”Extraordinary”
sir David Attenborough
Cover image: Octopus spring in Yellowstone National Park, USA. Photo
taken during the International Geobiology course 2008.
List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I
Lindås, A.-C.(*); Karlsson, E.A.(*); Lindgren, M.T.; Ettema
T.J.G. and Bernander, R. (2008) A unique cell division machinery in the Archaea. Proc Natl Acad Sci USA 105:18942-18946.
II
Bize, A.; Karlsson, E.A.; Ekefjärd, K.; Quax, T.E.; Pina, M.;
Prevost, M.C.; Forterre, P.; Tenaillon, O.; Bernander, R. and
Prangishvili, D. (2009) A unique virus release mechanism in the
Archaea. Proc Natl Acad Sci USA 106:11306-11311.
III
Andersson, A.F.; Pelve, E.A.; Lindeberg, S.; Lundgren, M.;
Nilsson, P. and Bernander, R. (2010) Replication-biased genome organisation in the crenarchaeon Sulfolobus. BMC Genomics 11:454.
IV
Pelve, E.A.(*); Lindås, A.-C.(*); Martens-Habbena, W.; de la
Torre, J.R.; Stahl, D.A. and Bernander, R. (2011) Cdv-based
cell division and cell cycle organization in the thaumarchaeon
Nitrosopumilus maritimus. Mol Microbiol. 82:555-66.
*) These authors contributed equally to these articles.
Reprints were made with permission from the respective publishers.
Contents
Introduction ..................................................................................................... 9
Archaea – Unique properties of ubiquitous organisms ............................ 10
Biology of the cell ............................................................................... 10
Evolution ............................................................................................. 11
Taxonomy ............................................................................................ 13
Ecology ................................................................................................ 15
Archaeal models .................................................................................. 17
Studies of the archaeal cell ........................................................................... 20
Cell division in the archaea ...................................................................... 20
Cell division in crenarchaea (Paper I).................................................. 22
Cell division in thaumarchaea (Paper IV)............................................ 28
Cell division – discussion .................................................................... 29
The archaeal cell cycle ............................................................................. 31
Cell cycle organization of thaumarchaea (Paper IV) ........................... 32
Cell cycle organization – discussion.................................................... 34
Chromosome organization of crenarchaea ............................................... 35
Transcription topology of Sulfolobus (Paper III) ................................. 36
Chromosome organization – discussion .............................................. 39
Viral release and infection in extremophilic environments ...................... 41
SIRV2 infection of Sulfolobus islandicus (Paper II) ........................... 42
Virus of the archaea – discussion ........................................................ 45
Conclusions ................................................................................................... 50
Perspectives .................................................................................................. 51
Methodology in microbiology .................................................................. 51
Archaea by name and nature .................................................................... 54
The universal through the particular ........................................................ 56
Final reflections ........................................................................................ 57
Svensk sammanfattning ................................................................................ 58
Acknowledgements ....................................................................................... 61
References ..................................................................................................... 63
Abbreviations
Cdv
CFU
DAPI
dN/dS
DNA
ESCRT
FtsZ
GC content
LUCA
MOI
OD
PCR
PFU
RNA
S-layer
SEM
SIRV2
STIV
TEM
VAP
Cell division system of many cren- and thaumarchaea
Colony forming units; quantification of viable cells
4',6-diamidino-2-phenylindole; DNA staining dye
Ratio of nucleotide substitution rates for non-synonymous
and synonymous mutations; Gene evolution estimation
Deoxyribonucleic acid; the genetic material in cells and
many viruses
Endosomal sorting complex required for transport; eukaryotic protein systems involved in cell sorting, viral release and
cytokinesis
Filamenting temperature-sensitive mutant Z; cell division
system of bacteria, eury- and korarchaea
Guanine/cytosine content; measurement of nucleotide composition of a genome
Last universal common ancestor; the postulated ancestor to
all now living cellular life
Multiplicity of infection; ratio of viruses to host cells
Optical density; measurement of cell culture turbidity
Polymerase chain reaction; method for DNA amplification
Plaque forming units; quantification of lytic viruses
Ribonucleic acid; information carrying and catalytic macromolecule
Surface layer; protein layer outside the archaeal and bacterial cell membrane
Scanning electron microscopy; visualization of surface
structures of prepared samples
Sulfolobus rod-shaped virus 2
Sulfolobus turreted isocahedral virus
Transmission electron microscopy; visualization of crosssections of prepared samples
Virus associated pyramids; the cell lysis system of SIRV2
and STIV
Introduction
There are three domains of life – three distinct paths of evolutionary history;
Bacteria, Eukarya and Archaea (Woese and Fox, 1977; Woese et al., 1990).
