir - - - + Orphan et al., 2000 pBA hot spring, Yellowstone National Park, USA - - - + Reysenbach et al., 2000a

VC2.1Arc in situ growth chamber, Mid-Atlantic Ridge hydrothermal vent

+ - + + Reysenbach et al., 2000b

SUBT seafloor hydrothermal vent, hot spring, Iceland - - - + Marteinsson et al., 2001a; Marteinsson et al., 2001b

pPACMA black smoker chimney, Manus Basin, Papua New Guinea + - - + Takai et al., 2001a clone A arsenite-oxidizing acid thermal spring, Yellowstone NP,

USA + - - + Jackson et al., 2001

Guaymas AT, CS deep-sea hydrothermally active sediment, Guaymas Basin + - + + Teske et al., 2002 IUA Lidy hot spring groundwater, Idaho, USA + - - + Chapelle et al., 2002

Nanoarchaeum equitans

submarine hot vent, Kolbeinsey ridge, north of Iceland - - - + Huber et al., 2002a

29

Table 2, continued Reported Group Clones name Environment description I II III other

Reference

Marine environments

WHAR Q, WHAR N

marine picoplankton, Atlantic ocean, USA + + - - Delong, 1992

NH marine picoplankton, Pacific Ocean, USA + - - - Fuhrman et al., 1992 SBAR, SB95 marine picoplankton, St.Barbara Channel, USA + + - - Delong, 1992; Massana et al., 1997 ANT, OARB marine picoplankton, Arthur Harbor, Antarctica + + - - Delong et al., 1994 Fosmid 4B7 marine picoplankton, Pacific Ocean + - - - Stein et al., 1996

pM, C marine plankton, Atlantic ocean + - - - McInerney et al., 1997 Mariana marine sediment, Mariana Trench + - - - Kato et al., 1997

pB1 marine picoplankton, Atlantic ocean + + - - Fuhrman and Davis, 1997 p712, pN1 marine plankton, Pacific ocean + + + - Fuhrman and Davis, 1997

BBA marine sediment, Cape Cod, USA + - + - Vetriani et al., 1998 TS, NS-TS suspended particulate matter, North Sea, Netherlands + + - + van der Maarel et al., 1999; van der

Maarel et al., 1998 ODPB-A methane hydrate containing marine fluids and sediments in

the Cascadia margin (ODP site 892B) + - - - Bidle et al., 1999

JBT, JTA, JTB marine sediment cold-seep area, Japan Trench + - - + Li et al., 1999 CRA, ACA, APA deep-sea sediments, Atlantic ocean + - + + Vetriani et al., 1999 Eel-BA, Eel-TA marine sediment, Eel river, California, USA - - + + Hinrichs et al., 1999

A2M marine sediment colonized by the phanerogam Zostera noltii

+ - + + Cifuentes et al., 2000

AT, ME, DN, DS, AM, CA, SB, DF

different marine provinces (Pacific and Atlantic oceans, Mediterranean sea, Antartic)

+ + + - Massana et al., 2000

A1, A2, B, M1- M7 Mediterranean and Antarctic waters at different depth + - - - Garcia-Martinez and Rodriguez-Valera, 2000

CRO coastal ocean near Columbia river, USA + - - - Crump and Baross, 2000 PENDANT, BUR-TON,CLEAR,ACE

anoxic sediments of marine salinity meromictic lake, Eastern Antarctica

- - + + Bowman et al., 2000b

30

Table 2, continued Reported Group Clones name Environment description I II III other

Reference

DH148-, GIV- deep-sea site, Antarctic Polar Front - + + + Lopez-Garcia et al., 2001b DH148- deep-sea site, Antarctic Polar Front - + + + Lopez-Garcia et al., 2001a

Car anoxic zone of the Cariaco Basin + - - + Madrid et al., 2001b Eel, SB anoxic marine sediments, Californian continental margin,

USA - - - + Orphan et al., 2001

VIARC, CIARC, CRARC, TOARC,

BANARC

planktonic assemblages of several anaerobic, sulfide-rich lakes

+ - + + Casamayor et al., 2001

AT 425 gas hydrate mineral, Gulf of Mexico - - + + Lanoil et al., 2001 Cas deep gas hydrate sediments from the Cascadia Margin - - - + Marchesi et al., 2001

ANME marine sediment, Aarhus Bay, Denmark - - - + Thomsen et al., 2001 33-FL, 33-P hydrothermal vent fluids, Juan de Fica Ridge, Oregon, USA + + + + Huber et al., 2002b

Hypersaline environments

HAC crystallizer ponds from a marine saltern - - - + Benlloch et al., 19951MT, 2MT, 2C salt marsh sediments, UK - - + + Munson et al., 1997

KTK highly saline brine sediments of Kebrit Deep, Red Sea - + + - Eder et al., 1999 MSP alkaline saternat Lake Magadi, Kenya, East Africa - - - + Grant et al., 1999

Dec., June hypersaline stratified lake Solar, Sinai, Egypt - - + + Cytryn et al., 2000 DEEP, ORGANIC three hypersaline Antarctic lakes - - - + Bowman et al., 2000a Nh.2, B1bra, 2Br ancient solt deposit - - - + McGenity et al., 2000

Env, CD,CLI, YELI crystallizer ponds from a solar saltern, Spain - - - + Benlloch et al., 2001 A Alpine Permo-Trassic rock salt - - - + Radax et al., 2001

31

Table 2, continued Reported Group Clones name Environment description I II III other

Reference

Soils

FIE16 soybean field soil, Japan + - - - Ueda et al., 1995 FFSB forest soil, Finland + - - - paper I, 1997

GU,GA,SC,SW,SO rice soil, Japan + - - + Kudo et al., 1997 P17, M17 forest soil, Brazil - - - + Borneman and Triplett, 1997

SCA agricultural soil, Wisconsin, USA + - - - Bintrim et al., 1997 KBS agricultural soil, Michigan, USA + - - - Buckley et al., 1998 ABS anoxic rice soil, Italy + - + + Grosskopf et al., 1998a; Grosskopf

et al., 1998b Arc. deep subsurface paleosol, Washington St. USA + - - - Chandler et al., 1998

FFSA, FFSC forest soil, Finland + - - - paper II, 1999 ST, S15, S30 anoxic rice soil, Italy + - - + Chin et al., 1999

SW, SC rice soil, Japan - - - + Kim et al., 2000 AS00, AS01, A08,

AS17 rice soil, Italia + - + + Lueders and Friedrich, 2000

Shen, Gap rice soil, China, The Philippines + - - + Ramakrishnan et al., 2001 ARS anoxic rice soil, Italy + - - + Weber et al., 2001

S agricultural soil and casts of earthworm + - - - Furlong et al., 2002 Symbionts and other environments

JM sea cucumber midgut, Atlantic ocean + - - - McInerney et al., 1995 R peat bogs - - - + Hales et al., 1996

C. symbiosum marine sponge tissue, Pacific Ocean + - - - Preston et al., 1996 Vadin (DA,DC,CA) biofilm of a fluidized-bed anaerobic digestor + + - + Godon et al., 1997

FIN flounder digestive tract + + - + van der Maarel et al., 1999; van der Maarel et al., 1998

FF flounder feces - + - + van der Maarel et al., 1999; van der Maarel et al., 1998

32

Table 2, continued Reported Group Clones name Environment description I II III other

Reference

GIN492 grey mullet digestive tract - + - - van der Maarel et al., 1998 ARR rice roots + - + + Grosskopf et al., 1998b

WCHD, WCHA hydrocarbon- and chlorinated-solvent-contaminated aquifer + - + + Dojka et al., 1998 MUG, TUG mesophilic and thermophilic granular sludges - - - + Sekiguchi et al., 1998

Cd30, MP, MN, MO, MH

intestinal microflora of the termite gut - - - + Ohkuma et al., 1999; Ohkuma and Kudo, 1998

Ar swine waste storage pit - - + + Whitehead and Cotta, 1999 H2, EtOH washed rice roots - - - + Lehmann-Richter et al., 1999

R associated with rumen ciliate - - - + Tokura et al., 1999 RS hindgut of the lower termite Reticulitermes speratus - - - + Shinzato et al., 1999

Aglo 120 copper commercial-scale bioleaching plant (mixed isolate) - - + - Vasquez et al., 1999 SJC, SJD anaerobic TCB transforming microbial consortium - - - + von Wintzingerode et al., 1999

TRC terrestrial plant roots + - - - Simon et al., 2000 SC lithotrophic biofilm at an extreme acid mine drainage site - - + - Bond et al., 2000

cM, c, cHole cow rumen - - + + Tajima et al., 2001 ARC bovine rumen - - - + Whitford et al., 2001

Cren21-, P-Ar- intestinal tract of temite Cubitermes orthognathus + - - - Friedrich et al., 2001 UASB-TA terephthalate-degrading anaerobic granular sludge system - - - + Wu et al., 2001

AC industrial dye effluent in an anaerobic baffled reactor - - - + Plumb et al., 2001 H1, H6, S3 ancient wall paintings - - - + Pinar et al., 2001

ZmrA roots of Zea mays L. + + - - Chelius and Triplett, 2001

33

Table 2, continued Reported Group Clones name Environment description I II III other

Reference

Freshwater and "mixed" environments

pLem, pGrf freshwater lake sediment, Lake Lemon and Griffy, USA + - - - Hershberger et al., 1996 pLaw freshwater lake sediment, Lawrence Lake, USA + - - + Schleper et al., 1997a LMA freshwater lake sediment, Lake Michigan, USA + - - + MacGregor et al., 1997

Winarc freshwater lake sediment, Lake Windermere, UK - - - + Miskin et al., 1998 Rot freshwater lake sediment, Lake Rotsee, Switzerland - - - + Zepp et al., 1999

VAL freshwater lake water, Valkea Kotinen Lake, Finland + - + + paper IV, 2000 CRE, CR Columbia river and it's estuary, USA + + - - Crump and Baross, 2000 N1d, N41r mobile deltaic muds of Southeastern Papua New Guinea + - - + Todorov et al., 2000

Soyang, SYA sediment of meso-eutrophic Lake Soyang, Korea - - - + Go et al., 2000 Green Bay, AR freshwater ferromanganous micronodules and sediments + - - + Stein et al., 2001

DOURO estuarine sediment, river Douro, Portugal + - - + paper III, 2001 CL500-AR bacterioplankton ultra-oligotrophic Crater Lake, USA + - - - Urbach et al., 2001

a50ev, a60av, mat from Lake Fryxell, McMurdo Dry Valleys, Antarctica + - - + Brambilla et al., 2001 SAGMA deep subsurface South African gold mine environments + - - + Takai et al., 2001b

KS mobile mud deposit, French Guiana + - - - Madrid et al., 2001a EHB freshwater sediment, River Colne estuary, UK - - - + Purdy et al., 2002

34

Studies of the phylogenetic identity, diversity, and distribution of the new uncultivated archaeal groups have revealed that with respect to their ecological distribution, the non-thermophilic Crenarchaeota from Group I seem to be the most widely distributed and abundant form of all known Archaea. They occupy many different habitats and ecological niches (Table 2). A recent study of Archaea in the mesopelagic zone of the Pacific Ocean shows that pelagic Crenarchaeota represent one of the ocean’s single most abundant cell types and, globally, the oceans harbor approximately 1.3x1028 archaeal cells (comparing to 3.1 x1028 of bacterial cells) (Karner et al., 2001). Current data suggests that several lineages of hyperthermophilic crenarchaeotes adapted to colder habitats independently (Pace, 1997; Hershberger et al., 1996; Barns et al. 1996) and their expansion into non-thermophilic habitats occurred during the mid-Cretaceous period (124 to 83 millions years ago) (Kuypers et al., 2001). Analysis of available sequences indicates that there is greater sequence divergence in euryarchaeal Group II rRNA genes isolated from the same biotop, relative to that of sympatric marine Group I rRNA genes. The significant rRNA sequence divergence among the new archaeal types probably reflects substantial physiological diversity too. On the basis of their phylogenetic position, it appears that the non-thermophilic archaea have thermophilic ancestries.

In the latest review “Exploring prokaryotic diversity in the genomic era” by Hugenholtz, 2002, 18 archaeal phylum-level lineages were described, 8 had cultivated representatives and 10 that did not.

♦ Biological characterization of the uncultivated Archaea

The ease and accessibility of gene amplification via the PCR approach has opened the

gates to a remarkable amount of comparative rRNA sequence data. Although necessary and extremely useful, gene phylogenies and associated phylogenetic probes do not provide comprehensive biological characterization. The recovery of rRNA and rRNA genes from the environment allows phylogenetic analysis and quantification of uncultivated Archaea in environmental samples. Reproducible patterns of distribution and variability monitored by phylogenetic probes can lead to clues about habitat preferences, probable energy sources, and physicochemical tolerances, but such conclusions are necessarily tentative and require further verification via more detailed physiological, biochemical and genetic characterization. What progress can be made in the characterization of these organisms in the absence of pure cultures? The development of general approaches for characterizing as yet uncultivated microbial species presents a major challenge for modern microbiologists. Uncultivated Archaea represent good test organism for new approaches, as their phylogenetic diversity is reasonably limited and unique archaeal biochemical signatures (such as specific rRNA sequence motifs, or cell membrane lipids) can be detected in mixed populations.

Advances in genomic analysis are providing new technologies that may be useful for characterizing uncultivated prokaryotes. The main requirement is the availability of pure, intact, high molecular weight genomic DNA. Large DNA fragments can be recovered from mixed microbial populations using modern genomic techniques. Analysis of these large fragments can yield information on gene organization, structure and content of uncultivated Archaea. The archived genome fragments of uncultivated microbes can be viewed as reagents, useful for expressing protein-encoding genes, determining enzyme structure and function, or dissecting metabolic pathways. Microbial groups previously defined solely by rRNA gene phylogeny can, at least partially, be characterized by genome content and biochemical characteristics. Examples of such an approach resulting in the genomic analysis of "non-thermophilic" crenarchaeotes are the fosmid DNA libraries prepared from a marine picoplankton assemblage (Stein et al., 1996) and the discovered archaeal symbiont

35

Cenarchaeum symbiosum (Preston et al., 1996; Schleper et al., 1997b). Two other examples are the construction of large-insert bacterial artificial chromosome (BAC) libraries from the genomic DNA of planktonic marine microbial assemblages (Beja et al., 2000) and from genomic DNA isolated directly from soil (Rondon et al., 2000).

36

2 AIMS OF THE STUDY

The increasing discovery of "non-extreme" Archaea in a wide variety of environments

raises important questions about their phylogenetic and systematic position and evolution. Most of these new Archaea are, as yet, uncultivated. As a result, their genomes, and biochemical and metabolic characteristics cannot be determined easily. However, a better understanding of their role in the environment is necessary in order to evaluate their importance in biogeochemical cycles, food web processes and interactions, and their contribution to ecosystem stability and flexibility. Due to their resistance to cultivation in vitro, new modern molecular and biochemical methods should be used for their identification and characterizations in situ.

The main aim of the research was to phylogenetically identify, characterize and compare yet unknown and uncultured indigenous Archaea found in boreal forest soil, temperate estuarine sediment and boreal freshwater lake using available modern molecular techniques. The second aim was to build and maintain an ARB database of aligned 16S rRNA archaeal sequences as an essential tool for analysis and systematization of novel archaeal sequences and a convenient instrument for designing/evaluating hybridization probes and PCR primers.

