ACTAUNIVERSITATISUPSALIENSISUPPSALA2007

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 300

Exploring the Cell Cycle ofArchaea

MAGNUS LUNDGREN

ISSN 1651-6214ISBN 978-91-554-6881-1urn:nbn:se:uu:diva-7848

List of papers

This thesis is based on the following papers, which are referred to in the text by their roman numerals.

I Robinson NP, Dionne I*, Lundgren M*, Marsh VL, Bernander R, Bell SD.Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus.Cell, 2004, 116:25-38.

II Lundgren M*, Andersson A*, Chen L, Nilsson P, Bernander R. Three replication origins in Sulfolobus species: synchronous initiation of chromosome replication and asynchronous termination.Proceedings of the National Academy of Sciences USA, 2004, 101:7046-7051.

III Majerník AI*, Lundgren M*, McDermott P, Bernander R, Chong JP.DNA content and nucleoid distribution in Methanothermobacterthermautotrophicus.Journal of Bacteriology, 2005, 187:1856-1858.

IV Lundgren M, Bernander R. A genome-wide transcription map of an archaeal cell cycle. Proceedings of the National Academy of Sciences USA, 2007, 104:2939-2944

V Lundgren M, Malandrin L, Eriksson S, Huber H, Bernander R. Cell Cycle Characteristics of Crenarchaea: Unity among Diversity. Manuscript

*These authors contributed equally

Reprints were made with the permission of the publishers

Papers by the author not included in this thesis

1. Lundgren M, Bernander R.Archaeal cell cycle progress. Current Opinion in Microbiology, 2005, 8:662-668.

2. Andersson AF, Lundgren M, Eriksson S, Rosenlund M, Bernander R, Nilsson P. Global analysis of mRNA stability in the archaeon Sulfolobus.Genome Biology, 2006, 7:R99

3. Brouns SJ, Walther J, Snijders AP, van de Werken HJ, Willemen HL, Worm P, de Vos MG, Andersson A, Lundgren M, Mazon HF, van den Heuvel RH, Nilsson P, Salmon L, de Vos WM, Wright PC, Bernander R, van der Oost J. Identification of the missing links in prokaryotic pentose oxidation pathways: evidence for enzyme recruitment. Journal of Biological Chemistry, 2006, 281:27378-27388

Contents

Introduction.....................................................................................................7Focus of this study......................................................................................8

The domain Archaea .....................................................................................10A brief history of archaea research...........................................................10Archaeal evolution and diversity..............................................................11The genus Sulfolobus ...............................................................................13

The cell cycle of Archaea .............................................................................15General cell cycle features .......................................................................15Replication ...............................................................................................16

Replication origins in the three domains .............................................16Replication initiation ...........................................................................18Replication elongation .........................................................................20Replication termination .......................................................................24

Genome segregation.................................................................................25Eukaryotic mitosis ...............................................................................25Bacterial genome segregation..............................................................26Archaeal genome segregation..............................................................27

Cell division .............................................................................................28Eukaryotic cytokinesis.........................................................................28Bacterial cell division ..........................................................................29Archaeal cell division ..........................................................................30

Flow Cytometry ............................................................................................31Principle of flow cytometry......................................................................31Flow cytometry in cell cycle analysis ......................................................32

Microarray technology..................................................................................33Principle of microarrays ...........................................................................33Design of spotted microarrays..................................................................34

Cell cycle studies of the archaea ...................................................................36Physiological analysis of archaeal cell cycles (Paper III and V).............36

Cell cycle features of M. thermautotrophicus .....................................37Conserved cell cycle characteristics in crenarchaea ............................39

Characterization of replication initiation in Sulfolobus (Paper I and II) ..41

Multiple replication origins in archaea ................................................41Cdc6 binds to replication origins .........................................................42Cell cycle specific expression and the different roles of the Cdc6 proteins ................................................................................................43

Comprehensive analysis of cell-cycle dependent transcription in Sulfolobus (Paper IV) ...............................................................................44

Synchronization method ......................................................................44Cyclic expression.................................................................................45

Concluding discussion and ideas for the future........................................48

Svensk sammanfattning ................................................................................51

Acknowledgements.......................................................................................53

References.....................................................................................................55

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Introduction

Microorganisms are the foundation of life on Earth; they are present in every environment, from inside Antarctic ice and saline lakes like the Dead Sea to deep inside the Earth’s crust and the superheated anoxic water of hot vents at the bottom of the oceans. They have created, and are maintaining, the environment necessary for organisms like ourselves (Madigan et al., 1997). Our own intestines and skin are also full of microorganisms, vital to our health and well-being, and our bodies harbor a large amount of microbial cells, up to 10 times the number of human cells (Savage, 1977). The exploration of the microbial world is essential to understanding ourselves, our planet and all life upon it.

The first microscope, built by Antoni van Leeuwenhoek in 1674, enabled us for the first time to study individual microbial cells, whose existence until then had only been speculated. In the mid 19th century, Louis Pasteur used cultivation methods to convincingly prove that microorganisms were abundant in nature and that they did not arise spontaneously. The study of microorganisms advanced further with the ability to grow microorganisms in pure culture, in particular on solid media, developed by Robert Koch. His work made it possible to separate microorganisms into species that could be studied individually in the same way as e.g. plants and animals. However, the small size and the low number of physiological characteristics of microorganisms limited the study of the microbial world. The study of microorganisms leaped forward with the onset of molecular biology in the 20th century. The identification of DNA, not protein, as the genetic material, was crucial. Avery, MacLeod and McCarty demonstrated that DNA was the “transforming principle”, endowing harmless bacteria with pathogenic capacity by exposing them to DNA from disease-causing bacteria (Avery etal., 1944). Later, Hershey and Chase proved that it was mainly DNA that was transferred to bacteria during phage infection (Hershey and Chase, 1952). In 1953, James Watson and Francis Crick solved the three-dimensional structure of the DNA polymer, using partly the DNA crystal refraction data from Rosalind Franklin (Watson and Crick, 1953). The structure elegantly revealed how the genetic material could be faithfully copied into two identical molecules. These findings laid the foundation for the central dogma of molecular biology, proposed by Francis Crick, which stipulates that DNA is the genetic material, transcribed to messenger RNA that in turn is translated into protein, the effecter molecule (Crick, 1958).

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The decades since the characterization of DNA as the genetic material have seen a formidable explosion in understanding of cells in the most profound sense. However, for a long time the evolutionary history (phylogeny) of microorganisms was a problem that seemed unsolvable. There were simply not enough characteristics such as shape, color, motility and metabolic properties which could be used for determining accurate relatedness. That problem was elegantly solved by Carl Woese and George Fox by direct investigation of the sequence of small subunit ribosomal RNA (rRNA). The principles of Woese’s method have since become the gold standard for determining relatedness in biology. Woese did his analysis by enzymatically digesting the rRNA molecule and analyzing the fingerprint that the pattern of pieces produced (Woese and Fox, 1977). The result rocked the foundation that biology had been built upon.

In history, there have been several different theories on how all living things are related and how they should be categorized. In the 18th century the first taxonomist, Linnaeus, divided the world into three kingdoms: plants, animals and minerals (Linnaeus, 1735). Microorganisms were inherently difficult to place within the kingdoms and caused controversies that led to various other models. In 1969, Whittaker proposed a five kingdom model consisting of monera (prokaryotes) and four kingdoms of eukaryotes: protista, plantae, fungi and animalia (Whittaker, 1969). Eukaryotes are the organisms whose cells have nuclear membranes, while prokaryote cells lack them. Woese challenged the view of prokaryotes and eukaryotes as the two most distantly related groups of life when he presented his study of little-explored methane producing bacteria, which were no more similar to other bacteria than they were to eukaryotes. Woese suggested the division of life into three domains: eubacteria, urkarya and archaebacteria, the last compromising the enigmatic methanogenic “bacteria”. Subsequent analysis of the archaebacteria revealed that they were actually more closely related to urkarya than eubacteria despite their prokaryotic appearance, prompting Woese to alter the nomenclature to Bacteria, Archaea and Eukaryotes (Woese et al., 1990). This system has proven reliable, and the immense amount of sequence data available, both from entire genomes and from environmental DNA, can be assigned to domains according to Woese’s model.

Focus of this study The work presented in this thesis is focused on analyzing the life cycle of archaeal microorganisms on both a physiological and a molecular level. The physiological analysis was aimed at characterizing the cell cycle of a range of species across the Archaea domain by measuring the length of the different phases of the cell cycle, analyzing the ploidy of the cells and

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studying the processes of genome segregation and cell division. This was done in part to understand the biology of the individual species, but also to enable comparative analysis of the variation of cell cycle modes in archaea.

The objective of the molecular analysis was to develop a mechanistic understanding of cell the cycle processes, and was focused on two species. There were two main aspects of this work: first, to characterize the process of replication initiation, the location of replication start points on the chromosome, what defined a start point, and to confirm and characterize the initiator protein function. Second: to amend the lack of candidates for proteins involved in the processes of chromosome segregation and cell division, but also to find novel factors in the replication process. This was to be done by studying the transcription pattern of all genes in a model organism and identifying which genes are activated at what point in the cell cycle. The second aspect was also focused at delineating regulatory mechanisms of the cell cycle and to map regulons and general mechanisms of regulation.

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The domain Archaea

A brief history of archaea research Archaea is one of the three domains of life on Earth. Like bacteria they are prokaryotes, i.e. they lack a nucleus of the kind that e.g. plant, animal and fungal cells have, though a few bacteria like planctomycetes actually possess membrane bounded nucleoids (Fuerst and Webb, 1991). Archaea and bacteria are also indistinguishable in their range of e.g. size and shape, which is one of the reasons that this domain of life, constituting a large proportion of all life on Earth in terms of both biomass and number of cells, has gone unrecognized until recently. The group of methane producing micro-organisms studied by Carl Woese was the first to be classified as Archaea. Once this new domain was described several characterized bacteria were reclassified as archaea, almost all of them preferring extreme conditions such as high temperature or acidic/alkaline pH.

Thermophilic life forms have for a long time triggered the curiosity of biologists and have been scientifically studied since the 19th century (Gaughran, 1947). Seminal work on thermophiles and the upper temperature limit of life was done by Thomas Brock, who sampled hot springs in Yellowstone National Park in the 1960s. Brock found an abundance of microorganisms not only tolerating, but requiring, temperatures close to the boiling point of water (Brock, 1967). Many of the extreme thermophiles Brock isolated were later reclassified as archaea, though some of them were bona fide bacteria, such as Thermus aquaticus (Brock and Freeze, 1969). The T. aquaticus DNA polymerase proves the industrial potential of these organisms, since it was central in improving the polymerase chain reaction (PCR) to the multi-billion dollar industry it is today (Mullis and Faloona, 1987; Saiki et al., 1988). Wolfram Zillig and Karl Stetter were other microbiological explorers who made important contributions to the emerging field of archaea biology by isolating large numbers of species, including strains able to grow well above 100°C in pressurized vessels (Blöchl et al.,1997). Research on archaea took off in the 1990s when biology entered the genomic era. At that time advancements in technology allowed entire genomes to be characterized rather than individual genes. Methods for large-scale studies of the function of genes, proteins and RNAs were developed in parallel. The archaeon Methanocaldococcus jannaschii became the fourth organism to have its entire genome deciphered (Bult et al., 1996). The

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genomic data provided further evidence of the unique nature of the archaea and their closer evolutionary relationship to eukaryotes compared to bacteria. Environmental analysis of archaea have led to the finding that they are true cosmopolitans, inhabiting environments all around our planet.

Archaeal evolution and diversity The name “Archaea” was selected to imply that archaea resemble the earliest forms of life on Earth. This notion was largely based on the fact that the first described archaea thrived in environments similar to that of the Archaean age (3.8–2.5 billion years ago): hot and oxygen free. Based on phylogeny of ribosomal RNA sequences, the archaea, eukaryotes and bacteria lineages developed from a common ancestor (Woese et al., 1990; Pace, 1997; Robertson et al., 2005). The nature of the cellular world at the time when the domains diverged was most likely quite different from what it is now. It has been suggested that RNA was both the genetic and the catalytic material, though the fragility of RNA has been used as an argument against this. This idea was suggested already by Francis Crick (Crick, 1968), and the time period has been named “the RNA world” (Gilbert, 1986). In the RNA world gene transfer between cells (horizontal transfer) could have been more important than inheritance and lineages with vertical, Darwinian, evolution developed only later. It has been proposed that several cell lineages were formed in the RNA world, prior to the introduction of DNA as genetic material, which is suggested to explain the differences in DNA replication between the domains (Olsen and Woese, 1996; Woese, 2002).

The phylogenetic distribution of thermophiles, most of which are archaea, has been used to suggest a thermophilic origin of life. Thermophiles tend to diverge deep in phylogenetic trees and form short branches (Stetter, 1994). This theory conflicted with the popular notion of early organisms as photosynthetic stromatolite-dwelling life forms (Schopf and Packer, 1987) and has met much criticism (e.g. Miller and Lazcano, 1995). When Schopf’s and Packer’s results were challenged (Brasier et al., 2002), the hot-origin-of-life hypothesis gained some terrain, but the issue is still far from settled.

The emergence of the domain Eukarya is another topic of great controversy. The oldest eukaryote fossils found are 2.1 billion years old (Han and Runnegar, 1992) suggesting that the divergence with archaea occurred at that time or even earlier. The information machinery (replication, transcription, recombination, DNA repair, etc.) of eukaryotes is more similar to the archaeal equivalent than to the bacterial, while the opposite is true for operational proteins (e.g. metabolic enzymes). This dual nature has been used as evidence for different endosymbiotic theories on the origin of the eukaryotes (Martin, 2005). These theories suggest that the eukaryotic nucleus is a result of an archaeon fusing with one or more other prokaryotic

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cells. There are however several other well-founded hypotheses and theories on the origin of eukaryotes, even suggesting that the last common ancestor was a proto-eukaryote, and that archaea and bacteria are reduced versions of that ancestor (Kurland et al., 2006).

The Archaea domain can be divided into two main phylogenetic groups, the Crenarchaeota and the Euryarchaeota phyla (Woese et al., 1990). The cultured euryarchaea include organisms growing at high or saturated salinity (halophiles) isolated from e.g. the Dead Sea in Israel and the Great Salt Lake in USA. The morphologically unique square-shaped Haloquadratum walsbyi(Walsby, 1980; Bolhuis et al., 2004; Burns et al., 2004) belongs to the halophilic euryarchaea. Several groups of methanogens can also be found among the euryarchaea. Methanogens are strict anaerobes described as the only life forms that actively produce methane, a powerful green-house gas, although plant material was recently also shown to produce methane by a so far unknown process (Keppler et al., 2006). Many euryarchaea also thrive at high temperatures. Crenarchaea in pure culture are almost exclusively sulfur metabolizing thermophiles. The Sulfolobus genus was isolated in 1972 (Brock et al., 1972) and is the most studied genus of the crenarchaea. The organisms tolerating the highest temperatures of all life forms are crenarchaea, with Pyrolobus fumarii thriving at 113°C and surviving autoclaving at 121°C (Blöchl et al., 1997). Reports of another crenarchaeon, preliminary named strain 121, grow at 121°C and survive 130°C (Kashefi and Lovley, 2003). The only cultured crenarchaeon preferring low temperatures has been preliminary named Nitrosopumilus maritimus(Könneke et al., 2005). N. maritimus is the first known archaeal nitrifier and the only cultured representative of Marine Group 1 (DeLong, 1992), which are low-temperature marine archaea estimated to constitute approximately one third of all prokaryotic cells in the oceans (Karner et al., 2001). Cenarchaeum symbiosum, another low-temperature crenarchaeon, can so far only be grown together with its host, an Axinella mexicana marine sponge, and is suggested to be an obligate symbiont (Preston et al., 1996).

There are a number of differences between the two main groups of archaea in addition to their phylogenetic separation. The unique metabolic feature of methane production is only present in euryarchaea. Cell division in euryarchaea is performed by a bacterial-type FtsZ protein, which is largely absent in crenarchaea (Bernander, 2000). Chromatin organizing histone proteins, long thought to be absent from crenarchaea, is present in euryarchaea. This has been interpreted as a support for the euryarchaea as an origin of the eukaryotic nucleus (Martin and Müller, 1998). Recent analysis of the Cenarchaeum symbiosum genome (Hallam et al., 2006) has identified histones, ftsZ and the previously euryarchaea-specific DNA polymerasepolD, in conflict with the former distribution of these genes. Analysis of environmental samples also indicates that histones were developed in ancestral archaea ( ubo ová et al., 2005).

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Other groups of archaea, diverging prior to the Euryarchaeota/Crenarchaeota split have been suggested. A group of DNA sequences from environmental samples have been described as a third branch, the Korarchaeota. Korarchaea have been sampled from geothermal environments (Barns et al., 1996), as well as from hydrothermal regions (Marteinsson et al., 2001; Auchtung et al., 2006). A fourth branch, Nanoarchaeota, has also been suggested (Huber et al., 2002). This group is composed of extremely small organisms, only 400 nm in diameter, which grows in a symbiotic/parasitic way on the surface of other archaeal species. Only one species of this branch, Nanoarchaeum equitans, has been cultivated to date (Huber et al., 2002), but sequences of others have been identified (Hohn et al., 2002). Recent studies have suggested that the korarchaea and nanoarchaea may represent fast evolving species within the Crenarchaeota and Euryarchaeota phylum, respectively (Brochier et al.,2005; Robertson et al., 2005).

Many biologists still regard archaea as obscure organisms only found in extreme environments but that notion have been contradicted by cultivation-independent methods for determining the microbial composition of environmental samples. Archaea was first discovered in non-extreme environments in 1992 (DeLong, 1992), and since then they have been found in virtually every environment studied (Schleper et al., 2005), even in our own intestines (Gill et al., 2006). The number of rRNA sequences from uncultivated archaea are several times larger than that of cultivated archaea (Robertson et al., 2005), implying that we have only begun to investigate the phylogenetic spread and environmental diversity of the archaea.

The genus SulfolobusSulfolobus are aerobic thermoacidophilic organisms, shaped as lobed spheres with a diameter of approximately 1 m (Fig. 1). Sulfolobus favours terrestrial geothermal environments with temperatures around 80°C and a pH of 2–3, but maintain an intracellular pH of around 5.5 (Grogan, 2000). They are capable of both autotrophic growth using CO2 as carbon source and sulfur as energy source, and heterotrophic growth on organic carbon compounds (She et al., 2001). Their membranes consist of ether-linked lipids, similar to other archaea but unlike bacteria and eukaryotes which use ester-linked lipids. Sulfolobus also have a protein shell, called the S layer, on the outside of the cell membrane, which increase the durability of the cells (Weiss, 1974).

Sulfolobus acidocaldarius and Sulfolobus solfataricus are the main model organisms of the genus. S. acidocaldarius was isolated from Yellowstone National Park, USA (Brock et al., 1972) and S. solfataricus from the Pisciarelli solfatara near Naples, Italy (de Rosa et al., 1975). Other cultivated

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Sulfolobus species are Sulfolobusshibatae (Grogan et al., 1990) and Sulfolobus tokodaii (Suzuki et al.,2002). Studies of geothermal solfataric environments have revealed a worldwide distribution of Sulfolobusspecies (Rice et al., 2001; Whitaker etal., 2003). The isolation of their environments has limited their community interaction, causing the formation of a biogeographic pattern (Whitaker et al., 2003), which conflicts with the idea that it is the environment, rather than the geographic location, that determines the microbial community structure (Finlay, 2002).

To date, genome sequences of three Sulfolobus species have been published: S. solfataricus (She et al., 2001), S. tokodaii (Kawarabayasi et al., 2001) and S. acidocaldarius (Chen et al., 2005). All Sulfolobus genomes analyzed consist of a single circular chromosome of 2.2–3.0 Mbp. The GC content is low, ranging from 33%–37%.

Figure 1. S. acidocaldarius cells at various cell cycle stages with DNA stained blue. Bar equals 1 m. FromLundgren and Bernander, 2005.

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The cell cycle of Archaea

General cell cycle features The cell cycle is the fundamental process of how cellular life generates offspring. The organisation of the cell cycle is highly complex and correct performance and timing of each stage is vital for duplicating the genetic material and producing daughter cells. The process is in essence the same for all life on Earth, though the variations on the theme are extensive (Angert etal., 2005).

With regard to the cell cycle of archaea, Sulfolobus has been the most extensively studied genus (Bernander, 1998; Bernander, 2000; Ber-nander, 2003; Lundgren and Bernander, 2005). In optimal conditions, the generation times of the model species S.solfataricus and S. acido-caldarius are 6 and 3.5 hrs, respectively. The studied Sulfolobus species all have a very short pre-replicative period, referred to as G1 phase or B period (Fig. 2; Bernander and Pop awski, 1997). There appears to be a tight coupling between cell division and replication initiation, both in terms of time and interdependence (Pop awski and Bernander, 1997). In order to manipulate the cell cycle, various chemicals and antibiotics have been used (Hjort and Bernander, 2001) and the result of that investigation and other experiments have shown that it is difficult to arrest cells in the pre-replicative phase, only hydroxyurea treatment has so far been able to arrest a small part of the population in G1. The major part of the Sulfolobus cell cycle is the G2 phase, which follows the replicative phase (S phase or C period) and precedes genome segregation. The entire post-replicative phase is also referred to as the D period. During the G2 phase, Sulfolobus grows

Figure 2. The DNA content distribution of S.acidocaldarius cells and the relative lengths of the cell cycle phases. Adapted from Hjort and Bernander, 1999.

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until both size and conditions are favourable for cell division, the cells then condense the DNA, segregate the genome copies and divide into daughter cells. The segregation is closely linked to cell division and can be observed a few minutes prior to cell division in a synchronized population (Hjort and Bernander, 1999).

The cell cycle has also been studied in a few euryarchaeal species. Archaeoglobus fulgidus, an anaerobic hyperthermophilic sulfate-reducer (Stetter et al., 1987), cycles between one and two copies of the genome and displays a cell cycle with a long post-replicative phase (Maisner-Patin et al.,2002). In contrast, Methanocaldococcus jannaschii contains 3–15 copies of the genome, suggesting asynchronous replication initiation of the different genome copies (Malandrin et al., 1999) similar to the case in Pyrococcusabyssi (Marie et al., 1996). Multiple genome copies have also been detected in the haloarchaea Haloferax volcanii and Halobacterium salinarum(Breuert et al., 2006). Interestingly, reduced growth rate does not affect the number of genome copies in M. jannaschii, H. volcanii or H. salinarum(Malandrin et al., 1999; Breuert et al., 2006), indicating that they do not have overlapping rounds of replication like fast-growing E. coli (Cooper and Helmstetter, 1968).

ReplicationWhen comparing proteins from the three domains carrying out the various stages of replication, two major groups can be seen, in accordance with molecular phylogeny. Bacteria contain one set of proteins, while archaea and eukaryotes contain another set, although the functional categories are basically the same. Some of the proteins carrying out analogous functions in bacteria and archaea have a striking structural similarity, despite little sequence homology. This has been shown for DnaA and Cdc6 replication initiation proteins (Erzberger et al., 2002), and for PolIII subunit and Pcna processivity factors (Kong et al., 1992; Krishna et al., 1994). These results suggest that original designs can be preserved over time despite divergence of primary sequence and reveal deep evolutionary relationships.

Below follows a description of the replication process in archaea. Bacterial and eukaryotic systems are also outlined for comparative analysis since archaeal replication has been described as “eukaryotic proteins in bacterial context” (Grabowski and Kelman, 2003).

Replication origins in the three domains In 1963, Francois Jacob and Sydney Brenner presented the replicator model to describe how bacterial replication was controlled (Jacob et al., 1963). They envisaged a DNA element, the replicator, which would be the target

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for a factor that initiates replication. The term “origin” is used for the starting point of replication, but as described for certain eukaryotes, the origin does not always contain a defined replicator element. Until the work presented in this thesis, all investigated prokaryotes were believed to use a single origin per chromosome while all eukaryotes used multiple origins (Klug and Cummings, 2002).

In bacteria the origin is known as oriC, which has been extensively studied, mainly in E. coli. The oriC contain several binding sites for the highly conserved protein DnaA (DnaA boxes or R boxes; Fuller et al.,1984), as well as I sites for DnaA in its ATP-bound form (McGarry et al.,2004) and an AT-rich DNA unwinding element (Bramhill and Kornberg, 1988; Hwang and Kornberg, 1992). The number of DnaA boxes and the arrangement of DnaA box clusters vary between bacteria and is even absent in some obligate intracellular bacteria (Mackiewicz et al., 2004).

In eukaryotes the situation is more complex and more varied than in bacteria (Cvetic and Walter, 2005). In the yeast Saccharomyces cerevisiae,the main model system for eukaryotic replication, most origins are characterized by the presence of an AT-rich autonomously replicating sequence (ARS; Stinchcomb et al., 1979), though not all ARS function as origins (Vasslev and DePamphilis, 1992). The S. cerevisiae ARS consensus sequence (ACS) is essential to ARS function and present in four similar versions in the ARS (Newlon and Theis, 1993). Schizosaccharomyces pombe, another yeast, also has an ARS in most of its origins, though it is not clear whether a consensus sequence like the ACS is present (Clyne and Kelly, 1995). In animal cells like the fruit fly Drosophila melanogaster and the frog Xenopus laevis, the origins vary with the developmental stage. In embryonic cells, it has been shown that replication initiate at non-specific sites (Harland and Laskey, 1980) and that more site specific initiation soon develops, primarily by transcription affecting origin usage (Hyrien et al., 1995; Sasaki et al., 1999). In humans and other mammals, no consensus sequence has been found and a sufficiently large piece of DNA seems to be the only requirement for autonomous replication (Heinzel et al., 1991). However, in chromosomal context several origins are described and they are grouped in clusters where location depends on chromatin structure, transcription level and other factors, rather than on DNA sequence (Cvetic and Walter, 2005).

The research on archaeal replication origins is in its infancy, compared to the work in bacteria and eukaryotes. The first replication origin described in archaea came from the euryarchaeon Pyrococcus abyssi, which contains a single origin (Matsunaga et al., 2001; Myllykallio et al., 2000). Pyrococcusorigins are highly conserved and contain several direct and inverted repeats of unknown function similar to the predicted origin in Methanothermobacter thermautotrophicus (Lopez et al., 1999). An autonomously replicating sequence has been isolated from the euryarchaeon Halobacterium sp. strain

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NRC-1 (Berquist and DasSarma, 2003). The studied archaeal origins all co-locate with genes encoding homologous to the eukaryotic Cdc6 and Orc proteins that bind to origins and initiate replication. Computational tools have been used to investigate origins and their location in archaea. Analysis of guanine/cytosine skew within each strand of DNA (Lobry, 1996) has shown single origins or inconclusive results in the species analyzed (Myllykallio et al., 2000). Z-curve representation of genome sequences has suggested one to three origins in archaeal species (Zhang and Zhang, 2005).

Replication initiation Eukaryal and bacterial initiation The identification of the origin sequence is the first step in the process of replication; a protein binds to a location on the DNA and then recruits the proteins needed to unwind the DNA. In E. coli, DnaA is the origin binding protein (Tomizawa and Selzer, 1979) and binds in several copies to the origin region (Messer et al., 2001; Carr and Kaguni 2001). When ATP-DnaA and other required factors binds negatively supercoiled DNA, several DnaA proteins form a right-handed helix that wraps DNA around it and enables DNA helix opening (Erzberger et al., 2006). The E. coli helicase DnaB is then recruited to the origin and loaded by DnaC as a hexameric ring around single stranded DNA (Bujalowski et al., 1994; Lanka and Schuster, 1983). The general E. coli mechanisms extend to other bacteria, but there are known differences, e.g. in B. subtilis where DnaI performs the helicase loader function (Imai et al., 2000).

In E. coli, binding of the initiators to the origin is controlled by three main processes listed below. First, non-origin R boxes around the chromosome block replication by binding DnaA until saturated, so called initiator titration (Hansen et al., 1991). Sequestration of the replication origin is a second mechanism, where SeqA protein binds hemimethylated sequences in oriC and inhibits initiation (Lu et al., 1994). The third process is regulation of DnaA activity (Katayama et al., 1998). Other bacteria have different or complementary systems; Bacillus subtilis lack the sequestration system and in C. crescentus replication can be blocked by CtrA, an origin binding protein that displays cyclic expression (Quon et al., 1998).