37

3 MATERIALS AND METHODS

3.1 Sampling and nucleic acid extraction 3.1.1 Soil sampling (Finland)

The humus samples were taken from a mixed forest in Northern Finland (65° 15´ N, 28°

50´ E) with a podzol on moraine soil type. The sampling area was covered with Norway Spruce and Scots Pine and consisted of two treatments: clear-cutting (A) and clear-cutting followed by prescribed burning two years later (B). The samples collected one year after the prescribed burning included a control soil (C) from an untreated standing forest. Each of the three bulk “composite samples” consisted of a mixture of twenty subsamples (twenty individual cores sampled with a 72-mm-diameter soil corer) (Pietikainen and Fritze, 1995). The composite samples were collected from the different sites on the same day. The area of each site was 50 m x 50 m, and the minimal distance between any of the 20 subsamples was 2 m. Crude DNA was isolated from a soil sample using proteinase K, cetyltrimethyl ammonium bromide and chloroform, and then applied to a Wizard DNA Clean-Up System Minicolumn (Promega, USA) with isopropanol purification (Saano et al., 1995).

3.1.2 Water sampling for microbial DNA extraction (Finland)

Lake water was taken from mesohumic boreal lake Valkea Kotinen which is located in

the Evo forest area of southern Finland (61°24` N, 24°07` E). Water was sampled using a 2.5 liter Limnos sampler from the surface (0.1 m) down to the deepest water layers (7 m) at 0.5 m intervals from the pelagial.

For microbial DNA extraction, integrated water samples (0-7 m) were taken from the pelagial in July and stored in pre-cleaned, sterile Nalgene bottles. The samples were then kept on crushed ice and in the dark prior to filtration. Cells from 250 ml of water were collected by filtration onto hydrophilic polycarbonate filters (2-µm pore size, 47 mm diameter (Whatman, UK)) and frozen immediately at -25°C until nucleic acids extraction.

Extraction began with the addition of 5 ml of lysis buffer (1 mg/ml lysozyme, 40 mM EDTA, 50 mM TrisHCl, 0,75 M sucrose, pH 8.0) and incubation at 37°C for 30 min (Massana et al., 1997). Proteinase K (0.5 mg/ml) and sodium dodecyl sulfate (1% w/v) were then added to the filter and incubated at 55°C for 2 h. The lysate was recovered from the filter and placed into a fresh tube. The filter was then rinsed with an additional 2 ml lysis buffer and incubated at 55°C for 10 min before pooling the lysates. To the pooled lysate, 5 M NaCl (final concentration 0.7 M) and hexadecyltrimethyl ammonium bromide (final concentration 1% w/v in the presence of 0.7M NaCl) were added. This mixture was incubated at 65°C for 20 min before extraction with chloroform-isoamyl alcohol (24:1). The upper aqueous-DNA phase was removed and placed into a fresh tube and DNA precipitated after the addition of 0.6 volumes of isopropanol. The pellet was washed with 70% (v/v) ethanol, dried and dissolved in 100 µl of water. This crude DNA sample was then further purified using a Wizard DNA clean up kit (Promega, USA) (Saano et al., 1995) before use as template DNA for PCR.

38

3.1.3 Estuarine sediment sampling (Portugal) Sediment cores of 25 cm (length) by 5 cm (diameter) were collected at several sampling

sites along the intertidal bank of the river Douro (Portugal) estuary (41º 08' N, 08º 40' W), where the salinity ranged from 1 to 5 practical salinity units. They were composed mainly of coarse sand (dominant particle size > 500 µm). One of the cores showed a clearly visible 2 cm-thick black layer of thin, compact sediment 10 cm from the surface, and a 3 cm-thick orange layer 19 cm from the surface. The cores were kept in the dark at 4ºC and processed within 1 hour. Core slice samples (4-5 mm) were taken at different depths of each core, including the top aerobic region of all samples and the 10 cm and 19 cm layers of the above mentioned core.

The samples were suspended in 4 ml SET-buffer (20% sucrose, 50 mM EDTA, 50 mM Tris-HCl pH 8.0) before the addition of 150 µl lysozyme (5 mg/ml SET), 400 µl SDS (10% w/v) and 100 µl proteinase K (20 mg/ml in SET-buffer). The samples were incubated at 60ºC for 30 min before centrifugation at 10,000 x g for 10 min. The supernatant fluids were then extracted twice with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1). To precipitate the DNA, 0.1 volumes of 3 mol/l sodium acetate (pH 5.2) and 2 volumes of 100% ethanol were added. The DNA was collected by centrifugation (14,000 x g, 10 min), washed in 70% ethanol, dried and resuspended in sterile distilled water.

3.2 PCR amplification, cloning and clones characterization Fragments of 16S rRNA gene (positions 7-927, Escherichia coli numbering) were

amplified using a nested PCR approach with primers described in Table 3. First round PCR was performed using two sets of primers: (i) Ar4F - Un1492R and (ii) Ar4F - Ar958R, producing amplicons approximately 1500 and 1000 bp in length, respectively. Both sets of first round PCR products were then used as template in a nested PCR using primer set Ar3F - Ar9R. These Archaea-specific primers hybridized at positions internal to those targeted by primers used in both first round PCR reactions, and generated fragments approximately 900 bp in length. DNA extracted from soil samples was amplified using a single round of PCR with the "internal" primer set Ar3F - Ar9R primers.

Reaction mixtures of 20 µl contained 1-5 ng of DNA template and 100 pmol of each primer. PCR was carried out using the following reaction conditions:

• for soil and water samples: 94°C for 4 min; 40 cycles of 94°C for 1 min, 55°C for 1

min, 73°C for 3 min. • for sediment samples: 94ºC for 4 min, 42 cycles of 94ºC for 1 min, 45ºC for 1 min,

and 73ºC for 2 min.

PCR products were only accepted for further analysis when a simultaneous negative control PCR (water instead of DNA) showed no amplification. PCR products were subject to agarose gel electrophoresis, and DNA of appropriate size was extracted from the gel and cloned into pGEM-T vector plasmid (Promega, USA). The archaeal origin of the inserts was checked by Southern blot hybridization using an Archaea-specific probe (the corresponding region of 16S rRNA from Halobacterium salinarum DSM 668 without primers). For hybridization conditions, see paper II. The inserts of the clones (74 clones, paper I; 138 clones, paper II; 235 clones, paper III; 160 clones, paper IV) were divided into groups according RFLP patterns using: AvaII (paper III); AvaII and MspI (paper IV); AvaII, MspI and RsaI (papers I and II) restriction enzymes.

39

Table 3. PCR primers and FISH probes used in this thesis. …………………………………………………………………………………………………………………………………………………………. a Modifications in primers Ar4F and Ar958R compared to reference’s primers were made by author b Target site (position numbers refer to the E.coli 16S rRNA) c FISH probes overlaps shown in bold d Formamide concentration in % (v/v) for the in situ hybridization buffer

Name Sequence (5'-3') Comments References PCR primers

Ar4F a (8-25) b TCY GGT TGA TCC TGC CRG forward primer, specific to Archaea 16S rRNA gene Hershberger et al., 1996 Ar958R a (958-967) YCC GGC GTT GAV TCC AAT T reverse primer, specific to Archaea 16S rRNA gene Delong, 1992

Ar9R (906-927) CCC GCC AAT TCC TTT AAG TTT C reverse “internal nested” primer, specific to Archaea 16S rRNA gene.

paper I

Ar3F (7-26) TTC CGG TTG ATC CTG CCG GA forward “internal nested” primer, specific to Archaea 16S rRNA gene.

paper I

Un1492R (1492-1510) GGT TAC CTT GTT ACG ACT T universal reverse primer, specific to all types of 16S/18S rRNA gene.

Delong, 1992

FISH probes c F d ARCH915 (915-934) GTG CTC CCC CGC CAA TTC CT specific to most Archaea sequences Stahl and Amann, 1991 20 CREN499 (499-515) CCA GRC TTG CCC CCC GCT specific to some Crenarchaeota sequences Burggraf et al., 1994 0 CREN512 (512-527) C GGC GGC TGA CAC CAG specific to most Crenarchaeota sequences in the

updated ARB database paper IV 0

CREN569 (569-585) GCT ACG GAT GCT TTA GG specific to most environmental Crenarchaeota sequences in updated database

paper IV 0

EURY496 (496-512) GTC TTG CCC GGC CCT TTC specific to only Methanomicrobiales group and new VAL 47, VAL 1, VAL 9, VAL 78 clones .

paper IV 20

EURY498 (498-511) C TTG CCC RGC CCT T specific to some Euryarchaeota sequences Burggraf et al., 1994 0 EURY499 (499-514) CG GTC TTG CCC GGC CCT specific to Methanosarcina, Methanosaeta,

Methanomicrobiales and new VAL 47, VAL 1, VAL 9, VAL 78 clones.

paper IV 20

EURY514 (514-528) G CGG CGG CTG GCA CC specific to most Euryarchaeota sequences in the updated ARB database.

paper IV 20

40

Several clones from each RFLP group (or just one when represented by a single clone) were chosen for sequence analysis. Altogether, the following were sequenced:

• from soil samples - 15 clones (accession numbers X96688 - X96696, Y08984, Y08985,

and AJ006919 - AJ006922); • from water samples - 28 clones (accession numbers AJ131263 - AJ131278 and

AJ131311 - AJ131322); • from sediment samples - 14 clones (accession numbers AF201355 - AF201368).

3.3 Conditions for in situ hybridization analysis 3.3.1 Sampling for in situ hybridization

Samples for fluorescent in situ hybridization were collected during a period from March

to September from three depths: the epilimnion (0-1 m depth), the metalimnion (1-3 m depth) and the hypolimnion (3-7 m depth, near the bottom sediment). Water samples were taken at 0.5 m intervals with a one-liter Limnos sampler in the pelagial of the lake at the deepest point. Replicate lake water samples were taken from the epi-, meta- and hypolimnetic strata, pooled and stored in sterile 5 liter Nalgene bottles. Samples were kept on crushed ice and in the dark during transportation to the laboratory. Within 2-3 hours after sampling, the microbial cells were concentrated from 15-20 ml of water by filtration onto polycarbonate filters (0.2 µm pore size, 47 mm diameter) (Whatman, UK) by vacuum filtration at < 100 P m-2. The filters were prepared and fixed for FISH as described previously (Amann, 1996; Glöckner et al., 1996) and stored at -25°C until further processing.

3.3.2 Hybridization conditions and probes description

Fluorescent in situ hybridization followed the protocol of Amann, 1996 and Glöckner et

al., 1996. Oligonucleotides labeled with the dye carbocyanine CY3 were purchased from INTERACTIVA (Germany). The specificity, nucleotide sequence and respective hybridization buffer formamide concentration for each probe is summarized in Table 3.

To detect Archaea in the samples, previously described 16S rRNA gene probes EURY498, CREN499 (Burggraf et al., 1994) and ARCH915 (Stahl and Amann, 1991) were used with new probes designed using the ARB "Probe_design" program (Strunk and Ludwig, 2000). In addition to the EURY498, three new probes for the Euryarchaeota (EURY514, EURY496 and EURY499) were designed taking into account new Archaea sequences including the VAL clones (paper IV). New probes CREN512 and EURY514 targeted sequences at the kingdom level and were specific to the Cren- and Euryarchaeota, respectively. New probe CREN569 was specific to most environmental sequences of Crenarchaeota. The optimal formamide concentration for hybridization with new probes CREN512, EURY499 and EURY514 were determined using pure cultures of Crenarchaeota (Pyrobaculum neutrophilum, Sulphobococcus zilligii, Thermoproteus tenax) and Euryarchaeota (Methanosarcina barkery, Methanobacterium formicium). Pure cultures of Bacteria (Escherichia coli and Sinorhizobium) were used as negative controls for all Archaea probes. Probe-specific cell counts were calculated taking into consideration counts obtained with negative control NON338 (Wallner et al., 1993).

For hybridization, a section of the filter was placed on a glass slide, overlaid with 20 µl of hybridization solution (0.9 M NaCl, 20 mM Tris/HCl (pH 7.4), 0.01% SDS w/v, 0-20% (v/v) formamide (Table 3) and 50 ng of probe) and incubated at 46°C for 2 h in an

41

equilibrated humid chamber (Glöckner et al., 1996). Optimal formamide concentrations for the new probes were determined empirically using a formamide series from 0% to 50% (v/v) at 10% intervals. The filters were transferred to a pre-warmed (48°C) vial containing 50 ml of washing solution (70 mM NaCl, 20 mM Tris/HCl (pH 7.4), 5 mM EDTA, and 0.01% SDS (w/v)) and incubated floating freely at 48°C for 15 min. Filters were dried on filter paper (Whatman, UK) and overlaid with 50 µl of DAPI solution (1 µg/ml in water) for 5-10 min at room temperature in the dark. The filters were briefly washed in 70% (v/v) ethanol and then in distilled water, and dried on filter paper before mounting on slides with Citifluor AF1 (Citifluor Ltd., Canterbury, UK). Epifluorescence microscopy was performed as described previously (Glöckner et al., 1996) using a Zeiss Axioplan microscope equipped with specific filter sets (DAPI: Zeiss01 and CY3: Chroma HQ41007, Chroma Tech. Corp., Brattleboro, VT, USA).

3.4 Phylogenetic analysis Programs CHECK_CHIMERA, RANK_SIMILARITY and SUGGEST_TREE from the

RDP (Ribosomal Database Project) (Maidak et al., 2001; Maidak et al., 1996) were applied to the studied sequences in order to detect possible chimeric artifacts, to find the most similar sequences from the RDP database, as well as place sequences on an existing phylogenetic RDP tree.

In the case of the FFSB soil sequences (paper I), reference 16S rRNA sequences were retrieved from GenEMBL and RDP databases and aligned manually according to their primary and secondary structures using Gelassembler sequencing editor (WISCONSIN PACKAGE, Version 8.1) (Wisconsin Package Software, 1994). Sites of uncertain alignment were excluded from the analysis. Three methods of phylogenetic analysis from PHYLIP package version 3.5c (Felsenstein, 1993) were used; neighbor-joining (using distances calculated with Jukes and Cantor correction), parsimony and maximum likelihood. Bootstrap support (Felsenstein, 1985) (PHYLIP package) was calculated to check the topology perturbation in the neighbor-joining and parsimony methods.

In the case of all water, sediment and all other soil sequences (paper II) phylogenetic analysis was performed using the package ARB (Strunk and Ludwig, 2000). Only sequences with sufficient length for phylogeny (min. 400 nucleotides) were included in the analysis. The ARB_EDIT tool was used first for automatic sequence alignment. The sequence alignment were then corrected manually. Regions of ambiguous alignment were excluded from the analysis. A 50% invariance criterion for the inclusion of individual nucleotide sequence position in the analysis was used to avoid possible treeing artifacts. During tree construction, several trees differing in the set of alignment positions as well as reference sequences were used. Phylogenetic trees were inferred by performing maximum likelihood (fastDNAml) (Olsen et al., 1994), maximum parsimony (Felsenstein, 1993) and neighbor-joining (with Jukes-Cantor distance correction (Jukes and Cantor, 1969)) analysis included in the ARB package.

42

4 RESULTS

4.1 ARB database and Archaea trees reconstruction Even if it were possible to construct a perfect tree, local topologies could be expected to

change as new data accumulated and was used to reconstruct the trees. In other words, phylogenetic trees are dynamic constructs that may change when new sequence information is included. New data confer additional information, new identities, and new differences resulting in branch attraction or repulsion. Formerly stable clusters may be dissected, while separated groups may be unified. The trees are "growing" and changing - very much like real ones. In general, for the definition, verification, and positioning of new phyla, one needs data collections containing a reasonable number of sequences representing different levels of relatedness.

As more 16S rRNA sequences accumulate from both cultured and uncultured archaea, the boundaries of existing phyla are being challenged and need to be revised. In order to evaluate the current (May 2002) state of archaeal phylogeny, the ARB software package was used. An ARB database consisting of more than 2500 complete and partial archaeal 16S rRNA sequences was built. Most of the sequences were imported from the EMBL database and about 80% of sequences were published in scientific journals (Table 2). Sequences were aligned and manually corrected taking into account the secondary structure of the rRNA molecule. This database is available upon request at German Jurgens' Website (ARB page http://honeybee.helsinki.fi/users/gjurgens/Arb/arb_page.htm).

Many entries retrieved from public sequence databases contain only partial sequences and some of them do not overlap. To avoid reconstruction artifacts arising from partial sequences, three separate “backbone” maximum-likelihood trees were constructed (Fig. 5): for Korarchaeota (A) (with novel clones, phylogenetically separate from all three phyla), Crenarchaeota (B), and Euryarchaeota (C). The partial sequences were then added, using the unique ARB program "parsimony" option, to backbone trees without changing the overall tree topology. This method allows the construction of big trees with sequences that do not overlap. The constructed trees give an overview of the "initial grouping" and relationship of sequences. These trees help in choosing groups of sequences with correct references which allow further work on more detailed and accurate tree reconstruction. Major lineages (phyla) on Crenarchaeota and Euryarchaeota trees are shown as wedges with horizontal dimensions reflecting the known degree of divergence within that lineage. Phyla with cultivated representatives are in grey and, where possible, named according to the taxonomic outline of Bergey’s Manual (Garrity, 2001). Phyla known only from environmental sequences are in white; because they are not formally recognized as taxonomic groups. They are usually named after the first clones found from within the group (Hugenholtz, 2002). Another interesting and important utilization of the constructed ARB database and trees are the “Probe design" and "Probe match” features of the ARB phylogenetic package. These features allow rapid searching of closest relatives or specific sequence signatures. Such signatures can be evaluated for taxon specificity against the full database and used as probes for hybridization and/or as PCR primers. This handy feature is unique to the ARB software package and allows quick design and evaluation of new and available probes and primers.

43

Euryarchaeotaphylum

Ko

rarc

ha

eo

tap

hylu

m

Crenarchaeotaphylum

BACTERIA DOMAIN

A Pyrococcus abyssi, Z70246 Thermococcus barossii, U76535

Methanopyrus kandleri, M59932 Nanoarchaeum equitans, AJ318041

Pyrodictium occultum, M21087 Sulfolobus acidocaldarius , U05018

Thermofilum pendens, X14835 pUWA43, acidic hot spring water, AB007306 pOWA54, marine hydrothermal vent water, AB007305 pOWA19, marine hydrothermal vent water, AB007304 pUWA9, acidic hot spring water, AB007315 C1_R025, hydrothermal vents sediment, Guaymas Bas, AF419639

pBA5, hot Calcite Springs, AF176347 pJP27, hot spring, L25852

OlA−431, Icelandic hot spring, AF311362 pJP78, hot spring, L25303 SRI−306, hot spring, AF255604

OlA−11, Icelandic hot spring, AF311360 SW1, Japanese paddy soils, D88483

SW14, Japanese paddy soils, D88481 SC1, Japanese paddy soils, D88482

SC7, Japanese paddy soils, D88484 SC6, Japanese paddy soils, D88485

SW9, Japanese paddy soils, D88487 SW3, Japanese paddy soils, D88488 SC4, Japanese paddy soils, D88489

SWY, Japanese paddy soils, D88486 SC2, Japanese paddy soils, D88480

pMC2A249, deep−sea hydrothermal vent, AB019714 pSSMCA1, deep−sea hydrothermal vent, AB019716 pMC2A14, deep−sea hydrothermal vent, AB019715 pMCA256, deep−sea hydrothermal vent, AB019717

pOWA133, marine hydrothermal vent water, AB007303 Escherichia coli, J01695

Thermotoga maritima, M21774 Aquifex pyrophilus, M83548

0.10

Figure 5. Phylogenetic trees showing present archaeal diversity derived from comparative analysis of 16S rRNA gene sequences. The trees were constructed using the ARB software package with an ARB sequence database consisting of more than 2500 complete and partial sequences. Major lineages (phyla) on Crenarchaeota and Euryarchaeota trees are shown as wedges with horizontal dimensions reflecting the known degree of divergence within that lineage. The numbers inside the wedges indicate total numbers of sequences included in each group. Phyla with cultivated representatives are in grey. Phyla known only from environmental sequences are in white and named after the first clones found from within the group. Detailed explanations of tree reconstruction are given in the text (see section 4.1). The scale bar represents 0.1 changes per nucleotide.

A. Tree for Korarchaeota and other novel clones separated from Cren-, Eury-, and Korarchaeota. B. Tree for Crenarchaeota. C. Tree for Euryarchaeota.

44

B

Korarchaeota phylumEuryarchaeota phylum

Uncultured Crenarchaeota

Group I −

338

SAGMA2

164

Soil group 1

SUBT−14, subterranean hot springs, AF361211 SUBT−13, subterranean hot springs, AF361212

46

pSL12, hot spring , U63343 pSL50, hot spring , U63342

Hot Spring 1

MarBenthGrB (DHVC)

Soil group 2

96

pJP89, hot spring, L25305 pSL123, hot spring , U63345

pSL17, hot spring , U63339

Archaeoglobus fulgidus, X05567 Y00275 Halobacterium halobium, M38280

Pyrococcus abyssi, Z70246

GroupI.2

GroupI.3a

GroupI.1a

GroupI.1b

GroupI.1c

Hot Spring 2

YNPFFA

GroupI.3b

Hot Spring 3

119

24

15

11

7

6

12

4

8

17

13

13

Cultured Crenarchaeota (Thermoprotei class)

0.10

45

Crenarchaeota phylum

Ther

mop

lasm

ata

clas

s

C Methanosacinaceae 219

122

34

197

72

291

179

89

Vadin 48

53

49

29

68

Thermococci class 83

Methanopyrus kandleri, M59932 Desulfurococcus mobilis , X06188

Pyrodictium occultum, M21087 Thermoproteus tenax, M35966

DHVEG

DH148

Rice II

Methanomicrobiales

ANME1

8

12

17

14

5

13

Methanosaetaseae

Group II

Group III

Thermoplasmatales

Archaeoglobi class

Sediment1

pMC2

Methanococcales

11

6

19

WCHA1

pMC1

SAGMA1

VAL2

Rice I

Halobacteria class

Methanobacteria class

Met

hano

saci

nacl

es

0.10

46

4.2 Phylogeny of Archaea from soil Soils are dynamic, complex systems of inorganic, organic and biotic components that

have the capacity to support plant life. Both culture-based and culture-independent approaches support the statement that soil represents one of the most diverse habitats for microorganisms (Hugenholtz et al., 1998). Pioneering studies by Torsvik and co-workers examined the diversity of natural communities in soil by DNA-DNA reannealing experiments. In these analyses, bulk DNA was isolated from a soil bacterial fraction obtained by differential centrifugation (Torsvik et al., 1990; Torsvik et al., 1996). Reannealing measurements revealed that the DNA isolated directly from soil was much more complex than expected and suggested that thousands of independent genomes were present in the sample. Extrapolation of the data suggests that there may be thousands of microbial types in a gram of soil, many of which are assumed to be uncultured.

Research on the diversity of "non-extreme" Archaea in soil (apart from euryarchaeotal methanogens) is a rather new sector in Archaea discipline and started with the discovery of the novel Crenarchaeota in Finnish forest soil (paper I). This was the first soil crenarchaeotal 16S rRNA group of sequences ever submitted to a public data bank (EMBL database AC X96688-X96696, submitted 20 March 1996). One short 287 nucleotide long sequence named "FIE16" was obtained by Ueda et al., 1995. The following submission of sequences and research papers (Bintrim et al., 1997; Kudo et al., 1997; Borneman and Triplett, 1997; Buckley et al., 1998; Bomberg et al., 2002) further demonstrated the diversity of Archaea in different types of soils and emphasized our lack of knowledge about them.

In paper I nine archaeal 16S rRNA gene sequences (named FFSB) were obtained by PCR amplification with Archaea-specific primers and template DNA extracted directly from a forest soil sample. The three used methods of phylogenetic analysis used undoubtedly placed eight of the obtained sequences in a group which was distantly related to the all other known crenarchaeotal sequences. This group was situated on the same lineage as the planktonic clade (low-temperature marine Archaea Group I (Delong, 1992)) together with other environmental sequences between the latter and the rest of the Crenarchaeota (Fig.1, paper I). One of the clones, FFSB6, was placed on an individual branch.

FFSB clones from paper I were divided into two clusters according to their hybridization with oligonucleotide probes FFS-Uni and FFS-6. These two probes were designed based on sequence data, as well as phylogenetic and secondary structure analysis of clear-cut followed by prescribed burning soil clones. Probe FFS-6 was designed to be specific to the phylogenetically distinct clone FFSB6.

The first cluster, including clones FFSB1 to 5, 7, 10, and 11 gave positive hybridization signals only with the FFS-Uni probe. The second cluster, consisting only of the clone FFSB6, gave positive signals only with the FFS-6 probe (probe and hybridization conditions are described in paper II).

In paper II the genetic diversity of Archaea in forest soil treated with clear-cutting and prescribed burning was described. The diversity of soil Archaea in intact (C), clear-cut (A) and clear-cut followed by prescribed burning (B) boreal forests was examined based on 19, 49 and 70 clones, respectively.

All obtained clones were subjected to RFLP analysis with AvaII, MspI and MspI/RsaI restriction enzymes, followed by Southern blotting and hybridization with the probes FFS-Uni and FFS-6.

47

70 clones from clear-cut followed by prescribed burning soil (type B) were divided into 9 classes according to restriction and hybridization patterns. The results shown include representatives of each of these classes (Fig. 1, paper II).

Altogether, 69 clones from clear-cut followed by prescribed burning soil (type B) hybridized only with the FFS-Uni probe and belong to the FFS-Uni cluster. Only one clone (FFSB6) gave a signal with the FFS-6 probe and thus belonged to the FFS-6 cluster.

49 clones from clear-cut soil (type A) were analyzed and fell into seven classes. Only two clones, FFSA8 and FFSA12 (Fig. 3, paper II), gave new restriction and hybridization patterns, not found among the FFSB clones. Nevertheless, they gave positive hybridization signals with the FFS-Uni probe and therefore were classified within the FFS-Uni cluster. 4 clones from clear-cut soil fell into the FFS-6 cluster (Table 1, paper II).

4 new classes among the 19 analyzed clones from intact soil (type C) were found. Their restriction and hybridization patterns differed from those isolated from clear-cut soil and clear-cut followed by prescribed burning soil. Only 2 clones showed a pattern identical to the existing FFSB classes. Clones from the 4 new classes showed homology with the FFS-Uni probe (FFS-Uni cluster).

Restriction patterns belonging to clones FFSB2 and FFSB3 were present in clone libraries from all three soils (A, B, and C). Clones from clear-cut soil and clear-cut followed by prescribed burning soil shared six common patterns, whereas the majority of clones from intact soil had patterns not found in either clear-cut soil and clear-cut followed by prescribed burning soil clones.

Phylogenetic analysis of six sequenced clones from clear-cut and intact soils (Fig 4., maximum likelihood tree, paper II) supported the paper I results. New sequences were affiliated with both the FFS-Uni and FFS-6 subgroups of soil Crenarchaeota. Results based on DNA hybridization and sequence analysis correlated with each other. Sequences FFSC1, 2, 3, FFSC4, from the FFS-Uni hybridization cluster (Table 1, paper II) were all placed within the FFSB group of sequences. Sequences FFSA1 and FFSA2 from the FFS-6 cluster (Fig.4, paper II) were affiliated with the sequence FFSB6. Thus, by using the RFLP-hybridization technique, clones belonging to different phylogenetic groups in the rDNA clone library were distinguished.

The FFS sequences were different (100% bootstrap value in maximum parsimony analysis) from two other soil Archaea groups of sequences isolated from Wisconsin (Bintrim et al., 1997) and Amazon soils (Borneman and Triplett, 1997). These two groups were similar to each other and were situated more closely to the planktonic clade of Crenarchaeota.

Currently all FFS sequences are placed in GroupI.1C on the latest Crenarchaeota tree (Fig. 5B). This group includes altogether 46 sequences isolated from terrestrial environments.

4.3 Phylogeny of Archaea from estuarine sediment Estuaries are the interfaces between freshwater and marine environments. Tidal

estuaries are extremely dynamic zones, with spatial and temporal gradients, due to the variability of several factors such as freshwater input, geomorphology, winds, tidal heights, as well as anthropogenic inputs. As expected, microorganisms play an important role in the dynamics of estuarine environments, particularly in biogeochemical cycles and food webs (Nickson, 1995). Therefore the biodiversity of estuarine microbial communities is an area of great interest in microbiological and ecological studies. Although reports on Archaea in estuarine environments are available (Munson et al., 1997; Crump and Baross, 2000), none have revealed the presence of Crenarchaeota organisms in brackish sediments.

48

In order to verify the presence and diversity of Archaea in the estuarine sediments of the river Douro (Portugal), total sediment DNA was isolated and archaeal 16S rDNA was selectively amplified by PCR, cloned, sequenced and phylogenetically characterized.

The 235 recombinant plasmids were screened with AvaII restriction enzyme and 14 were selected according to their original restriction pattern and sequenced (DOURO 1 to 14). Phylogenetic analysis of the sequences revealed the presence of Crenarchaeota 16S rRNA gene sequences in the sediment samples. These sequences were recovered mainly from the surface layer of the cores. However, sequences DOURO10 and 11 were retrieved from a depth of 10 cm, corresponding to a region of thin grain and compact sediment, while DOURO 12 and 13 were found at a depth of 19 cm from the same core. Comparison of 12 of the DOURO sequences (1-6, 8, 9, and 11-14) with existing sequences in public databases (EMBL and GenBank), revealed 95-98% identity to uncultured environmental archaeal sequences retrieved from marine ecosystems in other studies (Delong, 1992; Delong et al., 1994; Fuhrman et al., 1993). Indeed, the phylogenetic analysis placed them within the so-called “marine cluster” (Buckley et al., 1998), which is made up of archaeal 16S rDNA sequences recovered from marine environments and lake sediments (Fig. 1, paper III). In the current Crenarchaeota tree, this group is named GroupI.1a (Fig. 5B).

The DOURO7 sequence presented 88-90% identity with Euryarchaeota 16S rRNA sequences from deep-sea sediments (Vetriani et al., 1999) and 82% with Methanobacterium formicicum, and was placed in the euryarchaeotal group named pMC1 (Hugenholtz, 2002, Fig. 5C). DOURO10 showed 89% identity with Halobacterium sp. and was placed in group VAL2 (Fig. 5C).

4.4 Archaea from lake water

4.4.1 Phylogenetic analysis Freshwaters and wetlands in boreal environments have been identified as important

sources of trace gases (Khalil, 1993) and can contribute up to 34% of the total CH4 fluxes of wetlands globally. Prokaryotes in soil, sediments and water play an essential role in this scenario by their contribution to CO2 and CH4 cycling and fluxes (Conrad, 1996). In addition to their global role in biogeochemical cycles, prokaryotes play a key role in the transformation of natural organic matter, which in many boreal freshwaters is of primarily allochthonous origin and recalcitrant (Münster et al., 1999).

A total of 28 clones (named VAL) recovered from lake Valkea Kotinen were sequenced. We found that (1) VAL sequences contained representatives of both Cren- and Euryarchaeota, (2) no cultivated Archaea were closely related to the VAL sequences, (3) the VAL clones were most closely related (with a similarity around 96%) to archaeal sequences inhabiting a very wide range of environments - marine and lake sediments, different types of soils, rice roots, and an anaerobic digestor. Two separate trees, one for Crenarchaea and one for Euryarchaea, were generated (Fig. 1 and 2, paper IV).