In eukaryotes, the six-subunit origin recognition complexes (Orc) binds the replication origin in an ATP dependent manner and was first characterized in S. cerevisiae (Bell and Stillman, 1992) but it has subsequently been shown to be a feature of all eukaryotes (Bell and Dutta, 2002). Orc usually binds to the origin throughout the cell cycle, but is only active in late mitosis and early G1 phase in recruiting the additional factors that together with Orc form the pre-replication complex (pre-RC). Cdc6 and Cdt1 are central to the pre-RC functions and bind independently to DNA-

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associated Orc (Maiorano et al. 2000; Nishitani et al., 2000) and are in turn essential for the recruitment of the Mcm helicase complex. The Mcm in all eukaryotes consists of six subunits: Mcm4, 6 and 7 are implicated in helicase activity while Mcm2, 3 and 5 are suggested regulatory factors (Ishimi, 1997).

Formation of the pre-RC in eukaryotes is a highly regulated process, and there are several seemingly redundant systems for avoiding re-initiation of replication, as outlined below. Cyclic expression of Cdc6 and Mcm subunits is an important regulatory mechanism (Piatti et al., 1995; Leone et al., 1998; Spellman et al., 1998). Chromatin reorganization, and inhibition, degradation or relocalization of pre-RC components, are other initiation control tools (Drury et al., 1997; Petersen et al., 2000; Jiang et al., 1999). Phosphorylation by cyclin dependent kinases (Cdks) is known to play a central role in the regulating these processes. The cyclins are essential in cell cycle processes and vary in expression over the cell cycle in a cyclic manner (Evans et al., 1983). Cdks have a dual role in replication regulation in eukaryotes as they both activate origins and prevent re-initiation by interaction with Orc, Cdc6 and Mcm (Bell and Dutta, 2002). Geminin is another, Cdk-independent, regulator of pre-RC formation in metazoa that act by binding to Cdt1 and thereby inhibiting Mcm loading (McGarry and Kirschner, 1998).

The initiator protein Cdc6 Genes homologous to eukaryotic orc1/cdc6 have been found in all archaea except M. jannaschii (Myllykallio and Forterre, 2000) but are commonly referred to as only cdc6 in archaea. Archaeal Cdc6 proteins can be divided into two sequence groups (Singleton et al., 2004) and species with several Cdc6, such as Halobacterium sp. NRC-1 with nine cdc6 genes, often have proteins from both groups (Ng et al., 2000). It is not clear whether each archaeal Cdc6 is able to perform the functions of both eukaryotic Cdc6 and Orc, or if the functions are separated, especially in the species that have several copies. Cdc6 from Archaeoglobus fulgidus (Grainge et al., 2003), Sulfolobus solfataricus (DeFelice et al., 2003) and Aeropyrum pernix(Singleton et al., 2004) have been shown to bind DNA. Structural analysis of Cdc6 from Pyrobaculum aerophilum (Liu et al., 2000) and Aeropyrum pernix (Singleton et al., 2004) reveals a winged helix DNA interaction domain. Comparison of the structure of archaeal Cdc6 and bacterial DnaA reveal a striking similarity, suggesting an evolutionarily conserved design (Erzberger et al., 2002). Cdc6 is autophosphorylated on serine residues in a DNA-inhibited process, which could serve as a regulatory mechanism (Grabowski and Kelman, 2001).

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The Mcm helicase All studied archaea have at least one mcm gene (Myllykallio and Forterre, 2000). The forms of Mcm complexes in solution vary within the Archaea domain. Most archaeal Mcm are single hexamers in solution (Haugland etal., 2006), but M. thermautotrophicus Mcm has been reported to form both double hexamers and single heptamers (Chong et al., 2000; Yu et al., 2002). The S. solfataricus Mcm has been suggested to form a double hexamer on DNA similar to eukaryotes (Lee and Hurwitz, 2001). Structure analysis of truncated M. thermautotrophicus Mcm also reveals hexameric rings and the central channel is wide enough to accommodate double-stranded DNA (Fletcher et al., 2003). Most archaeal Mcms display helicase activity on linear double-stranded DNA, only Thermoplasma acidophilum seems to require a forked structure like eukaryotic Mcms do (Haugland et al., 2006; Lee and Hurwitz, 2001). Cdc6 interacts with Mcm by the winged helix domain but the response of Mcm to Cdc6 interaction varies. Both stimulation and inhibition of helicase activity have been reported, in different species (Haugland et al., 2006; Shin et al., 2003). Other eukaryotic-like proteins regulating Mcm activity and loading such as Cdk or Geminin have not yet been described in any archaeal species.

Replication elongation Eukaryal and bacterial elongation In eukaryotes, the pre-RC remains inactive until start of S phase (Tye, 1999). At that time the pre-RC unwinds the origin DNA and load the polymerase and associated DNA synthesis factors onto the opened origin. A large protein complex called the preinitiation complex initiates these processes and displaces Cdc6 and Cdt1 as a response to cellular signals by S phase active Cdk and Cdc7. The standard preinitiation complex consist of Mcm10, Cdc45-Sld3 dimer, Gins tetramer, Dbp11-Sld2 dimer and DNA polymerases , and /primase (Bell and Dutta, 2002; Takayama et al., 2003). The Rpa

single stranded DNA binding protein protects the unwound DNA and stabilizes the Pol /primase activity (Maga et al., 2001). Pol /primasesynthesize an RNA primer and extend a short stretch with DNA, which in turn triggers the Rfc clamp loader to recruit the processivity factor (the Pcna clamp) to the replisome which then starts DNA synthesis along the leading strand by extending the primer. The Mcm complex is strongly implicated as the replicative DNA helicase since it moves away from the origin as a part of the replisome (Bell and Dutta, 2002). Replication of the lagging strand is then initiated in a similar manner, but since replication can only proceed in the 5’-3’ direction and the DNA strands are anti-parallel the DNA synthesis there is discontinuous. Lagging strand replication is more complex and forms so called Okazaki fragments, requiring further components for

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completion. Pol /primase is error prone, so after primer extension Fen1, RNase HI and Dna2 remove the primer. The missing nucleotides are then added by Pol together with assisting factors and DNA ligase seals the DNA backbone gap (Hübscher and Seo, 2001). The processes of leading and lagging strand replication are controlled by Cdk and Cdc7 activity, but also by e.g. DNA damage checkpoint proteins of the Rad family (Bell and Dutta, 2002; Hübscher and Seo, 2001). Replication is initiated from the different pre-RC throughout the S phase, but the process that controls late-firing origins is not well understood. Pre-RCs are formed in G1 for all origins, but the preinitiation complex does not assemble at a given origin until at the appropriate time point.

The bacterial process is in essence similar to the eukaryotic, but far less complex. E. coli uses the Pol III core as replicative polymerase, to which several other factors are associated to form the complete Pol III holoenzyme (Johnson and O’Donnell, 2005). The Pol III core is a heterotrimer consisting of the polymerase, the 3’–5’ proofreading exonuclease and the subunit with no described function (McHenry and Crow, 1979). The Pol III dimer is a functional and structural homolog of the eukaryotic Pcna sliding clamp that encircles DNA and is together with vital for replisome speed and processivity (Wickner and Hurwitz, 1976; Maki and Kornberg, 1988). The clamp is loaded by the complex (Stukenberg et al., 1991), analogous to the Rfc and sometimes referred to as the preinitiation complex, though there is little similarity to the eukaryotic namesake. Other replisome proteins in bacteria are Ssb that protect single stranded DNA and melts secondary structures, DnaG that synthesizes primers and the PolIII holoenzyme subunit that coordinates the helicase with leading and lagging strand polymerases (Kornberg and Baker, 1992; Kim et al., 1996).

Functional differences between bacterial and eukaryotic replication are the 10–20 fold faster replication fork, and the 5 times longer Okazaki fragments, 1000 bp vs. 125 bp (Kornberg and Baker, 1992; Blumenthal and Clark, 1977). The bacterial helicase DnaB translocates 5’-3’ on the lagging strand, as opposed to the 3’–5’ leading strand translocation of eukaryotic Mcm (Ishimi, 1997), and the two helicases are suggested to have different evolutionary origins (Johnson and O’Donnell, 2005).

Single strand binding proteins The single strand binding protein in archaea, Rpa, is similar to the eukaryotic counterpart. Rpa has also been shown to stabilize the primer extension of DNA polymerase BI in the euryarchaeon Methanosarcina acetivorans(Robbins et al., 2004). Euryarchaea have several versions of Rpa assembly: single subunit (Kelly et al., 1998; Kelman et al., 1999), homodimers (Robbins et al., 2004) or heterotrimers (Komori and Ishino, 2001). The only characterized crenarchaeal Rpa is from S. solfataricus, which have been observed to form both single subunits in solution and homotetramers on

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DNA (Wadsworth and White, 2001). The tetramer form and the single DNA binding domain is similar to SSB from E. coli, but the protein structure of S. solfataricus Rpa has a striking similarity to human Rpa (Kerr et al., 2003).

PrimaseArchaeal polymerases cannot initiate DNA synthesis de novo, similar to polymerases from the other domains. They require a starting point, which in most cases is an RNA primer synthesized by a primase protein (Kornberg and Baker, 1992). Two archaeal primase proteins, p41 and p46, have been identified and are homologous to the two catalytic primase subunits in eukaryotes, p48 and p58 (Grabowski and Kelman, 2003). The archaeal primase heterodimer from Pyrococcus furiosus and S. solfataricus appears to combine the functions of the eukaryotic polymerase and primase and can produce both the RNA primer and extend it with DNA to form a pre-Okazaki fragment (Liu et al., 2001; Lao-Sirieix and Bell, 2004). The p41 is believed to perform the DNA/RNA synthesis (Desogus et al., 1999; Bocquier et al., 2001) while p46 is suggested to have a regulatory role in reducing the DNA synthesis by the error-prone p41 (Liu et al., 2001). The p41 subunit in S. solfataricus interacts with the Rfc clamp loader, suggesting a mechanism of how DNA synthesis is coupled to primase activity (Wu etal., 2007). Homologs of the bacterial primase DnaG have been detected in S.solfataricus but are reported to be involved in RNA metabolism rather than DNA replication (Evguenieva-Hackenberg et al., 2003).

The replicative polymerases The DNA polymerase is the central player in replication and carries out the synthesis of DNA. There are numerous types of DNA polymerases, grouped in the families A, B, C, D, E, X, and Y, of which D is found almost exclusively in euryarchaea and E only in a single crenarchaeon (Hübscher et al., 2002; Lipps et al., 2003; Ishino et al., 1998). In eukaryotes the , and replicative polymerases all belong to family B. In archaea the B and D families are thought to be involved in replication, and only those polymerase families will be discussed here.

All archaea contain at least one family B (PolB), but e.g. S. solfataricushave three (Grabowski and Kelman, 2003; She et al., 2001). The PolD polymerases have been found in all euryarchaea studied to date and also in the crenarchaeal sponge symbiont C. symbiosum (Hallam et al., 2006). PolD has two subunits, unlike PolB which are single subunit enzymes with the exception of M. thermautotrophicus PolB (Kelman et al., 1999). The large PolD subunit (DP2) is the catalytic subunit while the small (DP1) is an accessory factor suggested to act as a proofreading exonuclease (Ishino etal., 1998; Jokela et al., 2004). PolD also interacts with Rad51 (a recombination protein) suggesting additional roles for PolD in DNA repair (Hayashi et al., 1999). Both PolB and PolD processivity increase in the

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presence of Rfc and Pcna, implicating them in replication (Cann et al., 1999; Grabowski and Kelman, 2003). It is not clear which of the polymerases is the primary replicative factor in the species that have both PolD and PolB, or several PolB. The enzymes could however have complementary roles as suggested for P. abyssi (Henneke et al., 2005), as separate leading/lagging-strand polymerases. Replisome DNA synthesis rate has only been determined for P. abyssi, which synthesizes ~330 bp/s (Myllykallio et al.,2000), similar to e.g. C. crescentus but significantly higher than the 30–50 bp/s for eukaryotes and lower than the 1000 bp/s for E. coli (Chandler et al.,1975).

The Pcna clamp The processivity of replicative polymerases increases by the addition of a ring-shaped sliding clamp, which tethers the polymerase to the DNA. The sliding clamp in archaea is formed by the Pcna protein (proliferating cell nuclear antigen). Euryarchaea contain a single Pcna while crenarchaea usually have three. In the crenarchaeon S. solfataricus Pcna is a heterotrimer (Dionne et al., 2003; Williams et al., 2006), while in A. pernix it has been suggested that the different Pcna can form different homotrimers that interact with different polymerases or are used during different conditions (Daimon et al., 2002). The Pcna is a nexus in the replisome and interacts with many components. In S. solfataricus Pcna have been shown to associate with DNA ligase, Fen1, PolB1, Holliday junction resolvase Hjc, Rfc, nucleotide excision repair protein Xpf and uracil DNA glycosylase (Dionne et al., 2003; Dionne and Bell, 2005; Dorazi et al., 2006; Doré et al., 2006, Pascal et al., 2006; Roberts et al., 2003). In the euryarchaea, Pyrococcus furiosus Pcna has been shown to interact with DNA ligase and Holliday junction migration helicase Hjm (Kiyonari et al., 2006; Fujikane et al., 2006) and Archaeglobus fulgidus Pcna displays interaction with Fen-1, PolB, PolD, Rad2, Rfc, RNase HII and Rpa (Motz et al., 2002).

The Rfc clamp loader The clamp is loaded onto the DNA by replication factor C (Rfc). Two subunits of Rfc have been found in all archaea studied (Cann et al., 2001) and form multimeric complexes. In S. solfataricus the Rfc complex is a pentamer consisting of a small subunit tetramer and a single large subunit, connected by a hinge (Pisani et al., 2000; Seybert et al., 2002). Other combinations have been reported, e.g. two large and four small subunits in M. thermautotrophicus (Kelman and Hurwitz, 2000). A loading mechanism has been suggested for S. solfataricus where the Rfc-Pcna complex opens in an ATP-dependent manner, with Pcna1-Pcna2 bound to Rfc small subunit tetramer and Pcna3 to the large Rfc subunit. The complex then closes on DNA when ATP is dephosphorylated (Dionne et al., 2003). A different mechanism has been shown in P. furiosus where the Rfc pentamer forms a

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spiral-shaped open structure together with Pcna, which then is believed to close on DNA in an ATP-dependant manner (Miyata et al., 2005).