12 freshwater VAL-sequences fall within a subcluster named “freshwater cluster” (Buckley et al., 1998), Group I.3 (Delong, 1998) or Group I.3a (Fig. 5B) of Group I uncultured non-thermophilic Crenarchaeota. Eight of the sequences form a tight VAL I lineage inside the cluster which also includes sequences previously detected in lake sediments (Hershberger et al., 1996; Schleper et al., 1997a), rice roots and soil (Grosskopf et al., 1998b), and an anaerobic digestor (Godon et al., 1997) (Fig. 1, paper IV).

Analysis of 16 VAL sequences identified four distinct lineages related to different orders of Euryarchaeota (Fig. 2, paper IV). Four clones were affiliated with environmental

49

sequences previously detected in anoxic sediments from Rotsee (Switzerland) (Zepp et al., 1999), post-glacial profundal freshwater sediment (Miskin et al., 1998) and peat bogs (Hales et al., 1996) within the order Methanomicrobiales. Another four clones formed a group among sequences inhabiting marine sediments (Vetriani et al., 1998; Munson et al., 1997), rice roots and soil (Grosskopf et al., 1998b) and the deep-sea (Fuhrman and Davis, 1997) (Group III, Figure 2, paper IV). Clone VAL147 clustered together with clone Eel-TA1c9 which was retrieved from a marine sediment (Hinrichs et al., 1999). Convincing evidence (albeit circumstantial) has suggested that organisms represented by this clone are methanotrophic anaerobes involved in the decomposition of methane hydrate. The remaining 7 clones formed two distinct lines of descent which all three methods of phylogenetic analysis (maximum likelihood, maximum parsimony and neighbor-joining) placed in the kingdom Euryarchaeota. However, the exact placement of these two clusters within this kingdom could vary due to the large phylogenetic distances between members of these clades and the rest of the sequences analyzed. Clade VAL II, consisting of four clones, was only distantly related to the family Methanosaeta (Fig. 2, paper IV). At the time of writing paper IV, all methods of analysis allowed sequence VAL33-1 to place this clade on the same branch as Rice cluster I (Fig. 2, paper IV) on the basis of 85.9% similarity between sequence and cluster (Grosskopf et al., 1998b). Levels of rDNA similarity between the other three VAL II clade sequences and sequences from the closest Rice cluster I were extremely low - 65.8-69.2 %.

A similar situation was observed for three clones from cluster VAL III, which were related to the order Thermoplasmatales, which include the Groups II and III (Delong, 1998) and Rice cluster V (Fig. 2, paper IV). The level of similarity between members of this clade, compared to members of orders Thermoplasmatales and Methanobacteriales, ranged from 71.3% (Thermoplasma acidophilum, Thermoplasmatales) to 76.3% (Methanothermus fervidus, Methanobacteriales). Rice cluster V, which was closest to the three VAL III clade sequences, is believed to comprise of non-methanogenic anaerobic Archaea (Grosskopf et al., 1998b). At present, these three clones are placed in group pMC2(Hugenholtz, 2002) on latest Euryarchaeota tree (Fig. 5C).

4.4.2 In situ hybridization analysis

In paper IV, new archaeal oligonucleotides were designed for use as PCR primers and

fluorescent in situ hybridization probes (Table 3). This allowed study of the distribution of the indigenous Archaea in the water column and their potential contribution to the microbial food web.

With general Archaea-specific probe ARCH915 (Stahl and Amann, 1991) it was possible to detect 7±2% of the DAPI stained cells (data for the hypolimnium layer). No significant differences were detected between samples taken at different times during the sampling period from spring to autumn.

All positive hybridization signals and cell morphologies visualized with the four Euryarchaeotal probes EURY498, EURY514, EURY496 and EURY499 were identical to the signals obtained with the general Archaea probe ARCH915 (Fig. 3, paper IV). EURY496 was the most specific of all the Euryarchaeota probes tested and covered the order Methanomicrobiales and related environmental sequences. It was therefore assumed that the Euryarchaeota clones detected in the FISH experiments belonged to the order Methanomicrobiales. The size and morphotypes of the cells detected (Fig. 3, paper IV) showed similarities to some methanogens (for example Methanospirillum hungatei). Despite a high background fluorescence, maximum intensity and highest cell counts with the EURY496 probe (7±2% of the DAPI stained cells) were observed in the hypolimnion samples. Cell counts in the epilimnion samples were below the detection limit (1%).

50

Although positive hybridization signals were obtained with the Euryarchaeota probes, none of the three Crenarchaeota probes used (CREN499, CREN512 and CREN569) gave a positive hybridization signal.

4.5 Diagnostic signature and feature analysis of the studied sequences Analysis of the diagnostic signatures and features of the cloned sequences (Winker and

Woese, 1991; Woese, 1987) revealed that they all belonged to the archaeal domain.

• soil sequences: 66 out of the 68 relevant positions that define Archaea from Bacteria and Eucarya (Woese, 1987) show features common to Archaea. Intradomain nucleotide signature comparison (Winker and Woese, 1991) of the FFSB group (shown in Table 1 of paper I) and other soil sequences supported their phylogenetic within the Crenarchaeota.

• estuarine sediment sequences: the intradomain nucleotide signature analysis of the DOURO sequences (Table 1, paper III) confirmed the affiliation of DOURO7 with the Euryarchaeota, as well as the phylogenetic placement of the DOURO1-6, 8, 9, 11-14 sequences within the Crenarchaeota kingdom. Sequence DOURO10 had both crenarchaeal and euryarchaeal features. The phylogenetic analysis assigned it to the Euryarchaeota, while the intradomain signature analysis revealed a mixture of diagnostic features for both Euryarchaeota and Crenarchaeota kingdoms.

• water sequences: novel VAL sequences shared major parts of the archaeal domain signatures at homologous and non-homologous positions. However, clones with an unstable phylogenetic placement (VAL90, VAL35-1, VAL31-1, VAL84, VAL125, VAL112) contained non-archaeal signatures at the positions 338, 367, 393 and 867 (E. coli numbering). Intradomain signature analysis confirmed affiliation of twelve VAL sequences with the Crenarchaeota, and sixteen VAL sequences with the Euryarchaeota.

51

5 DISCUSSION

The results of all four original articles demonstrate that a large number of previously

“unknown” Archaea exist in "non-extreme" habitats, i.e. boreal forest soils, pelagic water of a boreal forest lake and temperate estuary sediment.

In all four articles the same PCR primers and similar PCR conditions were used. However, the rRNA sequences obtained were phylogenetically very diverse. For example, no euryarchaeotal sequences were found in any of the soil samples, whereas half of the sequences retrieved from the water samples were euryarchaeotal. This would indicate that the results obtained were not biased towards particular groups, but reflected the actual diversity and distribution of Archaea in these environments.

All 57 sequenced clones were aligned using ARB editor which found only some non-complementary secondary structure helix positions. This observation supports the results of the CHECK_CHIMERA program and suggests that the obtained sequences are not chimeras.

Paper I was among the very first reports describing the existence of Crenarchaeota in soil. The results of papers I and II provide evidence that boreal forest soil contains a diverse Crenarchaeota community. They also show that Crenarchaeota are found in soils, which have been subjected to strong ecological and physico-chemical treatments such as clear-cutting and burning. The results indicate that diverse representatives of the Crenarchaeota form a stable part of the highly variable microbial community in boreal forest soil, which provides a nutritionally rich, but physically labile environment, with periods of fast freezing and thawing. Very little is known of the role or metabolic activity of Archaea in soil, except for methanogenic genera belonging to the Euryarchaeota. The novel Archaea found in soil are phylogenetically distant from the Euryarchaeota, and form a cluster distinct from any other cultured Crenarchaeota. It is therefore not possible to infer their ecological role from other known Archaea.

In paper III, new non-thermophilic Crenarchaeota 16S rRNA gene sequences were recovered from estuarine sediment samples. Phylogenetic analysis revealed that these sequences were closely related to marine and freshwater-lake Crenarchaeota 16S rRNA gene sequences retrieved from a diverse range of geographical sites, such as North America (MacGregor et al., 1997), the Antarctic Ocean (Delong et al., 1994) and Japan (Li et al., 1999). Most of the sequences were isolated from the top layer of the sediment cores sampled and may suggest an aerobic metabolism. However, the phylogenetic distance between these novel sequences and the cultivated members of the Crenarchaeota prevents any inference of their physiological properties.

Two sequences were isolated from a layer of active iron oxidation, suggesting that these microorganisms might be involved in biogeochemical processes that occur in this zone of the sediments. Two other sequences from this study were affiliated with euryarchaeotal sequences retrieved from deep-sea sediment (Vetriani et al., 1999) indicating that these microorganisms are present in a wide range of environments. Although the molecular and phylogenetic data collected in this study cannot help in inferring an ecological role for these microorganisms in the environment, these findings are of fundamental value for understanding the complexity of estuarine ecosystems.

Sequences detected in the pelagic water of a boreal forest lake (paper IV) using a rRNA approach belonged to members of the Crenarchaeota and Euryarchaeota. Using fluorescent in situ hybridization, it was possible to detect euryarchaeotal microorganisms and quantify their abundance in the water samples. An important step forward in paper IV (comparing to papers I, II and III) was the visualization of euryarchaeal organisms by FISH. These results indicated

52

that euryarchaeal sequences obtained by PCR did not originate from DNA extracted from dead or dormant cells, but from active cells present in the water samples.

Positive hybridization signals were not obtained using Crenarchaeota-specific probes CREN499, CREN512 and CREN569 probes. Hybridization with the Euryarchaeota specific probes, on the other hand, detected as many cells as the general Archaea probe. The available probes appeared to detect only Euryarchaea species and the total number of Archaea in the samples is therefore probably underestimated. In theory, PCR can identify a single copy of a gene with appropriate condition and primers (Saiki et al., 1988) and FISH can detect a single cell (Poulsen et al., 1993). However, both methods have limitations when analyzing environmental samples. For example, humic compounds present in sediment and soil samples can decrease the efficiency of the PCR by inhibiting DNA polymerase activity (Fritze et al., 1999). Several factors can affect successful fluorescence in situ hybridization as well. The contrasting FISH and PCR results could be due to a low numbers of Crenarchaeota cells in the samples, and/or low ribosome contents in the cells (Roszak and Colwell, 1987; Kotler et al., 1993). Other possible reasons include physical-chemical factors, e.g. reduced penetration of oligonucleotide probes through the cell walls of Crenarchaeota; or probes targeting regions of 16S rRNA which are inaccessible due to secondary structure conformation. The permeation process during hybridization is difficult to adjust so that organisms with different cell envelope structures are simultaneously detected. Improvement of this process might reveal more Crenarchaeota in lake water samples.

The existence of Archaea in the freshwater environment was expected as methanogens have been found previously in freshwater ecosystems. However, the large diversity of retrieved archaeal 16S rRNA sequences, and the low sequence similarity to previously recovered sequences from pelagic interfaces, was surprising.

Original papers I, II and III have shown that representatives of the Crenarchaeota inhabit boreal forest soils and estuarine sediment. Paper IV have shown that they also occupy pelagic habitats in a boreal forest lake. It was found that in all three biotopes, the crenarchaeotal 16S rRNA gene sequences were not only diverse, but formed novel phylogenetic clusters distinct from sequences recovered from other environments. The Crenarchaeota found in the boreal forest lake water were significantly different from Crenarchaeota found in the boreal forest soil and estuarine sediment samples. This may be due to the different geographical location of each sampling site, or inherent differences in the properties of each biotope examined.

The Euryarchaeota detected in water samples occupy a very specific niche. As many of the sequences were only distantly related to any of the main metabolic subgroups of Euryarchaeota (namely the methanogens or halophiles), their metabolic properties and role in the environment could not be inferred.

53

6 SUMMARY AND CONCLUSIONS

Despite our increasing knowledge of the scale of microbial diversity, most microbes

observed in natural environments remain uncultivated. Subsequently, their ecology and functional roles are largely unknown and our understanding of the composition of the natural microbial world is therefore rudimentary (Torsvik et al., 2002). However, modern molecular methods provide a way of surveying biodiversity rapidly and comprehensively. Ribosomal RNA genes retrieved from the environment are snapshots of organisms and represent all the different types of genomes which could be targeted for further characterization if they appeared interesting or useful. If we want to understand the biosphere, it is essential that we create a representative survey of microbial diversity in the environment. A complete catalogue of the Earth's microbial biota is needless and, of course, impossible. A representative survey, however, would be useful and could be achieved with modest effort using automated sequencing technology. Analysis of 1000 clones (to detect the most abundant genome types) from each of 100 chemically, physically and biologically different environments would be comparable to the effort in sequencing a single microbial genome. The questions are large and there are many: for example, what kinds of organisms do we depend on and share this planet

with? How extensive is the reservoir of biodiversity from which we can draw useful lessons and resources? Can we use the distribution of microbes as a biosensor array to map and monitor the chemistries of this planet ?

The opportunities for the discovery of new organisms and the development of resources based on microbial diversity are greater than ever before. Molecular sequences have finally given microbiologists a way of defining microbial phylogeny. The sequences are the basis of tools that will allow microbiologists to explore the distribution and function of microbes in the environment.

One of the most significant findings from the application of new molecular approaches is the discovery of high numbers of novel and unexpected "non-extreme" archaeal phenotypes. Until these findings, it was considered that Archaea were restricted to specialized environments of high temperature, high salinity, extremes of pH, or strict anoxic. Over the last decade, researchers have demonstrated a ubiquitous distribution of "non-extreme" Archaea in terrestrial and aquatic habitats. These discoveries mark the beginning of a new era for investigating Archaea and in particular their physiological and ecological roles in complex microbial communities.

Results presented in this thesis include the discovery of unexpected and novel members of the Archaea in forest soil. The diversity of non-thermophilic crenarchaeota in this habitat has been described.

It was revealed that mesophylic Archaea were present in temperate brackish estuarine sediments. These findings are of fundamental value for understanding the complexity of estuarine ecosystems.

The presence of organisms belonging to both main archaeal phyla - the Crenarchaeota and the Euryarchaeota, was discovered in forest lake water samples. Members of the Euryarchaeota were visualized by fluorescent in situ hybridization. These results suggest that freshwater Archaea may have a specific role in the biogeochemical cycling of carbon in aquatic food webs.

The increasing number of archaeal sequences in the ARB database constructed for this thesis will facilitate analysis of novel archaeal sequences and may serve as a convenient tool for the accurate design and evaluation of hybridization probes and PCR primers.

The results of the research presented in this thesis, together with the large number of recent publications on Archaea in aquatic, terrestrial and other environments, supports the

54

hypothesis that these organisms inhabit a wider range of environments and suggests that Archaea are ecologically more successful then previously thought. I expect that a variety of new physiological phenotypes will be discovered as soon as some of these organisms are successfully cultivated, allowing their metabolic and genetic potentials to be characterized further.

Future studies, supported by already accumulated knowledge, will follow two major directions: (1) the continuation of cultivation attempts and (2) the creation of large DNA fragment libraries (for example, in BAC vectors). Analysis of such libraries can yield information on gene organization, structure and content of uncultivated Archaea and Bacteria.