GinsHomologs of the eukaryotic Gins have recently been described in S.solfataricus (Marinsek et al., 2006). Archaeal Gins23 is similar to eukaryotic Gins subunits Psf2 and Psf3, while Gins15 is similar to Psf1 and Sld5. Gins 23 has been shown to interact directly with the Mcm and primase, possibly acting as a bridge between them via Gins 15 and another protein (RecJdbh) coordinating leading and lagging strand replication.

Okazaki fragment maturation The archaeal repertoire of enzymes involved in Okazaki fragment maturation is similar to that of eukaryotes, with Fen-1, RNase H and DNA ligase as the main factors. These have all been shown to interact with Pcna, albeit not in the same model system, suggesting that it is a coordinating structure. Several archaeal Fen-1 have been characterized, degrading branched DNA structures and also functioning as a 5´-3´exonucleases (Grabowski and Kelman, 2003). RNase H is an enzyme that degrades the RNA in RNA/DNA hybrids, such as the RNA primers formed by primase (Frick and Richardson, 2001). There are two families of RNase H: type 1 (RNaseHI) and 2 (RNase HII and RNase HIII). Most archaea have a single RNase HII, but in a few species also an RNase HI has been found (Ohtani et al., 2004). A single DNA ligase has been found in all archaea to date, and they are ATP dependent like the eukaryal counterparts, rather than NAD+ dependent as in bacteria. The Pcna association of DNA ligase is suggested to direct the enzyme to the gap between adjacent Okazaki fragments (Dionne et al., 2003).

Replication termination Nothing is known about the process of replication termination in archaea. In E. coli termination is a highly controlled event (Neylon et al., 2005): when the replisome encounters a Tus protein bound to a ter site the progression of the DnaB helicase is blocked and the replisome stops. There are several tersites in the E. coli genome and the direction specific Tus-ter complexes form a fork trap around the terminus region, letting replisomes in but not out. This means that if replication is initiated at a site different from oriC, the termination still takes place in the same location (Louarn et al., 1977). A similar system is described in B. subtilis, but with Rtp protein, unrelated to Tus, binding ter sites with different sequence than the E. coli version (Hyrien, 2000). In eukaryotes the termination often takes place in random regions between origins but specific termination sites also exists, e.g.downstream of ribosomal genes to avoid collision of replication forks and transcription complexes (Hyrien, 2000). It is an intriguing question whether

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termination of replication in archaea will be of highly controlled bacterial type, or more prone to random fork collision, as in eukaryotes, or if a unique archaeal mechanism remains to be discovered.

Genome segregation When replication has finished the next main step of the cell cycle is chromosome segregation, called mitosis in eukaryotes. The process of eukaryotic mitosis is well characterized, and recently active genome segregation has also been described for bacteria. However, the process in archaea is once again largely unknown. Below follows a brief description of the process in both eukaryotes and bacteria, and a summary of the knowledge of genome segregation in archaea. There is no process equivalent to meiosis in archaea, where haploid gamete cells involved in sexual reproduction are produced.

Eukaryotic mitosis The mitosis phase is divided into several stages that can be seen clearly in a microscope: prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis (Morgan, 2007), the last stage is discussed further in the cell division section. This process applies widely to e.g. plants and animals, but in other eukaryotes such as S. cerevisiae the organization can be slightly different.

After replication the eukaryotic cell contains a duplicate set of intertwined chromosomes called sister chromatids. The sister chromatids are held together by DNA catenation introduced during replication, and by cohesin complexes and other proteins (Lee and Orr-Weaver, 2001). During prophase the sister chromatids are condensed by coiling and folding on several levels (Belmont, 2006). The chromosome segregation structure, the spindle, assembles as a result of reorganization of the microtubule cytoskeleton by e.g. katanin (McNally et al., 2006). The spindle radiates from two centrosomes that act as opposing poles in segregation (Kline-Smith and Walczak, 2004). In prometaphase the nuclear envelope dissolves and the sister chromatids are attached to the spindle at a structure on the chromosome called the kinetochore. The chromatids are aligned at the center of the cell in metaphase and bound to the spindle from both centrosomes. During the first phases sister chromatid cohesion is gradually resolved as cohesins are released and DNA topoisomerase II decatenates DNA (Losada et al., 2002). The separated sister chromatids are then pulled apart during anaphase and in telophase nuclear envelopes reform around the chromatids, and two daughter nuclei are formed.

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Entry into and progression of mitosis is regulated by mitotic cyclin B- Cdk1 complexes and other kinases, a system conserved among eukaryotes (Nurse, 1990). The cyclin B-Cdk1 regulates many proteins involved in forming the spindle by phosphorylation (Ubersax et al., 2003). Cdk1 activity is regulated by relocalization of cyclin B and Cdk, and proteins such Wee1 and Cdc25 (Gould and Nurse, 1989; Kumagai and Dunphy, 1991; Nurse, 1975; Pines and Hunter, 1991). The anaphase-promoting complex (APC) is a central factor in mitosis progression and a target for cyclin-Cdk phosphorylation. The APC has many functions and trigger degradation of cyclins, which inactivates the Cdks, necessary for progression of late mitotic steps (Morgan, 1999).

Bacterial genome segregationThe bacterial chromosome, previously thought to “float around” in the cytosol, has now been shown to be highly structured. Segregation of chromosomes after replication is to a large extent an active process (Thanbichler and Shapiro, 2006a), though not yet characterized to the same level of detail as for eukaryotes. The chromatin in bacteria is compacted by DNA supercoiling and DNA-binding proteins. These proteins include Smc proteins, similar to subunits of cohesin and condensin (Losada and Hirano, 2005; Nasmyth and Haering, 2005). Smc proteins are common among bacteria and are together with other proteins implicated as important DNA organizing factors (Hirano and Hirano, 2006; Thanbichler and Shapiro, 2006a).

Newly replicated chromosome regions are often the first to move towards the cell poles during genome segregation, even before replication is completed in some species, in clear contrast to eukaryotic mitosis (Thanbichler and Shapiro, 2006a). In C. crescentus, it has been shown that unreplicated loci remain in their position until the replisome has passed (Viollier et al., 2004). In E. coli the situation is less clear, it has been suggested that chromosomes stick together and are separated as a whole (Sunako et al., 2001), but recent findings describe that the chromosomal loci are segregated directly after replisome passage, with the exception of the oriC region which behaves differently (Fekete and Chattoraj, 2005). The last step of chromosome segregation takes place as cells divide, when the chromosomes are decatenated by topoisomerase IV together with FtsK and cleared of dimer structures by XerCD. DNA remaining at division plane is pumped to the respective daughter cell by FtsK (Thanbichler and Shapiro, 2006a). Chromosome segregation is an active process since chromosomes move faster than the cell grows (Viollier et al., 2004). Segregation is not coupled to a membrane anchor as the first model of chromosome separation suggested (Jacob et al., 1963). There are several theories on the mechanism of chromosome segregation: pushing from central replisome to cell pole and

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pulling from condensing DNA (Lemon and Grossman, 2000), pushing from many fixed RNA polymerase during transcription (Dworkin and Losick, 2002; Kruse et al., 2006), transertion mediated segregation (Norris, 1995; Woldringh, 2002) and movement generated by the thermodynamics of entangled DNA separation (Jun and Mulder, 2006). None of them explains the complete picture of chromosome segregation, especially the clear direction of origin movement (Viollier et al., 2004; Webb et al., 1998). The identification of the migS sequence in E. coli which enables origin segregation suggests that a functional equivalent of the eukaryotic centromere exist in bacteria (Fekete and Chattoraj, 2005; Yamaichi and Niki, 2004). The main candidate for the chromosome segregation machinery is the actin homolog MreB, initially characterized as a cytoskeletal element involved in cell-shape determination (Jones et al., 2001) but subsequently also shown to play an important role in origin segregation in C. crescentus(Gitai et al., 2005). MreB forms a helical structure in the cell and has been shown to be vital to segregation of origins and bulk chromosome together with RNA polymerase (Kruse et al., 2006). The ParA (Soj) and ParB (Spo0J) proteins, widely distributed in bacteria, also have a role in chromosome segregation (Lee and Grossmann, 2006). The ParA and ParB function is unclear but possibly analogous to the plasmid-encoded ParM actin homolog that is shown to assemble into polymers (van den Ent et al.,2002). In vitro experiments have demonstrated the ability of ParM to segregate DNA by pushing apart beads coated with ParR and parC (Garner et al., 2007).

Archaeal genome segregation The main clues to the segregation machinery in archaea come from presence of genes homologous to known genome segregation factors, such as parA.When it comes to overall chromatin organization several smc genes are present in archaea, possibly encoding proteins involved in cohesion (Hirano, 1998). It has also been suggested that Sulfolobus enable chromatid cohesion and pairing by DNA bridges, hemicatenanes, though it is not clear if this has any role in mitotic chromosome alignment (Robinson et al., 2007). There are also small DNA binding proteins found in archaea, grouped according to their molecular weight: 7, 8 and 10 kDa (Grote et al., 1986). The group 7 proteins in Sulfolobus (e.g. Sac7 and Sso7) introduce negative supercoils and bend DNA (Lopez-Garcia et al., 1998) suggesting a role in chromatin structure. The group 10b proteins (Alba) have been found in all thermophilic archaea. In vitro analysis has suggested that Alba is a main chromatin protein in archaea (Bell et al., 2002), though in vivo analysis has revealed that it can bind both RNA and DNA (Guo et al., 2003; Marsh et al., 2005). Another chromatin protein is the eukaryotic-like histone, which has been

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found in all euryarchaea, as well as in the crenarchaeon C. symbiosum and crenarchaeal metagenomes ( ubo ová et al., 2005; Venter et al., 2004).

Sulfolobus cells have been described to align their chromosomes during segregation, analogous to the mitotic process in eukaryotes (Pop awski and Bernander, 1997). There is little experimental data on proteins involved in genome segregation in archaea, though archaeal hp24, ScpA and Smc homolog Sph1 are implicated (Bernander, 1998; Soppa, 2001; Herrmann and Soppa, 2002). Insertional deletion of the smc gene in Methanococcus voltaecauses aberration in chromosome segregation (Long and Faguy, 2004), though it is not clear if it could be an indirect effect due to chromatin structure changes. A pumping ATPase, HerA, analogous to FtsK has also been suggested to be involved in segregation, based on e.g. mutually exclusive distributions of the two genes (Iyer et al., 2004). Repetitive patterns of short regularly spaced repeats (SRSR/CRISPR) have been found in all archaea, and they have been suggested to have a role in chromosome segregation (Peng et al., 2003), possibly as centromeres, though alternative roles have also been proposed (Makarova et al., 2006). Needless to say, the characterization of the principles and mechanisms of chromosome segregation in archaea still lies ahead, though several putative factors are presented in paper IV.

Cell division The last stage of the cell cycle is division, when daughter cells are produced. The common view of cell division is a spherical or rod-shaped cell that divides through invagination at mid-cell position, producing two identical daughter cells. There are many exceptions to this, such as budding in S. cerevisiae, filamentous growth with branching and multiple spore formation in Streptomyces coelicolor, single spore formation in B. subtilis, snapping division in Thermoproteus tenax, differentiation between daughter cells in C. crescentus, and division of flat square cells in H. walsbyi (Angert, 2005), but the focus here will be on the binary fission form of cell division.

Eukaryotic cytokinesisCell division and chromosome segregation are tightly coupled in many eukaryotes, and often regarded as the last stage of mitosis. However, in some specialized developmental stages, multinucleate cells are known to form (Mazumdar and Mazumdar, 2002). In metazoan cells the division plane is not set until anaphase, in contrast to e.g. S. cerevisiae where the bud starts growing out from the mother cell already in G1 phase. Below is an outline of cytokinesis in eukaryotes, focused on the system in metazoans.

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The molecular mechanism of division-plane determination is still unresolved, and varies between cell types. However, in response to some signal, a contractile ring consisting of actin and myosin (in metazoa), or only myosin (in e.g. S. cerevisiae), assembles at the division plane. The formation and contraction of the actin-myosin ring is dependent on several other proteins (Glotzer, 2005). Septin proteins are believed to act as a scaffold for actin via anillin proteins (Kinoshita et al., 2002). The actin filament formation depends on nucleation from formin proteins, and profilin is required for actin filament elongation (Evangelista et al., 2003).

The actin filaments are attached to the membrane and then pulled together pairwise by myosin II motor protein. The constriction is signalled by activation of myosin II by phosphorylation. Both formation and contraction of the actin-myosin ring is controlled by Rho GTPases that interact with multiple targets, e.g. formin and Rho-activated kinase that phosphorylates myosin II (Glotzer, 2005). Surplus actin filaments are removed by the action of e.g. cofilin (Maciver and Hussey, 2002).

As the ring contracts, new membrane material and cell wall components are inserted at the cleavage site. This insertion has an active role in cytokinesis, especially in plants, where no contractile ring is used (Albertson et al., 2005). New membrane material is transported to cleavage site via microtubuli. The contractile ring eventually meets the mitotic spindle which is compacted into a structure called the midbody, which is dismantled or cut to allow the cells to be parted. The mechanism of this last step is not entirely clear, but delivery of membrane vesicles to the division plane completes the separation (Baluška et al., 2006).

Bacterial cell division The key player in bacterial cell division is FtsZ, a structural homolog of tubulin found in most bacteria (Löwe and Amos, 1998; Nogales et al., 1998; Margolin, 2005). It is interesting to note that while actin performs division and tubulins form the mitotic structure in eukaryotes, the roles are reversed for the bacterial homologs, MreB and FtsZ.

FtsZ assembles as one of the first proteins at the division plane just after replication is finished and forms a ring around the circumference of the cell (Bi and Lutkenhaus, 1991). The FtsZ ring is stabilized by the membrane associated proteins FtsA and ZipA, which appear to have partially redundant functions (Pichoff and Lutkenhaus, 2002). In vitro, FtsZ polymerizes in a GTP dependent manner, similar to tubulin, but do not form the hollow tubes of microtubuli (Margolin, 2005). The initiation of FtsZ ring contraction is dependent on cellular signals, but the nature of these signals is not known. E. coli FtsZ associates with several other proteins, e.g. FtsA, FtsI, FtsQ, FtsL and FtsW, but their roles are not completely discerned.

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The positioning of the FtsZ ring is dependent on several factors. Nucleoid occlusion prevents formation of FtsZ rings over the nucleoid (Mulder and Woldringh, 1989), mediated by the Noc protein in B. subtilis (Wu and Errington, 2004) and SlmA in E. coli (Bernhardt and de Boer, 2005). Formation of FtsZ rings at the cell poles is in turn blocked by the combined action of MinC and MinD (de Boer et al., 1989), leaving only the midcell as possible site of FtsZ assembly. MinC binds to FtsZ and destabilizes ring formation but has no location specificity (Hu et al., 1999). MinD is the cell-pole membrane anchor that MinC interacts with, coordinated by MinE in E. coli and DivIVA in B. subtilis. Interestingly, in E. coli (but not in e.g. B. subtilis) MinCDE oscillates between cell poles, in a cycle that is less than a minute long (Raskin and de Boer, 1999), maintaining high average MinC levels at the cell poles. The MinCDE/DivIVA system is widely conserved in bacteria, even in chloroplasts (Aldridge et al., 2005), though a few species lack the proteins, e.g. C. crescentus where MipZ performs a similar role (Thanbichler and Shapiro, 2006b).

Archaeal cell division Euryarchaea contain the FtsZ gene and it has been shown to form a ring at constriction site in Haloferax volcanii and Haloferax mediterranei (Wang and Lutkenhaus, 1996; Pop awski et al., 2000). However, the gene is notably absent in crenarchaea, with the exception of C. symbiosum (Hallam et al.,2006). FtsZ from M. jannaschii was used in the study that revealed the similarity of FtsZ and tubulin, providing structural insight into the archaeal protein (Löwe and Amos, 1998). Most euryarchaea, and C. symbiosum,contain a MinD gene (Bernander, 2003), further suggesting the presence of a bacterial-like cell division system in euryarchaea. The components of cell division in crenarchaea are not known, though cell constriction can be seen in e.g. Sulfolobus (Pop awski and Bernander, 1997). An unusual mode of cell division is seen in the crenarchaeon Thermoproteus tenax, which divides by cell vibration followed by snapping (Horn et al., 1999), similar to coryneform bacteria (Krulwich and Pate, 1971).

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Flow Cytometry

Principle of flow cytometry Cytometers are in principle light microscopes but with automated detection of specified parameters. Scanning cytometers analyze a static microscopy sample while flow cytometers analyze cells as they pass a detection window. Flow cytometers of today come in many flavors and can detect and quantify virtually anything in a cell by fluorescence from specifically stained cellular constituents. Flow cytometry is used in many aspects of both basic biological and medical science as well as in clinical settings for diagnostic purposes.

The first cytometers were constructed in the 1930s and 1940s, and were used to detect bacteria used as biological weapons in World War II (Gucker et al., 1947). The developers concluded that the technology “may have wide applications in bacteriology,” which it certainly did. Much of the early work on analytical cytometry was done in Stockholm, Sweden, by Caspersson and coworkers in the 1930s–1960s. They determined e.g. that the amount of nucleic acids doubled during the cell cycle (Caspersson and Schultz, 1938), before Avery et al. concluded that DNA was the genetic material. In parallel, clinical cytometry was developed, the first use being diagnosis of uterine cancer (Mellors et al., 1952). Flow cytometers used for medical purpose were also developed, particularly for counting blood cells, based on light scatter of the cells (Crosland-Taylor, 1953) or conductivity change as cells passed a small orifice (Coulter, 1956). Important flow cytometer developments, such as multi-parameter analysis and sorting capability were developed by Kamentsky and coworkers (Kamentsky and Melamed, 1967), as well as by other groups. The first commercial flow cytometers became available in the 1970s, and their potential was quickly realized in immunological research, but soon also in other areas. The development since has been rapid in terms of detection sensitivity, resolution, the number of parameters, sorting capability etc. Specific staining methods have also greatly improved cytometry. The idea of using dyes to stain cells, or parts of them, was in essence developed by Nobel laureate Paul Ehrlich in the late 19th century. In the wake of his work a range of specific dyes was developed, especially fluorescent labels for nucleic acids. Great development also came with the use of antibodies, especially monoclonal antibodies (Köhler and Milstein, 1975), which can specifically target virtually any structure of the

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cell, and is detected either directly by a coupled dye, or indirectly with labeled generic secondary antibodies against the primary antibody.

Flow cytometry in cell cycle analysis Staining of DNA is an important tool in e.g. diagnosis of certain cancers and for performing cell cycle analysis. Early labeling methods, e.g. Feulgen staining, used chemical modifications of DNA but were replaced by simpler procedures with ethidium bromide (Dittrich and Göhde, 1969), propidium iodide (Crissman and Steinkamp, 1973) and mithramycin (Crissman and Tobey, 1974). Analysis of DNA content could provide detailed information on the proliferation of the cells and on the duration of the different cell cycle phases by determining the relative number of cells in the different cell cycle phases. The method also proved important in diagnosis of e.g. cancer cells that often display aberrant DNA content distributions. The use of flow cytometers in microbiology in general and cell-cycle studies in particular has been limited. The cause of this has been the small size of e.g. bacteria, roughly about 0.1% of the volume and DNA content of a mammalian cell. However, instruments that were specifically built or modified for high resolution analysis of small particles produced good results (Steen and Lindmo, 1979) and have since been applied in many different areas of microbiology and virology (Steen, 2000). Analysis of the E. coli cell cycle using flow cytometers was introduced in the 1980s by Boye and his colleagues and proved highly useful in e.g. determination of replication initiation timing (Skarstad et al., 1986). Important contributions to the understanding of the E. coli cell cycle using flow cytometry also came from the work of Nordström and coworkers, e.g. the functional separation of replication and cell division (Bernander and Nordström, 1990).

Flow cytometer analysis of archaea and their cell cycle was started in the mid 1990s by Bernander using a commercial dark-field version of Steen’s instrument with low-angle light scatter detection and ethidium bromide/mithramycin staining for DNA quantification (Bernander and Pop awski, 1997). The result demonstrated a linear correlation between fluorescence and DNA content as well as a light scatter signal that was proportional to cell size. Recently, other groups have also begun flow cytometry analysis of archaea, though only two other reports using flow cytometry have been published (Breuert et al., 2006; Robinson et al., 2007). We are still far from seeing the full potential of flow cytometry applied to archaeal research. The results presented in this study apply the method used by Bernander to various other archaea, as well as for further analysis of the cell cycle of Sulfolobus.

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Microarray technology

The DNA microarray is a tool for measuring the abundance of DNA molecules, usually cDNA for transcript analysis. They consist of large collections of DNA probes deposited in an ordered way on a solid support to which the samples are hybridized. Microarray technology is one of the most commonly used high-throughput methods as it can generate data from all genes in a species in one experiment. Microarray was the buzz-word of late 1990s and early 2000s, especially in the first years when there were about as many reviews as there were primary studies. Today the technology has more or less been integrated into the standard toolbox of molecular biology and novel applications of the technology are continuously being developed.

Principle of microarrays Microarray technology is based on several important scientific findings. David Gillespie and Sol Spiegelman developed the technique of nucleic acid immobilisation and hybridization (Gillespie and Spiegelman, 1965), and Edwin Southern was the first to develop a method for detecting nucleic acids using a molecular probe when he labeled RNA fragments and hybridized them to DNA sequences on a solid support: the Southern blot technique (Southern, 1975). The method was subsequently developed for macroarrays, filters with libraries of deposited sequences. The definitive step towards one of the major types of microarrays came through the innovations of Patrick Brown and colleagues (Schena et al., 1995) who deposited cDNA on glass slides using a robotic printer to produce so-called spotted microarrays. The methods used in the work presented in this thesis are modified versions of Brown’s protocols (http://cmgm.stanford.edu/ pbrown/protocols/index.html). Brown had a do-it-yourself attitude toward microarrays, and published complete blueprints for how to build your own array production robot (http://cmgm.stanford.edu/pbrown/mguide/), but both arrayer robots and microarrays can now be purchased from a range of suppliers. A standard spotted array contains probes against most genes in an organism. Due to the fact that the amount of probe material in each spot can vary, single samples cannot readily be analyzed. Instead, two samples (or occasionally more) are hybridized together in each experiment to achieve a measurement of the ratio of relative abundance between the two samples for each spot. The ratio is

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independent of absolute intensity, probe length, labeling efficiency, etc. The standard way of measuring relative abundance is by scanning the array with two lasers, which each excite a fluorophore that label one of the DNA samples. The total detected signal from a fluorophore in one spot is proportional to the amount off DNA hybridized to that spot. The analysis of microarrays is highly automated, allowing rapid extraction and processing of data.

The method of printing suspended DNA sequences onto solid support is only one of several ways of producing microarrays. The major producer of microarrays is the Affymetrix corporation who employ a method where probes are synthesized in situ, i.e. directly on the microarrays (called GeneChip by the company) by photolithography (Fodor et al., 1991). This method enables production of microarrays with millions of probes per array (compared to tens of thousands for spotted arrays) but each probe is very short, about 25 nucleotides compared to ~80 nt for an oligonucleotide probe and several hundred basepairs for a PCR product probe. The Affymetrix probes may also have imperfect sequences but this is compensated by having several probes against the same gene or sequence. Only a single sample is analyzed on each GeneChip, since each spot contain approximately the same amount of probe. Another microarray manufacturer that uses an in situ probe synthesis is the Agilent Corporation, the second largest microarray producer, who uses ink-jet depositing of nucleotides for synthesis of probes (Blanchard et al., 1996). These probes are longer than the Affymetrix probes, usually about 60 nucleotides, and can be synthesized to a density of several hundred thousand per array. Experiments using Agilent microarrays are performed similar to experiments using spotted arrays.

Microarrays were initially developed to analyze mRNA transcript abundance as a measure of gene expression in order to characterize gene function, operon structures, regulatory pathways and mechanisms, pathogenicity, etc. However, the number of novel uses of DNA microarray technology increases continuously with applications such as gene content and genome rearrangement analysis by comparative genome hybridization, transcription factor regulon analysis by chromatin immuno-precipitate hybridization (ChIP-chip; Buck and Lieb, 2004), mRNA half-life (Selinger et al., 2003), single nucleotide polymorphism (SNP) analysis (e.g. Wang etal., 1998), and replication pattern analysis by marker frequency (Khodursky et al., 2000; Raghuraman et al., 2001).

Design of spotted microarrays As explained above, spotted microarrays consist of probe DNA sequences deposited on solid support, usually glass. Production of spotted microarrays is the most common method used for in-house designed arrays. The spotted

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material can be of several types, e.g. PCR-amplified gene specific tags (GSTs), oligonucleotides, cDNA catalogues or shotgun libraries. The deposition is done by robots with pin sets that are dipped into the probe solution and then stamped on the array surface. The surface is modified to enhance nucleic acid binding, usually with aminosilane or polylysine. The negatively charged backbone of the probe DNA binds to the positively charged surface. The arrays are dried after printing, and the spotted material is linked to the surface by methods depending on the surface chemistry and the material that has been deposited. A common method to cross-link probes to aminosilane or poly-lysine coated surfaces is by exposure to UV-radiation. If PCR products are used for the probes, heating of the probes is required to denature the double stranded sequences. There is a high initial cost for production or purchase of the probes, but the cost per array is then relatively low, compared to commercially available arrays. The arrays used in the work presented in this thesis are non-commercially produced spotted arrays with mainly PCR products as probes.

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Cell cycle studies of the archaea

Physiological analysis of archaeal cell cycles(Paper III and V) Knowledge of overall organization of the cell cycle of a species is a vital foundation for understanding the molecular features of an organism, and the very life processes of the cell. As mentioned earlier, physiological characteristics of the cell cycle of archaeal species, such as ploidy and lengths of cell-cycle phases, have only been described for three species: A. fulgidus, S. solfataricus and S. acidocaldarius, with partial information available for an additional four species. It was an interesting discovery that Sulfolobus had an uncommon cell cycle with a short G1 and a long G2, similar to e.g. S. cerevisiae and S. pombe (Bernander and Pop awski, 1997). Alterations of Sulfolobus growth conditions that lead to longer generation times cause an extension of the G2 phase, while the other phases remain relatively constant. Entry into the stationary phase and stressful conditions commonly lead to arrest in G2 phase. There is no clear understanding of the preference for having two genome copies, but a second copy could be used as template for repair of DNA damage accumulated in the harsh environment that Sulfolobus species inhabit. Another possible advantage would be that in the case of damage occurring to one of the copies, there would still be one intact copy of the genome that could be used to generate viable offspring.

Characterization of the cell cycle of additional archaeal species was one of the objectives of this thesis. The reason was the possibility of performing comparative studies and the aid such information is in understanding the respective species. The study of the cell cycle requires controlled growth conditions and often large amounts of cells, which is most easily obtained for organisms in pure culture. This introduces a bias in the species possible to analyze, as most microorganisms cannot be grown in pure culture, either because they require conditions not yet tested, or that they require the actual presence of the complex composition of microbes that is the natural habitat for most species. Longer generation times in natural conditions, low cell concentrations and the large amount of debris are factors that limit analysis of environmental samples. The cell cycle is easily affected by the composition and pH of growth medium, temperature, oxygen access etc., so this should be taken into consideration when interpreting data. Furthermore, the cell cycle in laboratory conditions should not automatically be taken as

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to reflect the growth in natural conditions. Nonetheless, laboratory conditions are what we have and under e.g. optimal growth conditions comparisons can be made for that state of growth. By altering the conditions important conclusions can be made on which cell cycle phases are flexible in length. Examples are the changing relative length of the G2 phase of Sulfolobus and the approximately constant C period of E. coli over different generation times (Helmstetter, 1996).