55

7 TIIVISTELMÄ

Suurin osa luonnossa havaitsemistamme mikrobeista on sellaisia, joita emme

edelleenkään osaa kasvattaa laboratorio-oloissa, vaikka tietomme mikrobien monimuotoisuudesta paranevat koko ajan. Luonnontilaisen mikrobieliöstön kokoonpano eri ympäristöissä on paljolti epäselvä ja ymmärrämme vielä hyvin puutteellisesti mikrobien ekologiaa ja niiden rooleja eliöyhteisöissä. Nykyaikaiset molekulaariset tutkimusmenetelmät auttavat selvittämään mikrobien monimuotoisuutta kokonaisvaltaisesti ja nopeasti. Ympäristöstä kemiallisesti puhdistetut ribosomaalista RNA:ta koodaavat geenit edustavat periaatteessa kaikkia eliöyhteisön geneettisesti toisistaan poikkeavia eliöitä. Niistä voidaan valikoida halutut genomit jatkotutkimuksia varten.

Uusien menetelmien käyttö on tuonut esiin sen merkittävän seikan, että ”tavanomaisten” elinympäristöjen eliöyhteisöihin kuuluu suuri joukko entuudestaan tuntemattomia arkkieliöitä. Aiemmin kuviteltiin, että arkkieliöt asuttavat vain sellaisia “epätavallisia” tai ”äärimmäisiä” elinympäristöjä, joita luonnehtii joku seuraavista ominaisuuksista: hyvin korkea lämpötila, korkea suolapitoisuus, korkea happamuus tai emäksisyys, hapettomuus. Tutkijat ovat viimeisen noin kymmenen vuoden aikana osoittaneet, että arkkieliöt asuttavat hyvin monenlaisia kylmän ja lauhkean vyöhykkeen ympäristöjä, yhtä hyvin maaperää kuin suolaisen ja makean veden pohjaa tai pintakerroksia. Nämä löydöt ovat avanneet uuden alun arkkieliöiden tutkimukselle, erityisesti sen selvittämiselle, mitkä ovat niiden fysiologiset ja ekologiset roolit monimuotoisissa mikrobiyhteisöissä.

Tämä väitöskirja kuvaa entuudestaan tuntemattomien arkkieliöiden löytymistä

havumetsävyöhykkeen metsämaasta. Arkkieliöitä löytyi myös lauhkean vyöhykkeen vuorovesialueelta, murtoveden

huuhtelemasta pohjasta. Nämä löydöt ovat perustavalaatuisia vuorovesialueen eliöyhteisöjen ymmärtämiseksi.

Suomalaisen metsäjärven vedestä määritettiin molempien arkkieliöiden pääryhmien - tieteellisiltä nimiltään Crenarchaeota ja Euryarchaeota - edustajia. Euryarchaeota-ryhmän edustajia voitiin havainnoida myös fluoresenssi-mikroskopoinnilla. Löydöt viittaavat siihen, että arkkieliöillä on oma biogeokemiallinen roolinsa makeanveden ravintoketjujen hiilen käytössä.

Tässä työssä määritetyt uudet arkkieliöiden genomien nukleotidisekvenssit on toimitettu

ARB-tietokantaan, jonka kasvava vertailuaineisto edelleen parantaa uusien arkkieliösekvenssien analyysiä ja auttaa hybridisaatiokoetinten ja polymeraasiketjureaktioalukkeiden suunnittelussa ja arvioinnissa.

Tässä väitöskirjassa esitellyt tulokset yhdessä lukuisien vesi-, maaperä- ja muiden

ympäristöjen arkkieliöitä käsittelevien julkaisujen kanssa osoittavat, että arkkieliöt asuttavat monia erilaisia elinympäristöjä ja että ne ovat ekologisesti paljon menestyneempiä, kuin tieteenalalla on kuviteltu. Voimme olettaa, että heti kun joitain näistä eliöistä onnistutaan kasvattamaan ja ylläpitämään laboratorio-oloissa, niiden joukosta löydetään aivan uusia, entuudestaan tuntemattomia fysiologisia fenotyyppejä, jotka avaavat mielenkiintoisia näkymiä aineenvaihdunnan ja perinnöllisten ominaisuuksien tutkimukselle.

56

8 ACKNOWLEDGEMENTS

This work was carried out at the Department of Applied Chemistry and Microbiology,

Division of Microbiology, University of Helsinki, and the Max Planck Institute, Bremen, Germany (during a three month visit).

I would like to express my warmest thanks to: Supervisor of this thesis - Dr. Aimo Saano, who fearlessly accepted me as PhD student

for his project and was the main creator of the great ideas, techniques and whole background of this thesis. We experienced together all the ups and downs of routine work, the shared happiness of success and the depression of failure when (sometimes) everything went wrong. He managed to teach me how to work independently (which is very important), but at any time, his useful advice was available to me. In my opinion, this is how an ideal supervisor should be. It is a real pleasure to work and communicate with such a polite, honest and open-minded person. I would also like to acknowledge his language abilities which were extremely useful, particularly in the beginning of my work and life in Finland.

“Co-supervisor” - Dr. Kristina Lindström, my first supervisor in Finland and at the

University of Helsinki, who kindly allowed me to begin my work in her lab. She has helped me greatly during all these years (special appreciation for the first, hardest year of accommodation in Finland) and has always been extremely supportive and nice to me. It was only her great help in science and “beyond science” life that I got the chance to initiate and complete this thesis. She is now “hosting” the “Archaea group” at the Department of Applied Chemistry and Microbiology.

Second “co-supervisor” - Dr. Uwe Münster, who generously accommodated me in his

project and let me continue to work with Archaea (and even became interested in this subject himself). His great support, huge work experience and patience were necessary for finishing this thesis.

Colleague and co-member of our small “Archaea group”, - Leone Montonen, for her

enormous everyday help in work and life, wise advice and tolerance of me as her “office mate”.

All present and former colleagues, permanent and “visiting” members of the N2 -group,

for their assistance and providing an excellent working atmosphere: Leena Suominen, Leena Räsänen, Seppo Kaijalainen, Zewdu Terefework, Minna Jussila, Aneta Dresler-Nurmi, Gilles Lortet, Petri Nowak, Kati Jyrkiäinen, Jyrki Pitkäjärvi, Giselle Nick-Mäenpää, Eva Tas, Kaisa Wallenius, Elena Lapina-Balk, Anna Mäkinen and all others.

Rudolf, L. Amann, Professor, Ph.D., leader of the Molecular Ecology group at the Max-

Planck-Institute for Marine Research, Bremen, Germany, for the excellent opportunity to stay in his lab and learn modern molecular techniques. I would like to thank the following people from the Molecular Ecology lab for their great help through my stay in Bremen: Bernhard Fuchs, Katrin Ravenschlag and Jakob Pernthaler. Special thanks to Frank-Oliver Glöckner and Wilhelm Schönhuber, who taught me how to do in situ hybridization and how to fight with the ARB computer program.

57

I am grateful to the staff of the Lammi Biological Station and, in particular, the head of the station Lauri Arvola for being very kind and supportive during my time working there.

I would like to acknowledge Dr. Maarit Niemi and Dr. Hannu Fritze for reviewing this

manuscript. They were extremely fast (sorry again for my rushing), but did the job very thoroughly. Their valuable comments improved the manuscript notably.

My appreciation and gratefulness to the friendly staff of the Department of Applied

Chemistry and Microbiology, and to Professor Mirja Salkinoja-Salonen for her kind support. A special warm thank you to Dr. Graeme Nicol from the University of Aberdeen, UK.

He did an enormous job reviewing the English in this manuscript. His significant scientific comments and suggestions enhance this manuscript a lot.

I sincerely thank my family - mother Ina, aunt Ada and daughter Kristina for their

encouragement through these years. Special thanks to my wife Natalia for her great support, patience and understanding.

This work was financially supported by grants from the Academy of Finland, the

University of Helsinki, the Maj and Tor Nessling Foundation and the Max Planck Institute (Bremen, Germany)

58

9 REFERENCES Amann,R.I. 1995. Fluorescently labeled, ribosomal RNA targeted oligonucleotide probes in the study of microbial ecology. Molecular Ecology 4: 543-553.

Amann,R.I. 1996. In situ identification of micro-organisms by whole cell hybridization with rRNA-targeted nucleic acid probes. Molecular Microbial Ecology Manual 3.3.6/1-3.3.6/15.

Amann,R.I., Binder,B.J., Olson,R.J., Chisholm,S.W., Devereux,R., and Stahl,D.A. 1990. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Applied and Environmental Microbiology 56: 1919-1925.

Amann,R.I., Ludwig,W., and Schleifer,K.H. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiological Reviews 59: 143-169.

Amann,R.I., Zarda,B., Stahl,D.A., and Schleifer,K.H. 1992. Identification of individual prokaryotic cells by using enzyme-labeled, rRNA-targeted oligonucleotide probes. Applied and Environmental Microbiology 58: 3007-3011.

Balch,W.E., Fox,G.E., Magrum,L.J., Woese,C.R., and Wolfe,R.S. 1979. Methanogens: reevaluation of a unique biological group. Microbiological Reviews 43: 260-296.

Barns,S.M., Delwiche,C.F., Palmer,J.D., and Pace,N.R. 1996. Perspectives on archaeal diversity, thermophily and monophyly from environmental rRNA sequences. Proceedings of the National Academy of Sciences of the United States of America 93: 9188-9193.

Barns,S.M., Fundyga,R.E., Jeffries,M.W., and Pace,N.R. 1994. Remarkable Archaeal diversity detected in a Yellowstone National Park hot spring environment. Proceedings of the National Academy of Sciences of the United States of America 91: 1609-1613.

Beja,O., Suzuki,M.T., Koonin,E.V., Aravind,L., Hadd,A., Nguyen,L.P. et al. 2000. Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage. Environmental Microbiology 2: 516-529.

Benlloch,S., Acinas,S.G., Anton,J., Lopez,L., Luz,S.P., and Rodriguez-Valera,F. 2001. Archaeal biodiversity in crystallizer ponds from a solar saltern: culture versus PCR. Microbial Ecology 41: 12-19.

Benlloch,S., Martinez-Murcia,A.J., and Rodriguez-Valera,F. 1995. Sequencing of bacterial and archaeal 16S rRNA genes directly amplified from a hypersaline environment. Systematic and Applied Microbiology 18: 574-581.

Beveridge,T.J. 2001. Archaeal cells, http://www.els.net. London, Nature Publishing Group.

Bidle,K.A., Kastner,M., and Bartlett,D.H. 1999. A phylogenetic analysis of microbial communities associated with methane hydrate containing marine fluids and sediments in the Cascadia margin (ODP site 892B). Fems Microbiology Letters 177: 101-108.

Bintrim,S.B., Donohue,T.J., Handelsman,J., Roberts,G.P., and Goodman,R.M. 1997. Molecular phylogeny of archaea from soil. Proceedings of the National Academy of Sciences of the United States of America 94: 277-282.

Blöchl,E., Rachel,R., Burggraf,S., Hafenbradl,D., Jannasch,H.W., and Stetter,K.O. 1997. Pyrolobus fumarii, gen. and sp. nov., represents a novel group of archaea, extending the upper temperature limit for life to 113 degrees Celsius. Extremophiles 1: 14-21.

59

Bomberg,M., Jurgens,G., Saano,A., Sen,R., and Timonen,S. 2002. Nested PCR detection of Archaea in defined compartments of pine mycorrhizospheres developed in boreal forest humus microcosms. Fems Microbiology Ecology in press.

Bond,P.L., Smriga,S.P., and Banfield,J.F. 2000. Phylogeny of microorganisms populating a thick, subaerial, predominantly lithotrophic biofilm at an extreme acid mine drainage site. Applied and Environmental Microbiology 66: 3842-3849.

Borneman,J. and Triplett,E.W. 1997. Molecular microbial diversity in soils from eastern Amazonia: evidence for unusual microorganisms and microbial population shifts associated with deforestation. Applied and Environmental Microbiology 63: 2647-2653.

Bowman,J.P., McCammon,S.A., Rea,S.M., and McMeekin,T.A. 2000a. The microbial composition of three limnologically disparate hypersaline Antarctic lakes. Fems Microbiology Letters 183: 81-88.

Bowman,J.P., Rea,S.M., McCammon,S.A., and McMeekin,T.A. 2000b. Diversity and community structure within anoxic sediment from marine salinity meromictic lakes and a coastal meromictic marine basin, Vestfold Hills, Eastern Antarctica. Environmental Microbiology 2: 227-237.

Brambilla,E., Hippe,H., Hagelstein,A., Tindall,B.J., and Stackebrandt,E. 2001. 16S rDNA diversity of cultured and uncultured prokaryotes of a mat sample from Lake Fryxell, McMurdo Dry Valleys, Antarctica. Extremophiles 5: 23-33.

Brock,T.D. 1978. Thermophilic microorganisms and life at high temperatures. New-York: Springer-Verlag.

Brock,T.D., Brock,K.M., Belly,R.T., and Weiss,R.L. 1972. Sulfolobus: a new genus of sulfur-oxidizing bacteria living at low pH and high temperature. Archives of Microbiology 84: 54-68.

Brown,J.R. 1999. Universal tree of Life in Encyclopedia of Life Sciences, http://www.els.net. London, Nature Publishing Group.

Brown,J.R. and Doolittle,W.F. 1997. Archaea and the prokaryote-to-eukaryote transition. Microbiology and Molecular Biology Reviews 61: 456-502.

Buckley,D.H., Graber,J.R., and Schmidt,T.M. 1998. Phylogenetic analysis of nonthermophilic members of the kingdom Crenarchaeota and their diversity and abundance in soils. Applied and Environmental Microbiology 64: 4333-4339.

Bult,C.J., White,O., Olsen,G.J., Zhou,L.X., Fleischmann,R.D., Sutton,G.G. et al. 1996. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science 273: 1058-1073.

Burggraf,S., Heyder,P., and Eis,N. 1997. A pivotal Archaea group. Nature 385: 780.

Burggraf,S., Mayer,T., Amann,R., Schadhauser,S., Woese,C.R., and Stetter,K.O. 1994. Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes. Applied and Environmental Microbiology 60: 3112-3119.

Casamayor,E.O., Muyzer,G., and Pedros-Alio,C. 2001. Composition and temporal dynamics of planktonic archaeal assemblages from anaerobic sulfurous environments studied by 16S rDNA denaturing gradient gel electrophoresis and sequencing. Aquatic Microbial Ecology 25: 237-246.

Chandler,D.P., Brockman,F.J., Bailey,T.J., and Fredrickson,J.K. 1998. Phylogenetic diversity of archaea and bacteria in a deep subsurface paleosol. Microbial Ecology 36: 37-50.

Chapelle,F.H., O'Neill,K., Bradley,P.M., Methe,B.A., Ciufo,S.A., Knobel,L.L., and Lovley,D.R. 2002. A hydrogen-based subsurface microbial community dominated by methanogens. Nature 415: 312-315.

Chelius,M.K. and Triplett,E.W. 2001. The diversity of archaea and bacteria in association with the roots of Zea mays L. Microbial Ecology 41: 252-263.

60

Chin,K.J., Lukow,T., and Conrad,R. 1999. Effect of temperature on structure and function of the methanogenic archaeal community in an anoxic rice field soil. Applied and Environmental Microbiology 65: 2341-2349.

Cifuentes,A., Anton,J., Benlloch,S., Donnelly,A., Herbert,R.A., and Rodriguez-Valera,F. 2000. Prokaryotic diversity in Zostera noltii-colonized marine sediments. Applied and Environmental Microbiology 66: 1715-1719.

Cole,S.T. and Saint,G., I 1994. Bacterial genomics. Fems Microbiology Reviews 14: 139-160.

Conrad,R. 1996. Soil microorganisms as controllers of atmospheric trace gases (H2, CO, CH4, OCS, N2O, and NO). Microbiological Reviews 60: 609-640.

Crump,B.C. and Baross,J.A. 2000. Archaeaplankton in the Columbia River, its estuary and the adjacent coastal ocean, USA. Fems Microbiology Ecology 31: 231-239.

Cytryn,E., Minz,D., Oremland,R.S., and Cohen,Y. 2000. Distribution and diversity of archaea corresponding to the limnological cycle of a hypersaline stratified lake (Solar Lake, Sinai, Egypt). Applied and Environmental Microbiology 66: 3269-3276.

Delong,E.F. 1992. Archaea in coastal marine environments. Proceedings of the National Academy of Sciences of the United States of America 89: 5685-5689.

Delong,E.F. 1998. Everything in moderation: Archaea as "non-extremophiles". Current Opinion in Genetics and Development 8: 649-654.

Delong,E.F., Wu,K.Y., Prezelin,B.B., and Jovine,R.V.M. 1994. High abundance of Archaea in Antarctic marine picoplankton. Nature 371: 695-697.

Dojka,M.A., Hugenholtz,P., Haack,S.K., and Pace,N.R. 1998. Microbial diversity in a hydrocarbon- and chlorinated-solvent-contaminated aquifer undergoing intrinsic bioremediation. Applied and Environmental Microbiology 64: 3869-3877.

Doolittle,W.F. and Brown,J.R. 1994. Tempo, mode, the progenote, and the universal root. Proceedings of the National Academy of Sciences of the United States of America 91 : 6721-6728.

Eder,W., Ludwig,W., and Huber,R. 1999. Novel 16S rRNA gene sequences retrieved from highly saline brine sediments of Kebrit Deep, Red Sea. Archives of Microbiology 172: 213-218.

Farrelly,V., Rainey,F.A., and Stackebrandt,E. 1995. Effect of genome size and rRNA gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species. Applied and Environmental Microbiology 61: 2798-2801.

Felsenstein,J. 1981. Evolutionary trees from DNA sequences: a maximum likelihood approach. Journal of Molecular Evolution 17: 368-376.

Felsenstein,J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791.

Felsenstein,J. 1993. PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author.

Fiala,G. and Stetter,K.O. 1986. Pyrococcus furiosus, sp.nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100 degrees Celsius. Archives of Microbiology 145: 56-61.

Fitch,W.M. and Margoliash,E. 1967. Construction of phylogenetic trees. Science 155: 279-284.

Franzmann,P.D., Liu,Y.T., Balkwill,D.L., Aldrich,H.C., deMacario,E.C., and Boone,D.R. 1997. Methanogenium frigidum sp. nov., a psychrophilic, H2-using methanogen from Ace Lake, Antarctica. International Journal of Systematic Bacteriology 47: 1068-1072.

61

Friedrich,M.W., Schmitt-Wagner,D., Lueders,T., and Brune,A. 2001. Axial differences in community structure of Crenarchaeota and Euryarchaeota in the highly compartmentalized gut of the soil-feeding termite Cubitermes orthognathus. Applied and Environmental Microbiology 67: 4880-4890.

Fritze,H., Tikka,P., Pennanen,T., Saano,A., Jurgens,G., Nilsson,M. et al. 1999. Detection of archaeal diether lipid by gas chromatography from humus and peat. Scandinavian Journal of Forest Research 14: 545-551.

Fuhrman,J.A. and Davis,A.A. 1997. Widespread archaea and novel bacteria from the deep sea as shown by 16S rRNA gene sequences. Marine Ecology-Progress Series 150: 275-285.

Fuhrman,J.A., McCallum,K., and Davis,A.A. 1992. Novel major archaebacterial group from marine plankton. Nature 356: 148-149.

Fuhrman,J.A., McCallum,K., and Davis,A.A. 1993. Phylogenetic diversity of subsurface marine microbial communities from the Atlantic and Pacific Oceans. Applied and Environmental Microbiology 59: 1294-1302.

Fuhrman,J.A. and Ouverney,C.C. 1998. Marine microbial diversity studied via 16s rRNA sequences: cloning results from coastal waters and counting of native Archaea with fluorescent single cell probes. Aquatic Ecology 32: 3-15.

Fuhrman,J.A., Comeau,D.E., Hagstrom,A., and Chan,A.M. 1988. Extraction from natural planktonic microorganisms of DNA suitable for molecular biological studies. Applied and Environmental Microbiology 54: 1426-1429.

Furlong,M.A., Singleton,D.R., Coleman,D.C., and Whitman,W.B. 2002. Molecular and culture based analyses of prokaryotic communities from an agricultural soil and the burrows and casts of the earthworm Lumbricus rubellus. Applied and Environmental Microbiology 68: 1265-1279.

Garcia-Martinez,J. and Rodriguez-Valera,F. 2000. Microdiversity of uncultured marine prokaryotes: the SAR11 cluster and the marine Archaea of Group I. Molecular Ecology 9: 935-948.

Garrity,G.M. 2001. Bergey's Manual of Systematic Bacteriology. Bergey's Manual Trust, Michigan State University, East Lansing, MI.

Giovannoni,S.J., Delong,E.F., Olsen,G.J., and Pace,N.R. 1988. Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells. Journal of Bacteriology 170: 720-726.

Glöckner,F.O., Amann,R., Alfreider,A., Pernthaler,J., Psenner,R., Trebesius,K., and Schleifer,K.H. 1996. An in situ hybridization protocol for detection and identification of planktonic bacteria. Systematic and Applied Microbiology 19: 403-406.

Go,Y.S., Han,S.K., Lee,I.G., and Ahn,T.Y. 2000. Diversity of the domain Archaea as determined by 16S rRNA gene analysis in the sediments of Lake Soyang. Archiv fur Hydrobiologie 149: 459-466.

Godon,J.J., Zumstein,E., Dabert,P., Habouzit,F., and Moletta,R. 1997. Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis. Applied and Environmental Microbiology 63: 2802-2813.

Golyshina,O.V., Pivovarova,T.A., Karavaiko,G.I., Kondrat'eva,T.F., Moore,E.R.B., Abraham,W.R. et al. 2000. Ferroplasma acidiphilum gen. nov., sp nov., an acidophilic, autotrophic, ferrous-iron-oxidizing, cell-wall-lacking, mesophilic member of the Ferroplasmaceae fam. nov., comprising a distinct lineage of the Archaea. International Journal of Systematic and Evolutionary Microbiology 50: 997-1006.

Goodman,M. 1982. Macromolecular sequences in systematic and evolutionary biology. New York: Plenum Publishing Corp.

Grant,S., Grant,W.D., Jones,B.E., Kato,C., and Li,L. 1999. Novel archaeal phylotypes from an East African alkaline saltern. Extremophiles 3: 139-145.

62

Grant,W.D. and Larsen,H. 1989. Extremely halophilic archaeobacteria. In Bergey's Manual of Systematic Bacteriology, 1st ed., Vol.3. Staley, Bryant, Pfennig, and Holt (eds). Baltimore: The Williams and Wilkins Co., 2216-2219.

Grosskopf,R., Janssen,P.H., and Liesack,W. 1998a. Diversity and structure of the methanogenic community in anoxic rice paddy soil microcosms as examined by cultivation and direct 16S rRNA gene sequence retrieval. Applied and Environmental Microbiology 64: 960-969.

Grosskopf,R., Stubner,S., and Liesack,W. 1998b. Novel euryarchaeotal lineages detected on rice roots and in the anoxic bulk soil of flooded rice microcosms. Applied and Environmental Microbiology 64: 4983-4989.

Gutell,R.R., Larsen,N., and Woese,C.R. 1994. Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective. Microbiological Reviews 58: 10-26.

Hafenbradl,D., Keller,M., Dirmeier,R., Rachel,R., Rossnagel,P., Burggraf,S. et al. 1996. Ferroglobus placidus gen. nov., sp. nov., a novel hyperthermophilic archaeum that oxidizes Fe2+ at neutral pH under anoxic conditions. Archives of Microbiology 166: 308-314.

Hahn,D., Amann,R.I., Ludwig,W., Akkermans,A.D., and Schleifer,K.H. 1992. Detection of microorganisms in soil after in situ hybridization with rRNA-targeted, fluorescently labelled oligonucleotides. Journal of General Microbiology 138 ( Pt 5): 879-887.

Hales,B.A., Edwards,C., Ritchie,D.A., Hall,G., Pickup,R.W., and Saunders,J.R. 1996. Isolation and identification of methanogen-specific DNA from blanket bog peat by PCR amplification and sequence analysis. Applied and Environmental Microbiology 62: 668-675.

Head,I.M., Saunders,J.R., and Pickup,R.W. 1998. Microbial evolution, diversity, and ecology: a decade of ribosomal RNA analysis of uncultivated microorganisms. Microbial Ecology 35: 1-21.

Hershberger,K.L., Barns,S.M., Reysenbach,A.L., Dawson,S.C., and Pace,N.R. 1996. Wide diversity of Crenarchaeota. Nature 384: 420.

Hinrichs,K.U., Hayes,J.M., Sylva,S.P., Brewer,P.G., and Delong,E.F. 1999. Methane-consuming archaebacteria in marine sediments. Nature 398: 802-805.

Huber,G., Spinnler,C., Gambacorta,A., and Stetter,K.O. 1989. Metallosphaera sedula gen. and sp. nov. represents a new genus of aerobic, metal-mobilizing, thermoacidophilic archaebacteria. Systematic and Applied Microbiology 12: 38-47.

Huber,H., Burggraf,S., Mayer,T., Wyschkony,I., Rachel,R., and Stetter,K.O. 2000. Ignicoccus gen. nov., a novel genus of hyperthermophilic, chemolithoautotrophic Archaea, represented by two new species, Ignicoccus islandicus sp. nov. and Ignicoccus pacificus sp. nov. International Journal of Systematic and Evolutionary Microbiology 50: 2093-2100.

Huber,H., Hohn,M., Rachel,R., Fuchs,T., Wimmer,V., and Stetter,K. 2002a. A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont. Nature 417: 63-67.

Huber,H. and Stetter,K.O. 1999a. Crenarchaeota in Encyclopedia of Life Sciences, http://www.els.net. London, Nature Publishing Group.

Huber,H. and Stetter,K.O. 1999b. Euryarchaeota in Encyclopedia of Life Sciences, http://www.els.net. London, Nature Publishing Group.

Huber,J.A., Butterfield,D.A., and Baross,J.A. 2002b. Temporal changes in Archaeal diversity and chemistry in a mid-ocean ridge subseafloor habitat. Applied and Environmental Microbiology 68: 1585-1594.

Huber,R. and Stetter,K.O. 1992. The order Thermoproteales. In The Prokaryotes. A handbook of Bacteria: ecophysiology, isolation, identification, applications, Vol.1. Balows, Trüper, Dworkin, Harder, and Schleifer (eds). New York: Springer-Verlag, 677-683.

63

Huber,R., Stohr,J., Hohenhaus,S., Rachel,R., Burggraf,S., Jannasch,H.W., and Stetter,K.O. 1995. Thermococcus chitonophagus, sp.nov, a novel, chitin-degrading, hyperthermophillic Archaeum from a deep-sea hydrothermal vent environment. Archives of Microbiology 164: 255-264.

Hugenholtz,P. 2002. Exploring prokaryotic diversity in the genomic era. Genome Biology 3: 0003.1-0003.8.

Hugenholtz,P., Goebel,B.M., and Pace,N.R. 1998. Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. Journal of Bacteriology 180: 4765-4774.

Iwabe,N., Kuma,K., Hasegawa,M., Osawa,S., and Miyata,T. 1989. Evolutionary relationship of archaebacteria, eubacteria, and eukaryotes inferred from phylogenetic trees of duplicated genes. Proceedings of the National Academy of Sciences of the United States of America 86: 9355-9359.

Jackson,C.R., Langner,H.W., Donahoe-Christiansen,J., Inskeep,W.P., and McDermott,T.R. 2001. Molecular analysis of microbial community structure in an arsenite-oxidizing acidic thermal spring. Environmental Microbiology 3: 532-542.

Jukes,T.N. and Cantor,C.R. 1969. Mammalian protein metabolism. In Evolution of protein molecules. Munro,H.N. (ed). New York: Academic Press, 21-132.

Kandler,O. and König,H. 1993. Cell envelopes of archaea: structure and chemistry. In The Biochemistry of Archaea (Archaebacteria). Kates,M., Kushner,D.J., and Matheson,A.T. (eds). Amsterdam: Elsevier Science Publishers, 223-259.

Karner,M.B., Delong,E.F., and Karl,D.M. 2001. Archaeal dominance in the mesopelagic zone of the Pacific Ocean. Nature 409: 507-510.

Kato,C., Li,L., Tamaoka,J., and Horikoshi,K. 1997. Molecular analyses of the sediment of the 11,000-m deep Mariana Trench. Extremophiles 1: 117-123.

Kawarabayasi,Y., Hino,Y., Horikawa,H., Yamazaki,S., Haikawa,Y., Jin-no,K. et al. 1999. Complete genome sequence of an aerobic hyper-thermophilic crenarchaeon, Aeropyrum pernix K1. Dna Research 6: 83-52.

Kawarabayasi,Y., Sawada,M., Horikawa,H., Haikawa,Y., Hino,Y., Yamamoto,S. et al. 1998. Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3. Dna Research 5: 55-76.

Kawashima,T. 2000. Archaeal adaptation to higher temperatures revealed by genomic sequence of Thermoplasma volcanium. Proceedings of the National Academy of Sciences of the United States of America 97: 14257-14262.

Khalil,M.A.K. 1993. Atmospheric methane: sources, sinks and role in global change. New York: Springer.

Kim,H., Honda,D., Hanada,S., Kanamori,N., Shibata,S., Miyaki,T. et al. 2000. A deeply branched novel phylotype found in Japanese paddy soils. Microbiology 146: 2309-2315.

Klenk,H.P., Clayton,R.A., Tomb,J.F., White,O., Nelson,K.E., Ketchum,K.A. et al. 1997. The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus. Nature 390: 364-370.

König,H. 2001. Archaea, http://www.els.net. London, Nature Publishing Group.

König,H., Kralik,R., and Kandler,O. 1982. Structure and chemical modification of psudomurein in Methanobacteriales. Zentralblatt Bakteriologie und Hygiene 1 Abstracts Original C 3: 179-191.

Kopczynski,E.D., Bateson,M.M., and Ward,D.M. 1994. Recognition of chimeric small-subunit ribosomal DNAs composed of genes from uncultivated microorganisms. Applied and Environmental Microbiology 60: 746-748.

64

Kotler,R., Siegele,D.A., and Tormo,A. 1993. The stationary phase of the bacterial life cycle. Annual Review of Microbiology 47: 855-874.

Kudo,Y., Shibata,S., Miyaki,T., Aono,T., and Oyaizu,H. 1997. Peculiar archaea found in Japanese paddy soils. Bioscience Biotechnology and Biochemistry 61: 917-920.

Kurr,M., Huber,R., Konig,H., Jannasch,H.W., Fricke,H., Trincone,A. et al. 1991. Methanopyrus kandleri, gen. and sp. nov. represents a novel group of hyperthermophilic methanogens, growing at 110 degrees Celsius. Archives of Microbiology 156: 239-247.

Kuypers,M.M., Blokker,P., Erbacher,J., Kinkel,H., Pancost,R.D., Schouten,S., and Sinninghe Damste,J.S. 2001. Massive expansion of marine archaea during a mid-Cretaceous oceanic anoxic event. Science 293: 92-95.

Langworthy,T.A. 1985. Lipids of archaebacteria. In The Bacteria - A treatise on structure and function, vol. VIII: Archaebacteria. Wolfe,R.S. and Woese,C.R. (eds). New York: Academic Press, 459-497.