The physiological analysis of archaeal cell cycle performed for this thesis incorporates two studies. In Paper III a representative euryarchaeon, Methanothermobacter thermautotrophicus was studied to expand the knowledge on cell cycle characteristics among euryarchaea. Paper V is a comparative study of cell cycle organization among cultivated crenarchaea, where species from three orders were analyzed: Desulfurococcales (Aeropyrum pernix and Ignicoccus islandicus), Thermoproteales (Pyrobaculum calidifontis and Pyrobaculum aerophilum) and Sulfolobales (Acidianus hospitalis and Sulfolobus tokodaii). Like all cultured crenarchaea except one, they are thermophilic but nonetheless prefer different temperatures, ranging from 80 to 100°C. Other properties are also different between the species, e.g. shape (cocci or rod), generation time (85–480 min), pH preference (2.5–7.0), oxygen tolerance and metabolic substrate preference.

Cell cycle features of M. thermautotrophicusM. thermautotrophicus is a strict anaerobic thermophile, and as its name implies, it can grow on inorganic compounds and produce methane (Zeikus and Wolfe, 1972). It was isolated from sewage sludge and has become one of the most widely studied euryarchaea especially when it comes to replication. It grows as flexible rods or curved filaments with an optimal generation time of 5 hrs at 65°C.

The cells were cultured in a bioreactor with CO2 as carbon source and H2as energy source. Samples were taken during growth from exponential to stationary phase. Flow cytometry analysis was performed on an Apogee A40 flow cytometer using the ethidium bromide-mithramycin A staining of DNA used on other archaea (Bernander and Pop awski, 1997). The analysis revealed longer cell filaments and more copies of the genome per filament in early stationary phase compared to later samples. In stationary phase two distinct fluorescence peaks could be observed, which were first interpreted as cells with one and two genome copies. However, estimation of DNA content in the peaks by comparison with rifampicin-treated E. coli MG1655 seqA::Tn10 cells, together with the knowledge that the genome size of M. thermautotrophicus was 1.75 Mbp (Smith et al., 1997) clearly showed that the stationary phase peaks corresponded to two and four genome copies, with the 2-genome peak dominating. In the early exponential phase some

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cells with four, but mainly eight genome copies, could be seen. As the culture matured a 4-genome peak became domi-nating until finally a 2-genome peak took over as the culture entered stationary phase. In early samples the peaks were wide, indicating replication, and a shoulder representing filaments with 12 genomes was observed. The variation in filament lengths and lack of resolution made estimation of the lengths of the cell cycle phases difficult. An experiment with exchange of H2 for N2 was performed to observe how cells react to stress (Fig. 3). Growth was rapidly discontinued after H2 depletion and replication often progressed to termination resulting in sharper fluorescence peaks observed by flow cytometry. A peak with six genome equivalents appeared, further suggesting that asynchronous initiation of replication occur in some of the filaments.

Phase contrast and fluorescence microscopy was performed using 4’,6 diamidino-2-phenylindol (DAPI) staining of DNA which revealed evenly separated nucleoids. The nucleoid distribution closely matched the DNA content distribution, i.e. there were almost as many cells with e.g. four nucleoids as there were cells with a DNA content equivalent to four genome copies. This indicates that the G2 phase is short, in contrast to the characteristic features of e.g. the Archaeglobus fulgidus and Sulfolobus cell cycles, and more similar to the E. coli cell cycle. It was also clear that under the conditions investigated the M. thermautotrophicus cells never contained a single genome copy, also in contrast to Sulfolobus. Close inspection of the microscopy images revealed that each filament often contained septations, i.e. each filament could contain several more or less separated cells but septations were never formed around single nucleoids. This result is consistent with the inter-filament crosswalls observed by electron microscopy (Zeikus and Wolfe, 1973) and the generation of several protoplasts from each filament by peptidase treatment (Kiener et al., 1987). Further, the presence of 12-genome filaments at low OD, and 6-genome filaments in the gas-exchange experiment, indicates that separation of cells, or initiation of replication, is not perfectly synchronized within a filament.

Figure 3. Flow cytometer analysis of M.thermautotrophicus during gas exchange experiment. Time between samples ~3 hrs.

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The results prompt a model of the M. thermautotrophicus cell cycle where each cell goes from two to four genome copies and then divides, with genome segregation being initiated shortly after or during replication.

Conserved cell cycle characteristics in crenarchaea

Length of cell cycle phases The six investigated crenarchaea were cultured heterotrophically at their respective optimal conditions. Five were analyzed by flow cytometry, and it was fascinating to see species after species, with very little in common apart for their phylum, displaying virtually identical cell cycle features. They all cycle from one to two genome copies, initiate replication shortly after cell division and remain in post-replicative phase for the majority of the cell cycle before initiating cell division again. The G1 phase ranged from 1%–4% of the cell cycle, the S phase from 14%–31% and G2/M from 64%–85%. For the sixth species, P. aerophilum, flow cytometry analysis remained inconclusive and microscopy was performed instead. The knowledge of S phase length together with genome size determination (below) enables calculation of replication rate if the number of replication origins is known or postulated. The replication rates were estimated to 210–1000 bp/s assuming bidirectional replication from a single origin. The first archaeal replication rate described prior to this thesis is from P. abyssi, which replicates at 330 bp/s (Myllykallio et al., 2000). The replication rate for Sulfolobus was determined to at least 250 bp/s for S. acidocaldariusassuming a single origin (Hjort and Bernander, 2001) but was changed to 80–110 bp/s in Paper II for all replication forks from all three origins. Based on this knowledge it can be assumed that S. tokodaii also contain multiple origins, otherwise the replication rate would be much higher than for its close relatives. Two origins have recently been demonstrated for A. pernix,in line with our proposal (Robinson and Bell, 2007). Multiple replication origins can also be speculated for the other species, especially I. islandicuswhich otherwise need a replisome ten times faster than that of Sulfolobus.

Stationary phase While the cell cycle organisation was highly similar between the crenarchaeal species, their stationary phase varied. I. islandicus and S.tokodaii displayed a stationary phase similar to characterized Sulfolobusspecies, with most cells containing two genome copies. In P. calidifontis the stationary phase DNA distribution was not very different from exponential growth, suggesting that the cells arrested at different cell-cycle stages, thus lacking a true stationary phase. This indicates that P. calidifontis does not have a special resting state in e.g. nutrient-poor environments. In A. pernixthe number of cells with a single chromosome increased in stationary phase,

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indicating a preferential state different from Sulfolobus, though the fact that some cells remained with two genome copies also suggest a non-programmed entry into stationary phase. In A. hospitalis the fluorescence decreased, even to below one chromosome equivalent. This suggests that the cells goes through DNA degradation and/or chromosome reorganization resulting in decreased dye binding as described for stationary phase Sulfolobus (Hjort and Bernander, 1999).

Genome size estimation As in Paper III, fluorescence signals were used to determine the number of chromosomes, the ploidy, of the cells investigated in Paper V. When calibrated against flow cytometer data from species with known genome sizes analyzed at the same time and conditions, the fluorescence data can be used to accurately estimate the genome size. The correlation between fluorescence signal and DNA content has proven remarkably linear (Paper III; Bernander et al., 2001) and the measured DNA content of organisms with known genome size rarely deviates more than a few percent from the correct value. Using A. pernix, E. coli, M. thermautotrophicus, S.acidocaldarius, S. tokodaii and S. solfataricus for calibration, the genome sizes were estimated to 1.4 Mbp for I. islandicus, 1.7 Mbp for P. calidifontisand 1.8 Mbp for A. hospitalis. Genome size data can facilitate genome sequencing and other genomic analysis. The results presented here show that the genome size of an organism can be determined using flow cytometer when the ploidy is known. The method is rapid and inexpensive (given availability to an instrument) and provides an alternative to other methods for estimating genome sizes, such as pulsed field gel electrophoresis.

Microscopy analysis of nucleoid and cell division structures I. islandicus, P. calidifontis and P. aerophilum were analyzed by phase contrast and fluorescence microscopy. In I. islandicus, with coccoidal cells, the nucleoids were often uniform and centrally located and division septas were observed at a very low frequency, approximately in 1.5% of all cells. This indicates that the long post-replicative period seen by flow cytometry consisted mainly of a G2 phase. In contrast, P. aerophilum and P. calidifontis displayed clearly separated nucleoids in 48% and 38% of the cells, respectively, suggesting that they have a long period with segregated nucleoids and only a short G2 phase. Further, no constrictions indicating closing division septas were observed. Instead, bent and partially split cells were seen, suggesting that cells divide by rapid splitting, presumably along a preformed internal crosswall similar to e.g. Arthrobacter crystallopoietes (Krulwich and Pate, 1971) and Thermoproteus tenax (Horn et al., 1999). The split was not always central, causing uneven lengths of daughter cells, and occasionally formation of small cells without DNA.

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Characterization of replication initiation in Sulfolobus(Paper I and II) Initiation of replication triggers the duplication of the genetic material, which is one of the key steps in the life cycle of a cell. Replication initiation has been found to be a complex, highly regulated feature in all studied eukaryotic and bacterial species. In archaea, knowledge of the process has mainly been inferred from the presence of genes encoding proteins homologous to e.g. eukaryotic replication initiators Orc1 and Cdc6. However, little functional information is available on the organization and mechanism of replication initiation in archaea. To remedy this situation, we initiated an investigation of the location of replication origins and the binding of initiator proteins. As a model system we chose Sulfolobus, easily cultivated organisms that are the most well characterized archaea in terms of cell cycle features. The investigation employed several methods. For overall mapping of replication initiation we used microarray-based marker frequency analysis (MFA). This technique measures copy numbers of genes, which are highest at the origin of replication in a growing culture. For high resolution mapping of origin we employed two-dimensional gel electrophoresis, which is based on the fact that DNA fragments containing the bubble structure of an origin migrates in a unique manner in an ethidium bromide agarose gel. Molecular characteristics of origin regions were investigated using sequence analysis and the binding of initiator proteins to the origins was studied using biochemical methods.

Multiple replication origins in archaea Using MFA it was shown in Paper II, to our great surprise, that both S.solfataricus and S. acidocaldarius contain three origins of replication. This was not anticipated since a single origin has been thought to be one of the defining differences between prokaryotes and eukaryotes (Klug and Cummings, 2002). The data displayed the same decrease in marker frequency on both sides of each origin, revealing that replication was initiated bidirectionally from each origin, similar to the single P. abyssiorigin (Myllykallio et al., 2000). Further, computer simulation of MFA data showed that a model with three origins used synchronously in each cell matched the experimental result perfectly, while simulations of different cells using different origins could not be reconciled with the results. The distribution of the three origins was different in the two species. If rapid replication was the only goal of multiple origins, then an even distribution would be a positively selected feature. It is interesting to note that in both species the spacing was uneven, and in S. acidocaldarius one origin could in theory be deleted without increasing the duration of the S phase to any great extent. This suggests that other factors are important, such as quick

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replication of essential or highly expressed genes. The uneven distribution of origins also causes the termination events to be asynchronous, raising questions on the nature and organization of these events in Sulfolobus.

To investigate the progress of replication, a series of samples for DNA extraction were taken on an acetate-synchronized culture of S. acido-caldarius as it progressed through S phase. The DNA samples were then analyzed by microarray-based MFA. The experiment clearly showed that replication initiates from all three origins in a simultaneous manner, and also enabled in vivo determination of replication rates, which were highly similar for the different replisomes and progressed at 80–110 bp/s. The rate is similar to eukaryotic replisomes and lower than that of the only other characterized archaeon, P. abyssi, and bacteria such as E. coli and C.crescentus. Two of the three origins, oriC1 and oriC2, were found to be co-located with a cdc6 gene, an organization first seen for the single origin in P.abyssi. In Paper I, the two origins adjacent to cdc6 genes were mapped in detail by our collaborators and it was also shown that the cdc6-2 gene was not associated with an origin. Due to limitations of the scanning ability of 2D-gel mapping of origins, the third origin, oriC3, was not mapped in detail at the time, but could be confined to an approximately 40 kb region. No known replication gene was found in this region but the origin was suggested to be located in the region of the gene Saci1405 in S.acidocaldarius and in the region of the ortholog Sso0867 in S. solfataricus.It is interesting to note that in paper IV Saci1405 is described to display an mRNA abundance pattern over the cell cycle highly similar to cdc6-1 and cdc6-3. Subsequent analysis of oriC3 in S. solfataricus has placed the origin in the intergenic region between Sso0866 and Sso0867 in S. solfataricus(Robinson et al., 2007) and shown that Sso0867 binds the origin regions (Robinson and Bell, 2007). The advantage of the co-localization of origins and cdc6 genes is not known, but with the recent finding of transcription-coupled translation in archaea (French et al., 2007) it seems plausible that such an organization would be beneficial since it would place the newly produced protein just where it is needed.

Cdc6 binds to replication origins Using recombinant Sulfolobus Cdc6 we ascertained that they are actual origin binding proteins. DNase I footprinting assays revealed three specific sequences that the Cdc6-proteins bind to, denoted origin recognition boxes (ORBs). The binding to the different ORB elements at the same origin is independent in vitro, as the mutation of one ORB does not inhibit binding of the others. Analysis of the ORB sequences revealed a short dyad repeat element. We searched for ORB elements in other archaea with wide evolutionary distribution and in many cases found similar sequences associated with known or suggested origins, e.g. in A. fulgidus,

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Halobacterium NRC-1 and P. abyssi. The repeats had been observed in P. abyssi earlier (Matsunaga et al., 2003) but no function was determined at the time. The conservation of the ORB sequences is demonstrated by the fact that Sulfolobus Cdc6-1 can specifically recognize the ORB sequences of both P. abyssi and Halobacterium. Thus, in contrast to the eukaryotic origins which are only rarely defined at sequence level, archaeal origins seem more similar to bacterial origins with their DnaA boxes. The structure of origin DNA being coiled on the outside of a helix of DnaA proteins in bacteria have been suggested to be applicable also to archaea (Erzberger et al., 2006), though experimental verification has not yet been published. Detailed mapping of oriC3 revealed that it also contains inverted repeat elements, although different in sequence from other ORB elements (Robinson et al.,2007). Analysis of the M. thermautotrophicus Cdc6 has shown that they also bind to specific 13 bp repeats at the origin (Capaldi and Berger, 2004), further showing the conserved nature of replication initiation in archaea.