Lanoil,B.D., Sassen,R., La Duc,M.T., Sweet,S.T., and Nealson,K.H. 2001. Bacteria and Archaea physically associated with Gulf of Mexico gas hydrates. Applied and Environmental Microbiology 67: 5143-5153.

Larsen,H. 1973. The halobacteria's confusion to biology. Antonie Van Leeuwenhoek International Journal of Microbiology Serology 39: 383-396.

Lee,S.H., Malone,C., and Kemp,P.F. 1993. Use of multiple 16S ribosomal RNA targeted fluorescent probes to increase signal strength and measure cellular RNA from natural planktonic bacteria. Marine Ecology-Progress Series 101: 193-201.

Lehmann-Richter,S., Grosskopf,R., Liesack,W., Frenzel,P., and Conrad,R. 1999. Methanogenic archaea and CO2-dependent methanogenesis on washed rice roots. Environmental Microbiology 1: 159-166.

Li,L., Kato,C., and Horikoshi,K. 1999. Microbial diversity in sediments collected from the deepest cold-seep area, the Japan Trench. Marine Biotechnology 1: 391-400.

Liesack,W., Weyland,H., and Stackebrandt,E. 1991. Potential risks of gene amplification by PCR as determined by 16S rDNA analysis of a mixed culture of strict barophilic bacteria. Microbial Ecology 21: 191-198.

Lopez-Garcia,P., Lopez-Lopez,A., Moreira,D., and Rodriguez-Valera,F. 2001a. Diversity of free-living prokaryotes from a deep-sea site at the Antarctic Polar Front. Fems Microbiology Ecology 36: 193-202.

Lopez-Garcia,P., Moreira,D., Lopez-Lopez,A., and Rodriguez-Valera,F. 2001b. A novel haloarchaeal-related lineage is widely distributed in deep oceanic regions. Environmental Microbiology 3: 72-78.

Ludwig,W., Rossellomora,R., Aznar,R., Klugbauer,S., Spring,S., Reetz,K. et al. 1995. Comparative sequence analysis of 23S ribosomal RNA from Proteobacteria. Systematic and Applied Microbiology 18: 164-188.

Ludwig,W. and Schleifer,K.H. 1999. Phylogeny of bacteria beyond the 16S rRNA standard. Asm News 65: 752-757.

Lueders,T. and Friedrich,M. 2000. Archaeal population dynamics during sequential reduction processes in rice field soil. Applied and Environmental Microbiology 66: 2732-2742.

MacGregor,B.J., Moser,D.P., Alm,E.W., Nealson,K.H., and Stahl,D.A. 1997. Crenarchaeota in Lake Michigan sediment. Applied and Environmental Microbiology 63: 1178-1181.

Madigan,M.T., Martinko,J.M., and Parker,J. 2003. Brock Biology of Microorganisms. Pearson Educations, Uper Saddle River, NJ, ed. 10.

65

Madrid,V.M., Aller,J.Y., Aller,R.C., and Chistoserdov,A.Y. 2001a. High prokaryote diversity and analysis of community structure in mobile mud deposits off French Guiana: identification of two new bacterial candidate divisions. Fems Microbiology Ecology 37: 197-209.

Madrid,V.M., Taylor,G.T., Scranton,M.I., and Chistoserdov,A.Y. 2001b. Phylogenetic diversity of bacterial and archaeal communities in the anoxic zone of the Cariaco Basin. Applied and Environmental Microbiology 67: 1663-1674.

Maidak,B.L., Cole,J.R., Lilburn,T.G., Parker,C.T., Saxman,P.R., Farris,R.J. et al. 2001. The RDP-II (Ribosomal Database Project). Nucleic Acids Research 29: 173-174.

Maidak,B.L., Olsen,G.J., Larsen,N., Overbeek,R., McCaughey,M.J., and Woese,C.R. 1996. The Ribosomal Database Project (RDP). Nucleic Acids Research 24: 82-85.

Manz,W., Szewzyk,U., Ericsson,P., Amann,R., Schleifer,K.H., and Stenstrom,T.A. 1993. In situ identification of bacteria in drinking water and adjoining biofilms by hybridization with 16S and 23S rRNA-directed fluorescent oligonucleotide probes. Applied and Environmental Microbiology 59: 2293-2298.

Marchesi,J.R., Weightman,A.J., Cragg,B.A., Parkes,R.J., and Fry,J.C. 2001. Methanogen and bacterial diversity and distribution in deep gas hydrate sediments from the Cascadia Margin as revealed by 16S rRNA molecular analysis. Fems Microbiology Ecology 34: 221-228.

Margulis,L. 1993. Symbiosis in cell evolution, microbial communities in the Archean and Proterozoic eons. New York, USA.

Marteinsson,V.T., Hauksdottir,S., Hobel,C.F.V., Kristmannsdottir,H., Hreggvidsson,G.O., and Kristjansson,J.K. 2001a. Phylogenetic diversity analysis of subterranean hot springs in Iceland. Applied and Environmental Microbiology 67: 4242-4248.

Marteinsson,V.T., Kristjansson,J.K., Kristmannsdottir,H., Dahlkvist,M., Saemundsson,K., Hannington,M. et al. 2001b. Discovery and description of giant submarine smectite cones on the seafloor in Eyjafjordur, northern Iceland, and a novel thermal microbial habitat. Applied and Environmental Microbiology 67: 827-833.

Massana,R., Delong,E.F., and Pedros-Alio,C. 2000. A few cosmopolitan phylotypes dominate planktonic archaeal assemblages in widely different oceanic provinces. Applied and Environmental Microbiology 66: 1777-1787.

Massana,R., Murray,A.E., Preston,C.M., and Delong,E.F. 1997. Vertical distribution and phylogenetic characterization of marine planktonic Archaea in the Santa Barbara Channel. Applied and Environmental Microbiology 63: 50-56.

McGenity,T.J., Gemmell,R.T., Grant,W.D., and Stan-Lotter,H. 2000. Origins of halophilic microorganisms in ancient salt deposits. Environmental Microbiology 2: 243-250.

McInerney,J.O., Mullarkey,M., Wernecke,M.E., and Powell,R. 1997. Phylogenetic analysis of Group I marine archaeal rRNA sequences emphasizes the hidden diversity within the primary group Archaea. Proceedings of the Royal Society of London Series B-Biological Sciences 264: 1663-1669.

McInerney,J.O., Wilkinson,M., Patching,J.W., Embley,T.M., and Powell,R. 1995. Recovery and phylogenetic analysis of novel archaeal ribosomal RNA sequences from a deep-sea deposit feeder. Applied and Environmental Microbiology 61: 1646-1648.

Miskin,I., Rhodes,G., Lawlor,K., Saunders,J.R., and Pickup,R.W. 1998. Bacteria in post-glacial freshwater sediments. Microbiology 144 ( Pt 9): 2427-2439.

More,M.I., Herrick,J.B., Silva,M.C., Ghiorse,W.C., and Madsen,E.L. 1994. Quantitative cell lysis of indigenous microorganisms and rapid extraction of microbial DNA from sediment. Applied and Environmental Microbiology 60: 1572-1580.

66

Moyer,C.L., Tiedje,J.M., Dobbs,F.C., and Karl,D.M. 1998. Diversity of deep-sea hydrothermal vent Archaea from Loihi seamount, Hawaii. Deep-Sea Research Part Ii-Topical Studies in Oceanography 45: 303-317.

Munson,M.A., Nedwell,D.B., and Embley,T.M. 1997. Phylogenetic diversity of Archaea in sediment samples from a coastal salt marsh. Applied and Environmental Microbiology 63: 4729-4733.

Münster,U., Salonen,K., and Tulonen,T. 1999. Decomposition. In Limnology of Humic Waters. Keskitalo,J. and Eloranta,P. (eds). The Netherlands: Backhuys, 225-264.

Muyzer,G., de Waal,E.C., and Uitterlinden,A.G. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Applied and Environmental Microbiology 59: 695-700.

Nei,M. and Kumar,S. 2000. Molecular evolution and phylogenetics. New York: Oxford University Press.

Ng,W.V., Kennedy,S.P., Mahairas,G.G., Berquist,B., Pan,M., Shukla,H.D. et al. 2000. Genome sequence of Halobacterium species NRC-1. Proceedings of the National Academy of Sciences of the United States of America 97: 12176-12181.

Nickson,S.W. 1995. Coastal marine eutrophication. A definition, social causes and future concerns. Ophela 41: 191-219.

Ohkuma,M. and Kudo,T. 1998. Phylogenetic analysis of the symbiotic intestinal microflora of the termite Cryptotermes domesticus. Fems Microbiology Letters 164: 389-392.

Ohkuma,M., Noda,S., and Kudo,T. 1999. Phylogenetic relationships of symbiotic methanogens in diverse termites. Fems Microbiology Letters 171: 147-153.

Olsen,G.J., Lane,D.J., Giovannoni,S.J., Pace,N.R., and Stahl,D.A. 1986. Microbial ecology and evolution: a ribosomal RNA approach. Annual Review of Microbiology 40: 337-365.

Olsen,G.J., Matsuda,H., Hagstrom,R., and Overbeek,R. 1994. fastDNAmL: a tool for construction of phylogenetic trees of DNA sequences using maximum likelihood. Computer applications in the biosciences : CABIOS 10: 41-48.

Olsen,G.J. and Woese,C.R. 1993. Ribosomal RNA: a key to phylogeny. Faseb Journal 7: 113-123.

Olsen,G.J. and Woese,C.R. 1997. Archaeal genomics: an overview. Cell 89: 991-994.

Oren,A., Ventosa,A., Gutierrez,M.C., and Kamekura,M. 1999. Haloarcula quadrata sp. nov., a square, motile archaeon isolated from a brine pool in Sinai (Egypt). International Journal of Systematic Bacteriology 49 Pt 3: 1149-1155.

Orphan,V.J., Hinrichs,K.U., Ussler,W., Paull,C.K., Taylor,L.T., Sylva,S.P. et al. 2001. Comparative analysis of methane-oxidizing archaea and sulfate-reducing bacteria in anoxic marine sediments. Applied and Environmental Microbiology 67: 1922-1934.

Orphan,V.J., Taylor,L.T., Hafenbradl,D., and Delong,E.F. 2000. Culture-dependent and culture-independent characterization of microbial assemblages associated with high-temperature petroleum reservoirs. Applied and Environmental Microbiology 66: 700-711.

Øvreås,L., Forney,L., Daae,F.L., and Torsvik,V. 1997. Distribution of bacterioplankton in meromictic Lake Saelenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA. Applied and Environmental Microbiology 63: 3367-3373.

Pace,N.R. 1997. A molecular view of microbial diversity and the biosphere. Science 276: 734-740.

Pace,N.R., Stahl,D.A., Lane,D.J., and Olsen,G.J. 1985. Analyzing natural microbial population by rRNA sequences. Asm News 51: 4-12.

67

Pace,N.R., Stahl,D.A., Lane,D.J., and Olsen,G.J. 1986. The analysis of natural microbial populations by ribosomal RNA sequences. Advances in microbial ecology 9: 1-55.

Palmer,J.D. 1997. Enhanced organelle genomes: going, going, gone. Science 275: 790.

Pietikainen,J. and Fritze,H. 1995. Clear-cutting and prescribed burning in coniferous forest: comparison of effects on soil fungal and total microbial biomass, respiration activity and nitrification. Soil Biology and Biochemistry 27: 101-109.

Pinar,G., Saiz-Jimenez,C., Schabereiter-Gurtner,C., Blanco-Varela,M.T., Lubitz,W., and Rolleke,S. 2001. Archaeal communities in two disparate deteriorated ancient wall paintings: detection, identification and temporal monitoring by denaturing gradient gel electrophoresis. Fems Microbiology Ecology 37: 45-54.

Pley,U., Schipka,J., Gambacorta,A., Jannasch,H.W., Fricke,H., Rachel,R., and Stetter,K.O. 1991. Pyrodictium abyssi, sp. nov. represents a novel heterotrophic marine archaeal hyperthermophile growing at 110 degrees Celsius. Systematic and Applied Microbiology 14: 245-253.

Plumb,J.J., Bell,J., and Stuckey,D.C. 2001. Microbial populations associated with treatment of an industrial dye effluent in an anaerobic baffled reactor. Applied and Environmental Microbiology 67: 3226-3235.

Poulsen,L.K., Ballard,G., and Stahl,D.A. 1993. Use of rRNA fluorescence in situ hybridization for measuring the activity of single cells in young and established biofilms. Applied and Environmental Microbiology 59: 1354-1360.

Preston,C.M., Wu,K.Y., Molinski,T.F., and Delong,E.F. 1996. A psychrophilic crenarchaeon inhabits a marine sponge: Cenarchaeum symbiosum gen. nov., sp. nov. Proceedings of the National Academy of Sciences of the United States of America 93: 6241-6246.

Purdy,K.J., Munson,M.A., Nedwell,D.B., and Embley,T.M. 2002. Comparison of the molecular diversity of the methanogenic community at the brackish and marine ends of a UK estuary. Fems Microbiology Ecology 39: 17-21.

Radax,C., Gruber,C., and Stan-Lotter,H. 2001. Novel haloarchaeal 16S rRNA gene sequences from Alpine Permo-Triassic rock salt. Extremophiles 5: 221-228.

Ramakrishnan,B., Lueders,T., Dunfield,P.F., Conrad,R., and Friedrich,M.W. 2001. Archaeal community structures in rice soils from different geographical regions before and after initiation of methane production. Fems Microbiology Ecology 37: 175-186.

Reysenbach,A.L., Ehringer,H., and Hershberger,K. 2000a. Microbial diversity at 83 degrees Celsius in calcite springs, Yellowstone National Park: another environment where the Aquificales and "Korarchaeota" coexist. Extremophiles 4: 61-67.

Reysenbach,A.L., Giver,L.J., Wickham,G.S., and Pace,N.R. 1992. Differential amplification of rRNA genes by polymerase chain reaction. Applied and Environmental Microbiology 58: 3417-3418.

Reysenbach,A.L., Longnecker,K., and Kirshtein,J. 2000b. Novel bacterial and archaeal lineages from an in situ growth chamber deployed at a Mid-Atlantic Ridge hydrothermal vent. Applied and Environmental Microbiology 66: 3798-3806.

Rieger,G., Rachel,R., Hermann,R., and Stetter,K.O. 1995. Ultrastructure of the hyperthermophilic Archaeon Pyrodictium abyssi. Journal of Structural Biology 115: 78-87.

Robb,F.T. and Place,A.R. 1995. Archaea: a laboratory manual. Cold Spring Harbor Laboratory Press.

Rochelle,P.A., Fry,J.C., Parkes,R.J., and Weightman,A.J. 1992. DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sediment communities. Fems Microbiology Letters 79: 59-65.

68

Rondon,M.R., August,P.R., Bettermann,A.D., Brady,S.F., Grossman,T.H., Liles,M.R. et al. 2000. Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms. Applied and Environmental Microbiology 66: 2541-2547.

Roszak,D.B. and Colwell,R.R. 1987. Survival strategies of bacteria in the natural environment. Microbiological Reviews 51: 365-379.

Ruepp,A., Graml,W., Santos-Martinez,M.L., Koretke,K.K., Volker,C., Mewes,H.W. et al. 2000. The genome sequence of the thermoacidophilic scavenger Thermoplasma acidophilum. Nature 407: 508-513.

Saano,A., Tas,E., Piippola,S., Lindstrom,K., and Van Elsas,J. 1995. Nucleic acids in the environment: methods and application. Trevors,J.T. and Van Elsas,J. (eds). Berlin: Springer Verlag, 49-67.

Saiki,R.K., Gelfand,D.H., Stoffel,S., Scharf,S.J., Higuchi,R., Horn,G.T. et al. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491.

Sako,Y., Nomura,N., Uchida,A., Ishida,Y., Morii,H., Koga,Y. et al. 1996. Aeropyrum pernix gen. nov., sp. nov., a novel aerobic hyperthermophilic archaeon growing at temperatures up to 100 degrees Celsius. International Journal of Systematic Bacteriology 46: 1070-1077.

Scherer,P. 1989. Vanadium and molybdenum requirement for the fixation of molecular nitrogen by two Methanosarcina strains. Archives of Microbiology 151: 44-48.

Schleper,C., Holben,W., and Klenk,H.P. 1997a. Recovery of Crenarchaeotal ribosomal DNA sequences from freshwater lake sediments. Applied and Environmental Microbiology 63: 321-323.

Schleper,C., Puehler,G., Holz,I., Gambacorta,A., Janekovic,D., Santarius,U. et al. 1995. Picrophilus gen. nov., fam. nov.: a novel aerobic, heterotrophic, thermoacidophilic genus and family comprising archaea capable of growth around pH 0. Journal of Bacteriology 177: 7050-7059.

Schleper,C., Swanson,R.V., Mathur,E.J., and Delong,E.F. 1997b. Characterization of a DNA polymerase from the uncultivated psychrophilic archaeon Cenarchaeum symbiosum. Journal of Bacteriology 179: 7803-7811.

Sekiguchi,Y., Kamagata,Y., Syutsubo,K., Ohashi,A., Harada,H., and Nakamura,K. 1998. Phylogenetic diversity of mesophilic and thermophilic granular sludges determined by 16S rRNA gene analysis. Microbiology 144 ( Pt 9): 2655-2665.

She,Q., Singh,R.K., Confalonieri,F., Zivanovic,Y., Allard,G., Awayez,M.J. et al. 2001. The complete genome of the crenarchaeon Sulfolobus solfataricus P2. Proceedings of the National Academy of Sciences of the United States of America 98: 7835-7840.

Shinzato,N., Matsumoto,T., Yamaoka,I., Oshima,T., and Yamagishi,A. 1999. Phylogenetic diversity of symbiotic methanogens living in the hindgut of the lower termite Reticulitermes speratus analyzed by PCR and in situ hybridization. Applied and Environmental Microbiology 65: 837-840.

Simon,H.M., Dodsworth,J.A., and Goodman,R.M. 2000. Crenarchaeota colonize terrestrial plant roots. Environmental Microbiology 2: 495-505.

Smith,D.R., Doucette-Stamm,L.A., Deloughery,C., Lee,H., Dubois,J., Aldredge,T. et al. 1997. Complete genome sequence of Methanobacterium thermoautotrophicum deltaH: functional analysis and comparative genomics. Journal of Bacteriology 179: 7135-7155.

Stackebrandt,E. and Woese,C. 1981. The evolution of prokaryotes. In Molecular and cellular aspects of microbial evolution. Carlise,M.J., Collins,J.R., and Moseley,B.E.B. (eds). Cambridge: Cambridge University Press, 1-31.

Stahl,D.A. and Amann,R. 1991. Development and application of nucleic acid probes. In Nucleic acid techniques in bacterial systematics. Stackebrandt,E. and Goodfellow,M. (eds). Chichester, United Kingdom: John Wiley & Sons Ltd., 205-248.

69

Stein,J.L., Marsh,T.L., Wu,K.Y., Shizuya,H., and Delong,E.F. 1996. Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment front a planktonic marine archaeon. Journal of Bacteriology 178: 591-599.

Stein,L.Y., La Duc,M.T., Grundl,T.J., and Nealson,K.H. 2001. Bacterial and archaeal populations associated with freshwater ferromanganous micronodules and sediments. Environmental Microbiology 3: 10-18.

Stetter,K.O. 1988. Archaeoglobus fulgidus gen.nov., sp..nov.: a new taxon of extremely thermophilic archaebacteria. Systematic and Applied Microbiology 10: 172-173.

Stetter,K.O. 1989. Order III. Sulfolobales. In Bergey's Manual of Systematic Bacteriology, 1st ed., Vol.3. Staley, Bryant, Pfennig, and Holt (eds). Baltimore: The Williams and Wilkins Co., 2250.

Stetter,K.O. 1998. Volcanoes, hydrothermal venting, and the origin of life. In Volcanoes and the Environment. Marti,J. and Ernst,G.J. (eds). Cambridge: Cambridge University Press.

Stetter,K.O., Thomm,M., Winter,J., Wildgruber,G., Huber,H., Zillig,W. et al. 1981. Methanothermus fervidus, sp.nov., a novel extremely thermophilic methanogen isolated from an icelandic hot spring. Zentralblatt Bakteriologie und Hygiene 1 Abstracts Original C 2: 166-178.

Strunk,O. and Ludwig,W. 2000. ARB: a software environment for sequence data, http://www.arb-home.de.

Swofford,D.L., Olsen,G.J., Waddell,P.J., and Hillis,D.M. 1996. Phylogeny reconstruction. In Molecular Systematics. Hillis,D.M., Moritz,C., and Mable,B.K. (eds). Sunderland, MA: Sinauer, 407-514.

Tajima,K., Nagamine,T., Matsui,H., Nakamura,M., and Aminov,R.I. 2001. Phylogenetic analysis of archaeal 16S rRNA libraries from the rumen suggests the existence of a novel group of archaea not associated with known methanogens. Fems Microbiology Letters 200: 67-72.

Takai,K. and Horikoshi,K. 1999. Genetic diversity of archaea in deep-sea hydrothermal vent environments. Genetics 152: 1285-1297.

Takai,K., Komatsu,T., Inagaki,F., and Horikoshi,K. 2001a. Distribution of archaea in a black smoker chimney structure. Applied and Environmental Microbiology 67: 3618-3629.

Takai,K., Moser,D.P., DeFlaun,M., Onstott,T.C., and Fredrickson,J.K. 2001b. Archaeal diversity in waters from deep South African gold mines. Applied and Environmental Microbiology 67: 5750-5760.

Takai,K. and Sako,Y. 1999. A molecular view of archaeal diversity in marine and terrestrial hot water environments. Fems Microbiology Ecology 28: 177-188.

Takai,K., Sugai,A., Itoh,T., and Horikoshi,K. 2000. Palaeococcus ferrophilus gen. nov., sp. nov., a barophilic, hyperthermophilic archaeon from a deep-sea hydrothermal vent chimney. International Journal of Systematic and Evolutionary Microbiology 50: 489-500.

Teske,A., Hinrichs,K.U., Edgcomb,V., Gomez,A.V., Kysela,D., Sylva,S.P., and Jannasch,H.W. 2002. Microbial diversity of hydrothermal sediments in the Guaymas basin: evidence for anaerobic methanotrophic communities. Applied and Environmental Microbiology 68: 1994-2007.

Thomas,N.A., Faguy,D., and Jarrell,K.F. 2001. Archaeal chromosome, http://www.els.net. London, Nature Publishing Group.

Thomsen,T.R., Finster,K., and Ramsing,N.B. 2001. Biogeochemical and molecular signatures of anaerobic methane oxidation in a marine sediment. Applied and Environmental Microbiology 67: 1646-1656.

Todorov,J.R., Chistoserdov,A.Y., and Aller,J.Y. 2000. Molecular analysis of microbial communities in mobile deltaic muds of Southeastern Papua New Guinea. Fems Microbiology Ecology 33: 147-155.

Tokura,M., Chagan,I., Ushida,K., and Kojima,Y. 1999. Phylogenetic study of methanogens associated with rumen ciliates. Current Microbiology 39: 123-128.

70

Torsvik,V., Daae,F.L., Sandaa,R.A., and Øvreås,L. 1998. Novel techniques for analysing microbial diversity in natural and perturbed environments. Journal of Biotechnology 64: 53-62.

Torsvik,V., Goksoyr,J., and Daae,F.L. 1990. High diversity in DNA of soil bacteria. Applied and Environmental Microbiology 56: 782-787.

Torsvik,V., Sorheim,R., and Goksoyr,J. 1996. Total bacterial diversity in soil and sediment communities - a review. Journal of Industrial Microbiology 17: 170-178.

Torsvik,V., Øvreås,L., and Thingstad,T.F. 2002. Prokaryotic diversity - magnitude, dynamics, and controlling factors. Science 296: 1064-1066.

Tsai,Y.L. and Olson,B.H. 1991. Rapid method for direct extraction of DNA from soil and sediments. Applied and Environmental Microbiology 57: 1070-1074.

Ueda,T., Suga,Y., and Matsuguchi,T. 1995. Molecular phylogenetic analysis of a soil microbial community in a soybean field. European Journal of Soil Science 46: 415-421.

Urbach,E., Vergin,K.L., Young,L., Morse,A., Larson,G.L., and Giovannoni,S.J. 2001. Unusual bacterioplankton community structure in ultra-oligotrophic Crater Lake. Limnology and Oceanography 46: 557-572.

van de Peer Y., Chapelle,S., and De Wachter,R. 1996. A quantitative map of nucleotide substitution rates in bacterial rRNA. Nucleic Acids Research 24: 3381-3391.

van der Maarel,M.J., Artz,R.R., Haanstra,R., and Forney,L.J. 1998. Association of marine archaea with the digestive tracts of two marine fish species. Applied and Environmental Microbiology 64: 2894-2898.

van der Maarel,M.J., Sprenger,W., Haanstra,R., and Forney,L.J. 1999. Detection of methanogenic archaea in seawater particles and the digestive tract of a marine fish species. Fems Microbiology Letters 173: 189-194.

Vasquez,M., Moore,E.R.B., and Espejo,R.T. 1999. Detection by polymerase chain reaction amplification and sequencing of an archaeon in a commercial-scale copper bioleaching plant. Fems Microbiology Letters 173: 183-187.

Vetriani,C., Jannasch,H.W., MacGregor,B.J., Stahl,D.A., and Reysenbach,A.L. 1999. Population structure and phylogenetic characterization of marine benthic archaea in deep-sea sediments. Applied and Environmental Microbiology 65: 4375-4384.

Vetriani,C., Reysenbach,A.L., and Dore,J. 1998. Recovery and phylogenetic analysis of archaeal rRNA sequences from continental shelf sediments. Fems Microbiology Letters 161: 83-88.

von Wintzingerode,F., Selent,B., Hegemann,W., and Gobel,U.B. 1999. Phylogenetic analysis of an anaerobic, trichlorobenzene transforming microbial consortium. Applied and Environmental Microbiology 65: 283-286.

Wallner,G., Amann,R., and Beisker,W. 1993. Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry 14: 136-143.

Wang,G.C. and Wang,Y. 1996. The frequency of chimeric molecules as a consequence of PCR co-amplification of 16S rRNA genes from different bacterial species. Microbiology 142 ( Pt 5): 1107-1114.

Ward,D.M., Bateson,M.M., Weller,R., and Ruff-Roberts,A.L. 1992. Ribosomal RNA analysis of microorganisms as they occur in nature. Advances in microbial ecology 12: 219-286.

Weber,S., Lueders,T., Friedrich,M.W., and Conrad,R. 2001. Methanogenic populations involved in the degradation of rice straw in anoxic paddy soil. Fems Microbiology Ecology 38: 11-20.

Whitehead,T.R. and Cotta,M.A. 1999. Phylogenetic diversity of methanogenic archaea in swine waste storage pits. Fems Microbiology Letters 179: 223-226.

71

Whitford,M.F., Teather,R.M., and Forster,R.J. 2001. Phylogenetic analysis of methanogens from the bovine rumen. BMC Microbiology 1: 5.

Whitman,W.B., Coleman,D.C., and Wiebe,W.J. 1998. Prokaryotes: the unseen majority. Proceedings of the National Academy of Sciences of the United States of America 95: 6578-6583.

Winker,S. and Woese,C.R. 1991. A definition of the domains Archaea, Bacteria and Eucarya in terms of small subunit ribosomal RNA characteristics. Systematic and Applied Microbiology 14: 305-310.

Wisconsin Package Software . 1994. Program Manual for the Wisconsin Package, Version 8.1. Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711.

Woese,C.R. 1987. Bacterial evolution. Microbiological Reviews 51: 221-271.

Woese,C.R. 1994. There must be a prokaryote somewhere: microbiology's search for itself. Microbiological Reviews 58: 1-9.

Woese,C.R. and Fox,G.E. 1977. Phylogenetic structure of the prokaryotic domain: the primary kingdoms. Proceedings of the National Academy of Sciences of the United States of America 74: 5088-5090.

Woese,C.R., Kandler,O., and Wheelis,M.L. 1990. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proceedings of the National Academy of Sciences of the United States of America 87: 4576-4579.

Wu,J.H., Liu,W.T., Tseng,I.C., and Cheng,S.S. 2001. Characterization of microbial consortia in a terephthalate-degrading anaerobic granular sludge system. Microbiology 147: 373-382.

Xin,H.W., Itoh,T., Zhou,P.J., Suzuki,K., and Nakase,T. 2001. Natronobacterium nitratireducens sp. nov., a haloalkaliphilic archaeon isolated from a soda lake in China. International Journal of Systematic and Evolutionary Microbiology 51: 1825-1829.

Zarda,B., Amann,R., Wallner,G., and Schleifer,K.H. 1991. Identification of single bacterial cells using digoxigenin-labelled, rRNA-targeted oligonucleotides. Journal of General Microbiology 137 ( Pt 12): 2823-2830.

Zeikus,J.G. 1977. The biology of methanogenic bacteria. Bacteriological Reviews 41: 514-541.

Zepp,F.K., Holliger,C., Grosskopf,R., Liesack,W., Nozhevnikova,A.N., Muller,B. et al. 1999. Vertical distribution of methanogens in the anoxic sediment of Rotsee (Switzerland). Applied and Environmental Microbiology 65: 2402-2408.

Zhilina,T.N. and Zavarzin,G.A. 1987. Methanohalobium evestigatus, gen. nov., sp. nov., the extremely halophilic methanogenic archaebacterium. Dokladi Akademii Nauk SSSR 293: 464-468.

Zillig,W., Holz,I., Klenk,H.P., Trent,J., Wunderl,S., Janekovic,D. et al. 1987. Pyrococcus woesei, sp.nov., an ultra-thermophilic marine archaebacterium, representing a novel order, Thermococcales. Systematic and Applied Microbiology 9: 62-70.

Zillig,W., Holz,I., and Wunderl,S. 1991. Hyperthermus butylicus, gen. nov., sp. nov., a hyperthermophilic, anaerobic, peptide fermenting, facultatively H2S generating archaebacterium. International Journal of Systematic Bacteriology 41: 169-170.

Zillig,W., Stetter,K.O., Prangishvili,D.A., Schäfer,W., Janekovik,D., Wunderl,S. et al. 1982. Desulfurococcaceae: the second family of the extremely thermophilic, anaerobic, sulfur-respiring Thermoproteales. Zentralblatt Bakteriologie und Hygiene 1 Abstracts Original C 3: 304-317.

Zillig,W., Stetter,K.O., Schäfer,W., Janekovik,D., Wunderl,S., Holz,I., and Palm,P. 1981. Thermoproteales: a novel type of extremely thermoacidophilic archaebacteria isolated from Icelandic solfataras. Zentralblatt Bakteriologie und Hygiene 1 Abstracts Original C 2: 205-227.

72

73

Zinder,S.Z. 1993. Physiological ecology of methanogens. In Methanogens. Ecology, physiology, biochemistry and genetics. Ferry,J.G. (ed). New York: Chapman & Hall, 128-206.

Zuckerkandl,E. and Pauling,L. 1965. Molecules as documents of evolutionary history. Journal of Theoretical Biology 8: 357-366.