Cell cycle specific expression and the different roles of the Cdc6 proteinsThe lack of origin activity associated with the Cdc6-2 paralog raised the question of the function of this protein in replication. In order to investigate that, and study the dynamics of Cdc6 expression, S. acidocaldarius cultures were synchronized and samples for protein extraction were taken at several time points in the cell cycle. The progression of the cell cycle was monitored by flow cytometer analysis of light scatter and fluorescence from labeled DNA. Western blot analysis of cell extracts with antibodies against the three Cdc6 proteins showed that Cdc6-1 and Cdc6-3 displayed identical expression patterns with induction in S phase, concurrent with a role in replication initiation. Cdc6-2, in contrast, displayed a reversed expression pattern; it was detected prior to cell division, disappeared during S phase and reappeared again in G2, after S phase was completed. Transcript profiles for the cdc6 genes in paper IV showed similar patterns. Analysis of batch cell cultures determined that Cdc6-2 was present in stationary phase, while Cdc6-1 and Cdc6-3 were not. UV treatment of Sulfolobus, which cause DNA damage and cell cycle arrest, also induce cdc6-2 transcription (unpublished information). Taken together, these results suggest a role for Cdc6-2 not associated with replication, or possibly as a negative factor inhibiting replication initiation in other cell cycle stages than S phase.

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Comprehensive analysis of cell-cycle dependent transcription in Sulfolobus (Paper IV) A primary intention of this thesis was to find a means to start unraveling the mechanisms of genome segregation and cell division in archaea. With inadequate genetic methods available for Sulfolobus, a genomics approach was chosen. Cell-cycle dependent transcription of genes has been observed in many organisms (Jensen et al., 2006; Laub et al., 2000) and is often indicative of the gene’s functional roles. An example is the eukaryotic cyclin cell cycle regulator which was detected based on its expression pattern (Evans et al., 1983). In order to perform a comprehensive transcription-based search for putative factors involved in different cell cycle processes, microarrays for both S. solfataricus and S. acidocaldarius were designed (Andersson et al., 2005).

Synchronization method In order to study cell cycle specific gene expression a synchronization method is needed. Several described methods for synchronizing Sulfolobus,such as dilution of stationary phase cells and daunomycin treatment (Hjort and Bernander, 1999; Hjort and Bernander, 2001), were compared to newly developed methods, before settling on the acetate treatment. A low dose of acetate, e.g. acetic acid or sodium acetate, resulted in cells arresting in G2 phase, as seen by discontinued cell density increase in the culture and accumulation of cells with two genome copies. The cause of the arrest is not known, but we speculate that it is an effect of the pH difference between the environment (pH 2–3) and Sulfolobus cytosol (pH 5.5). Acetate is in acid form in the low pH environment and we suggest that it penetrates the membrane where it dissociates and acidifies the cell, causing resources to be diverted to pumping out protons. This results in a lack of energy available for normal growth, which the cells respond to by proceeding to the G2 phase and arresting there. A concentration of 2–3 mM acetate causes all cells to enter G2 in about 4 hours, indicating that S phase proceeds slower than the normal 90 minutes (Hjort and Bernander, 2001). A lower acetate concentration causes an incomplete block and a higher concentration kills cells. The G2 arrest generated by acetate is readily reversed by collecting the cells by centrifugation and resuspending them in preheated acetate-free medium. The growth rate of treated cells recover almost immediately after resuspension (as measured by optical density) compared to untreated cells. A large proportion of the released cells (approximately 50% after division) initiate a wave of synchronous progression through G2, which is then followed by mitosis, cell division, G1, and S phase before returning to G2 again, as determined by flow cytometry. The fact that the synchronization is imperfect indicates that all cells are not in the same state when they are

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resuspended in fresh medium. The cells are probably of different sizes after treatment, which could cause the large cells to initiate the synchronous wave. The cells may also arrest at different positions in G2 due to unknown checkpoint mechanisms. A hypothesis would be that cells that were in G1 or S phase when acetate was added arrest at early G2, while cells already in G2 arrest at a later stage. Another interpretation is that the cells that are in earlier phases when treated are more severely affected by the chemical (directly or indirectly) which causes them to recover more slowly. The partial synchronisation also means that the observed expression change levels do not correspond to the average changes in individual cells, which are greater than shown by the experimental data. Furthermore, since the pulse of synchronized cells is not perfect, the expression changes in individual cells are more distinct than observed, with more rapid production and quicker degradation of mRNA. The synchronisation methods developed as part of this project were also used in Paper I and II.

Cyclic expression Three individual biological replicates of the entire experiment were performed. Samples for total RNA extraction were taken at eight time points with 20–30 min interval during the synchronous progression through the cell cycle. The mRNA abundance ratios for genes were measured by pairwise microarray hybridizations through the time series. The data was normalized to compensate for systematic errors, and the ratios for each gene were sequentially added to generate a final abundance profile over the cell cycle. The abundance patterns can readily be interpreted as changes in mRNA synthesis since most transcripts in archaea have a short half-life with a median of 5 min (Andersson et al., 2006). The mRNA detected at each time point must hence, to the large majority, be newly synthesized. Many adjacent genes displayed highly similar transcription patterns, enabling prediction of operon structures. Co-transcription of genes also aid in functional prediction of uncharacterized factors based on the notion that genes in the same operon are often involved in the same processes. The dataset was searched for genes displaying cyclic mRNA abundance patterns by clustering genes with similar expression profiles. We detected more than 160 genes displaying clear cyclic transcription patterns showing that transcription control is a major regulatory function of the cell cycle. Another set of gene transcripts seemed to increase or decrease constitutively during the experiment. These transcripts constitute genes that are responding to the change in environment, rather than to the progression of the cell cycle, or genes that are cyclic but not observed as such over the limited time period studied. We could see cyclic genes with peak expression in all cell cycle phases, indicating active control at all stages. The quality of the expression patterns generated by microarrays was investigated by quantitative PCR

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analysis of several genes. The agreement of the two methods was very good with a Pearson correlation of 0.98. Most genes that code for proteins known to be involved in the cell cycle (mainly replication factors) displayed cyclic expression. Principal component analysis of the entire dataset also showed a cyclic pattern, indicating progression through the cell cycle as the major cue for expression change in the experiment. Below follows a description and discussion of the functional groups of genes that display cyclic expression.

Cell cycle regulation At least seven transcription factors with cyclic expression were detected, with peak expression in different cell cycle phases, indicating putative transcription control components of each cell cycle phase. One of the transcription factors is the basal promoter recognition protein Tfb-2. The differential transcription of tfb-2 shows that there are separate specificities for the three tfb paralogs in Sulfolobus, which suggests that Tfb-2 is an important regulator of many genes involved in the cell cycle. Tfb has a protein fold similar to that of cyclins (Noble et al., 1997), suggesting a possible alternative role of Tfb-2. Another interesting transcription factor that displays cyclic transcription is the recently characterized Sta1 which has been found to be used by the SIRV1 virus during infection to regulate transcription of viral genes (Kessler et al., 2006). If Sta1 trigger production of components needed for e.g. replication that would suggest another reason for SIRV1 to induce transcription of the gene. Production of viral particles requires replication of the viral genome and Sta1 activation would then provide the building blocks for the process. The genes that displayed cyclic induction were searched for common upstream sequence motifs, in order to detect possible co-regulated groups. Two candidate motifs were detected, cell cycle regulon (CCR) box 1 and 2. The consensus sequences were CCTCTCCCTA for CCR-1 and TGTATTAT for CCR-2. The CCR-2 sequence has a high AT content, which could suggest another role of the sequence as a BRE/TATA element, i.e. a non-specific transcription regulator. It will be of key interest to determine if transcription factors bind to the CCR boxes, and if so, what their identity is. Transcription control is not the only level of cell cycle regulation as shown by the cyclic expression of two kinases. They phosphorylate proteins on serine and threonine residues, similar to the eukaryotic Cdks. Possibly the kinases could be involved in the described phosphorylation of Cdc6 (Grabowski and Kelman, 2001).

ReplicationReplication is the best characterized phase of the cell cycle in archaea. Many known replication genes displayed cyclic expression, but it was with special interest we observed the clear cyclic expression of the cdc6-1 and cdc6-3replication initiators, similar to the observed protein level changes in Paper I.

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The induction of the initiator proteins might even occur before cell division, indicating coordination of cell division and replication initiation. The result showed a clear correlation between mRNA and protein levels, which functions as a control for the experiment and suggests close coupling of transcription and translation in Sulfolobus. No known replication-associated gene was seen in the oriC3 region (Paper II), so it is tempting to speculate that the immediately adjacent and cyclically expressed gene Saci1405 could function as the initiator, as suggested also by others (Robinson and Bell, 2007), which would give the oriC3 region an organization similar to the other origins.

As mentioned, several replication-associated genes displayed expression in S phase, including the three pcna, mcm, gins23, ribonucleotide reductase, deoxycytidine deaminase and thymidylate synthase. There are four different DNA polymerases encoded in the S. acidocaldarius genome, and the roles of the three B-type polymerases have not been elucidated. Pol I was the only polymerase that displayed induction in S phase, which strongly implicates it as the in vivo replicative polymerase. Genes involved in the synthesis of purines (DNA precursors) display expected induction in S phase. However, their expression did not decrease in expression during the investigated time period, making determination of cyclic induction inconclusive. In contrast, several genes associated with lagging strand replication, including fen-1,DNA ligase, rfc, ribonuclease HII, ssb and the large subunit of DNA primase, did not display any expression change in the experiment. This is interpreted as that these genes also have a function in DNA repair in other stages of the cell cycle. A set of genes coding for proteins involved in DNA repair also displayed cyclic induction, such as xerD, ogg, radA and exonuclease III, implicating them in replication or replication-associated repair. Several uncharacterized genes, e.g. Saci0002 and Saci0721, physically associated with known replication genes also displayed cyclic expression similar to their neighbours, suggesting that they are novel replication factors.

Genome segregation and cell division Given the unknown nature of genome segregation and cell division of archaea, it was fascinating to see many genes with increased transcription in those two cell cycle phases. However, the close timing of the two events makes it difficult to clearly separate their expression response, whereas their detachment from replication is clear. Marker genes induced in late G2 and M phase, such as the parA gene, can be used to guide the analysis. ParA is most likely involved in the same processes as its bacterial orthologs, which makes it a strong candidate for a role in the genome segregation machinery of Sulfolobus. An uncharacterized gene, Saci0203, located adjacent to parAdisplayed a similar transcription profile, which implicates it as a novel co-transcribed genome segregation factor. Another putative operon that

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displayed a transcription pattern similar to parA was the Saci1372–1374 region. It caught our attention since it encodes a protein similar to the eukaryotic tubulin-interacting katanin, a spindle remodelling factor. The presence and transcription pattern of katanin in Sulfolobus suggest that may have a structure analogous to the eukaryotic spindle in archaea. The two other genes in the putative katanin operon are expected to be involved in similar processes, though little information is available on them. More than ten other genes displayed a transcription pattern similar to parA and katanin, and are suggested to have a role in genome segregation or cell division, but their specific functions are not yet known. Another eukaryotic-like gene with a possible role in genome segregation is pelota, induced in the post-replicative phase in Sulfolobus and involved in spindle formation in Drosophila (Eberhart and Wasserman, 1995). Several factors involved in chromatin organization, such as sac7 and an un-annotated topR-2 gene, displayed cyclic transcription with induction in G2/M. This could implicate involvement in e.g. the described condensation of nucleoids prior to segregation (Pop awski and Bernander, 1997; Hjort and Bernander, 1999) by altering the supercoiling and packaging of DNA.

Concluding discussion and ideas for the future The work presented in this thesis was aimed at the exploration of the cell cycle of archaea at several levels. The main goal was to provide a comprehensive map of cell cycle regulated genes in Sulfolobus by describing the transcription pattern over the cell cycle. These results give an understanding of the nature of cell cycle regulation in archaea in general, and Sulfolobus in particular. This worked out well with the overall findings that the production of cell cycle components was induced when they were needed, but also by characterization of transcription factors, kinases and regulatory DNA motifs. Equally, if not more important, was the finding of cyclically expressed genes that lacked functional annotation. These genes provide a short list of candidates for novel components of the archaeal cell cycle machinery, in particular of the uncharacterized chromosome segregation and cell division processes. Biochemical and genetic investigations of some of these genes are already underway and will most likely provide further insights into the archaeal cell cycle. Cloning and expression of the genes will provide material for a range of assays, such as protein interaction studies, catalytic function determination and structure analysis. Identification of the interaction targets for e.g. archaeal katanin can provide a way to find possible analogs of spindle forming proteins. Structure analysis can reveal mechanistic information, as well as deep evolutionary connections of proteins with similar designs even when amino acid sequence

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homology is very low, as in the case of e.g. bacterial FtsZ and eukaryotic tubulin.

The molecular similarities of archaea and eukaryotes make the findings presented in this thesis even more interesting, and it is my belief that they will provide novel insights into the cell cycle processes of eukaryotes. The presence of multiple replication origins in Sulfolobus is a key example, which was unexpected since it requires a whole new level of replication organization, previously thought to be exclusive to eukaryotes. Investigation of the coordinated initiation of replication in the limited three-origin system of Sulfolobus can provide essential information on regulation of replication in eukaryotes, where such investigations can be hampered by the presence of hundreds (or thousands) of origins initiated throughout the S phase. Elucidation of the cellular location of the different replisomes, the in vivofunction of the different initiator proteins, and spatial and temporal coordination of initiation will be of high interest. Genetic tools for Sulfolobus are being developed (Albers et al., 2006; Worthington et al.,2003), and movement, deletion and duplication of origins are obvious experiments to perform, where the effects on replication organization can be rapidly investigated by MFA and flow cytometry. Determination of autonomous replication capability of the different origins will also be of high interest, and can possibly be used in the improvement of genetics systems. Origin alteration experiments would also provide information on the completely uncharacterized process of replication termination in archaea. It is e.g. not known whether archaea use defined termination sites, like e.g. E. coli, or if they use random termination when replisomes meet.

Antibodies against cell cycle regulated genes will provide further means to investigate their function, e.g. by visualizing structures such as the segregation machinery and the cell division apparatus. This would compensate for the lack of proteins tagged with fluorescent markers such as GFP or mCherry, which have provided spectacular visualization of the positions and dynamics of cellular components in bacteria and eukaryotes. Such methods have been difficult to implement in archaea due to lack of adequate genetic tools or the presence of unfavourable environment, such as high temperature, extreme pH or high salt concentrations. Antibodies can also be used to perform immuno-precipitation analysis on microarrays, ChIP-chip, to define the targets of DNA binding proteins such as the different cell cycle regulated transcription factors. Finding phosphorylation targets for the differentially expressed kinases will also be highly interesting. Tests of the kinases’ phosphorylation ability on Cdc6 would be a good starting point when recombinant proteins are available.

An interesting observation in the microarray dataset was the occurrence of highly similar expression pattern of many genes, some of them also with adjacent positions in the genome. In a single experiment the majority of genes do not usually display great changes in expression, and there is always

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a chance that a similar expression pattern occurs by chance. However, this can be overcome by combining the expression patterns generated from several microarray studies from different conditions. Such combined data can be used to construct reliable transcription and operon maps for the genome. Such maps would greatly aid the understanding of transcription organization and regulation in Sulfolobus and other archaea, and can be searched for e.g. common transcription factor binding sites, BRE and TATA boxes, leader sequences and Shine-Dalgarno elements. Several Sulfolobusmicroarray datasets are already published (Paper IV; Brouns et al., 2006; Snijders et al., 2006) and more are forthcoming, providing the raw material for such analysis.

The detection of conserved cell cycle features during optimal growth for a range of thermophilic crenarchaea raises questions about the driving force behind the observation. It remains to be determined if the design provides a competitive advantage in the extreme environment, and if that design has been reached independently by the various species. Some euryarchaeal thermophiles, such as M. thermautotrophicus, do not have a cell cycle similar to the thermophilic crenarchaea. This suggests that the conserved cell cycle mode is not of outstanding benefit to thermophiles. Instead, the design could be an evolutionary conserved feature, and it will be interesting to see whether non-thermophilic crenarchaea like N. maritimus share the same cell cycle characteristics. Given the availability of enough N. maritimus cells, a test is rather straightforward, though their long generation time may limit the conclusions that can be drawn from such an analysis.

Each finding presented in this thesis has inevitably led to a multitude of new questions, a process common for all science that often drives research forward by the sheer curiosity of finding out how things work. The vast complexity of living organisms, with thousands of processes interacting and regulating each other, sometimes feels as an insurmountable obstacle to understanding life. However, the large number of contributions by scientists around the world, using the tools at hand and the full inventiveness of women and men, are pieces in the great puzzle which step by step increase the understanding of how the world around us works. It has been my great privilege and joy as a PhD student to fit a few more pieces into this puzzle.

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Svensk sammanfattning

Allt liv på jorden har ett gemensamt ursprung. Släktskapet mellan de livsformer vi kan se, som t.ex. växter och djur, har studerats sedan Carl von Linnés dagar på 1700-talet. Linné använde framförallt organismernas morfologi för att avgöra vilka familjer de tillhörde. Redan innan Linné hade Antoni van Leeuwenhoek, en holländsk vetenskapsman och mikroskop-byggare, upptäckt livsformer som var så små att de inte gick att se med blotta ögat. Dessa mikroorganismer hade dock så få utseendemässiga och andra egenskaper att det länge ansågs omöjligt att avgöra deras släktskap. Det var först Carl Woese på 1970-talet som lyckades studera mikroorganismernas familjeträd på ett systematiskt sätt. Han analyserade själva grunden för släktskap, arvsmassan (DNA), särskilt gener som utvecklas mycket långsamt. Innan Woeses arbete trodde man att den äldsta uppdelningen av liv var mellan de organismer som hade en cellkärna, eukaryoter, och de som inte har det, prokaryoter. Eukaryoterna omfattar alla växter, svampar och djur, men även många mikroorganismer som t.ex. amöbor och jäst, medan prokaryoterna utgjordes av bakterier. Woese gjorde en revolutionerande upptäckt: några få metanproducerande ”bakterier” visade sig vara närmare släkt med eukaryoterna än de var med de andra prokaryoterna, vilket betydde att de utgjorde en tredje gren på livets familjeträd, arkéerna. Jämfört med bakterier och eukaryoter vet vi fortfarande mycket lite om arkéerna. De första arkeérna som upptäcktes föredrog extrema miljöer som var t.ex. starkt sura, syrefria, mycket salta eller mycket heta. Idag har dock arkeér hittats i stora mängder i de flesta miljöer som studerats, och de har visats utgöra en viktig del av jordens ekologiska system. Märkligt nog har ännu inga sjukdomsframkallande arkéer beskrivits, trots att de finns i t.ex. vårt matsmältningssystem.

Det arbete som presenteras i denna avhandling beskriver arkéernas cellcykeln, den centrala livsprocessen som beskriver hur en cell växer, kopierar sin arvsmassa och delar sig. De gener som arkeér använder för att utföra t.ex. kopieringen av arvsmassan, replikationen, är mycket lika de som våra egna celler använder, men betydligt mindre komplicerade. Detta betyder att undersökningar av arkéer kan ge oss en förståelse för hur våra egna celler fungerar. För att få en överblick över de gener som används i cellcykeln studerade jag en art av arkéer, Sulfolobus acidocaldarius, i detalj. Sulfolobus hittas vanligen i vulkaniska områden, särskilt heta källor, och trivs bäst vid c:a 80°C och pH 2–3. Vi utvecklade en metod för att studera

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vilka gener som slås på (uttrycks) under specifika delar av cellcykeln hos S.acidocaldarius. Studien bekräftade och klargjorde rollen för flera gener som tros vara inblandade i cellcykeln, och flera gener som används vid regleringen av cellcykeln hittades också. Ännu mer intressanta var de många gener som uttrycktes särskilt i de faser då kromosomkopiorna skiljs från varandra och då cellen delar sig, processer som är praktiskt taget okarakteriserade hos arkéer. Resultatet utgör en karta över de olika delarna av cellcykeln hos arkéer och möjliggör detaljerade studier av mekanismerna bakom de olika processerna.

Vi studerade starten av replikationen hos två Sulfolobus-arter för att lära oss mer om denna process i dessa märkliga celler och våra resultat var mycket överraskande. En av de största skillnaderna mellan eukaryoter och prokaryoter har länge ansetts vara att eukaryoter, med stora mängder DNA, använder många startpunkter på kromosomen för att påbörja replikation medan prokaryoterna med sina små arvsmassor använder en startpunkt per kromosom. Vi hittade tre startpunkter som replikationen initierade synkront från hos Sulfolobus. Denna upptäckt visar att Sulfolobus kan användas för att studera principerna för samordning av replikationstartpunkter i t.ex. våra egna celler som har tusentals startpunkter. Vi karakteriserade även proteiner som binder till startpunkterna och därmed möjliggör replikation. Dessa proteiner visade sig binda till särskilda sekvenser i arvsmassan, och vi kunde hitta liknande sekvenser i många andra organismer. Dessa sekvenser kan därmed användas som markörer för att identifiera replikationstartpunkter i arkéer. Replikationshastigheten uppmättes och var likvärdig med den för eukaryoter, men betydligt långsammare än den för bakterier.

Den övergripande utformningen av cellcykeln, som de olika fasernas längd och antalet kopior av arvsmassan, har endast studerats hos ett fåtal arkeér. Vi utförde en jämförande analys av crenarkéerna, en av de två huvudgrupperna av arkéer. Vi upptäckte att alla hade ungefär samma organisering av cellcykeln, ett fenomen som inte observerats hos någon annan motsvarande stor grupp av organismer. Den gemensamma organiseringen kan ha visat sig fördelaktig, och uppnåtts av de olika organismerna oberoende av varandra eftersom de studerade crenarkeérna växer i heta miljöer (80–100°C). Som jämförelse studerades cellcykeln hos en euryarké, dvs. från den andra huvudgruppen, och den uppvisade inte samma utformning av cellcykeln. Detta visar på att andra mikroorganismer som växer vid höga temperaturer kan ha en annan organisering av cellcykeln, och att crenarkéernas cellcykelorganisationen kan ha ett gemensamt ursprung och vara bevarad sedan lång tid.

Sammanfattningsvis ger dessa upptäckter en förståelse för de grund-läggande livsprocesserna hos några av de mest fascinerande organismerna på jorden, och visar även att arkéer kan användas som modeller för att analysera våra egna cellers mycket mera komplexa system.

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Acknowledgements

The past five years as a PhD student have been very rewarding and I have learned the joys and sorrows of being a scientist. None of the results presented in this thesis would have been possible without help, and support of colleagues, family and friends. I could write a book of thanks, so please forgive the briefness, you mean more to me than I could express in words.

Special thanks go out to Rolf, my supervisor. It’s always fun when you come running with new results or publications that just have to be discussed. Your knowledge and fascination for science have always impressed me. I’m deeply grateful for your friendship and for the opportunity to work with you.

The long days in the lab would definitely not been as fun without the past and present people in the Archaea group. Karin H: thanks for teaching me to the art of Sulfolobus cultivation; I’ve tried my best to fill your shoes. Anders A: Thanks for the travel company, HKMF, teaching me microarrays and for being a good friend. Karin E: Thanks for all the fun, the lab is not the same without you practising aerobics there. Erik K: Thanks for being such good room mate and lab companion. Maria L: You’re the protein expert we need, thanks for your constant good mood. Stefan E: Thank you for the music, the magic touch in the lab and all the discussions. Ulrika: You also taught me the basics of the Sulfolobus work and helped me find my way around the BMC lab. Erika A: Thanks for great help and good company in the lab. Linus and Petra: I had great fun while we were testing out the microarrays, my degree project took just another five years to complete. Bas,Erika G, Hanna N, Marianne, Ovidiu and Ying: thanks for being such good and fun project students working hard on the more or less strange ideas we came up with. Peter: Thanks for a good collaboration in designing and producing the microarrays. Special thanks to the Graduate Research School in Genomics and Bioinformatics (FGB), and to all our collaborators without whom a lot of this thesis work would not have been possible: Steve Bell, Jean Carr, Roger Garrett, Dennis Grogan, Dorothee Götz, Harald Huber, Robert Huber, John van der Oost, Neil Raven,Jasper Walther and Malcolm White.

Thanks to all people that worked at Molecular Evolution, I can’t thank you all enough, these years have been great! Siv, thanks for bringing together such a great department. Håkan, you’re the best computer-guy imaginable, and a good climbing buddy. Karin O, simply nothing would work without you. Hillevi L, extra special thanks for constant cheerful help

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with arrays, their analysis, all those KEGG exercises, thesis hints and so on, if only all people were as kind as you. Your next in line, I wish you luck! Thanks also to all other grad students for help, and making each department party, beer-club and coffee break something to look forward to: Björn B,Björn C, Björn S, Boris, Cessie, Cissi, Caroline F, Haleh, Hans-Henrik,Idress, Ivica, Johan, Jonas S2, Lisa, Mats, Olga, Olof, Sean, Wagied.Very special thanks to Eva, for everything. Thanks also to all you other people working at the department Alex M, Alexandra, Alistair, Ann-Sofie,Dave, Dirk, Joakim, Jonas S1, Kristina, Micke, Ola, Otto, Robban,Thijs, Ylva. I also have to thank the cell-cycle gang: Alex E, Bhupender,Heléne, Janne, Klas, Kurt, Nina, Nora, Santanu, Sonchita, Staffan,Stefan B. A lot of good ideas and discussions came from those meetings.

Jag vill också tacka alla i min familj för allt stöd som ni visat genom åren och för att ni faktiskt försökt begripa vad det är jag gör: Mamma och Pappa, det var tack var er som intresset för vetenskap vaknade. Åsa, Matsoch Fredrik, de bästa syskon jag kan tänka mig! Inga-Lill och Lorens, nya tillskott till familjen som bara gör den bättre.

Ett stort tack till alla vänner som har följt mig genom åren och delat min glädje och sorg. Det är ni som har fyllt mitt liv med fester, skratt, resor, äventyr och insikter om mig själv och livet i allmänhet. Stort tack till alla gamla (jag menar förstås unga) gymnasiekompisar, Fredrik: vem trodde att vi skulle hamna här när vi började vid teleskopen, tack för vänskapen genom alla åren. Denise, Erik, Mårten, Anna och alla andra Vasaiter, NFare och FAAiter för experiment, fester, resor och allt annat som gjorde gymnasietiden fantastiskt kul. Tiden här i Uppsala har också varit fantastiskt kul tack vare alla nya vänner. Redan i studentkorridoren snubblade jag över trevligt folk: Daniel, Elin, Gian, Hans, Helena W, Natascha, Pontus, SaraB. Studentvägen 30 kommer alltid ha en särskild plats i mitt hjärta. Studierna på Polacksbacken var tuffa men också roliga: Emma K, tack för sydamerika- och sydostasienresor, dykning, vandring, och alla brev och samtal. Särskilt tack också till Ioanna, Kicki, Robert och Sophia, och andra alla X-are, Sfinxare och glada teknologer. Tusen tack till alla andra goda vänner: Anders B, Arzu, Birgitta, Brian (för korekturlesningen!), David,Emma B, Emma R, Florian, Hanna S, Jonas W, Karin T, Linus, Martin,Mikael, Patrick, Sofie och Vanna. Lots of arigato, merci and danke to my friends from Japan: Nina, Sophie, Jean-Gabriel, Jacqueline, I wish everyone has a year like that in their lifetime.

Forskning är inte det enda som kommer att rädda världen, och jag vill tacka alla aktivister i Amnesty International i Uppsala som blivit mina vänner genom åren. Agnes, André, Annika, Caroline S, Frida H, HannaA, Helén, Hillevi G, Ingrid, Jan, Jenny W, Johanna, Jussi, Helena H,Karin A, Karin P, Kristina, Maja, Mattias, Maria M, Mounir, Rikard,Roni, Sara E, Torbjörn och sist men inte minst Åsa V.

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