Organisms of each of these three branches of the tree of life have been
shaped by similar evolutionary forces and, with a few exceptions for the
most extreme of environments, molded by the same habitats. In many aspects the bacterial, archaeal and eukaryotic cells are very similar, utilizing
the same macromolecules for the same tasks, deploying similar strategies for
similar situations, but there are crucial differences between them which reveal their separate evolutionary histories. Some such solutions are the subject of this thesis.
Since 1977 when the first study of systematic phylogenetic comparison of
the ribosomal RNA was published our understanding of the three domains
and their relationship to each other has exploded. Classical microbiology
where model organisms are cultivated and studied in laboratories has been
given a much needed phylogenetic framework in which to place its observations, and application of the new techniques directly to environmental samples has made it possible to study organisms that cannot be cultivated. Also,
considering the technological revolution the biological sciences are currently
experiencing – particularly in the fields of DNA sequencing – this is an exciting time to study microbiology.
Among the lessons learned during the last three decades is the realization of
just how large the tree of life is and what a small part of it has been available
for study through conventional means. The majority of described bacterial
phyla are known only by DNA sequencing (Chaban, 2006), and for all microbial life only a small subset of the known diversity has ever been studied
in the laboratory (Rappé and Giovannoni, 2003). The same is true for our
understanding of the internal workings of the cell. Even for the few designated model organisms such as the bacterium Escherichia coli, the eukaryote
Saccharomyces cerevisiae or, to a lesser extent, the archaeon Sulfolobus
acidocaldarius where a large body of collected experimental data has given
us a detailed map of their cell functions, fundamental questions remain unanswered. It is also becoming increasingly clear that even in the basic organ9
ization of the cell, the model organisms are not universally representative.
Newly sequenced genomes reveal a lack of genes that were thought to be
essential and familiar systems are shown to be used for unexpected tasks. In
a related process, DNA sequencing of novel organisms and environmental
samples has enormously expanded our list of genes without an assigned
function, again highlighting how much is still unknown in modern biology.
The archaea are the focus of a remarkably large number of highly interesting
stories. Archaea are studied in the context of the life under extreme conditions, life on early Earth and putative extracellular life, the function of the
eukaryotic cell, the evolutionary relationship between the three domains and
the last universal common ancestor (LUCA), of global carbon- and nitrogen
metabolism and on a more fundamental scale, our basal understanding of
biological life. My research is primarily focused on cellular properties of
cren- and thaumarchaea, but within the scope of this thesis I will touch upon
the eukaryotic cell, viral ecology, archaeal phylogenetics and the fundamental relationship between the three domains themselves.
Archaea – Unique properties of ubiquitous organisms
The archaea are unicellular prokaryotes, adapted to a range of habitats and
life styles. They are a vast and diverse group of organisms that represent a
third of the tree of life as we know it and – given their numerous representations in some of the largest ecosystems of the biosphere (e.g. Karner et al.,
2001) – they also represent a significant portion of the Earth’s biomass.
While best known for living as extremophiles, the archaea are found in all
environments and are major players in some of the fundamental biogeochemical cycles of the biosphere. The archaea have similarities to both bacteria and eukaryotes, and also have unique properties not found in any of the
other domains of life.
Biology of the cell
Under the microscope, archaea are very hard to distinguish from bacteria.
They both share the same general morphology; small, single-celled organisms that lack a cell nucleus. In contrast to eukaryotes, their DNA is organized in nucleoids that are not separated from the rest of the cytosol. This
permits transcription and translation to occur without an intermediate
transport step. Similarly to bacteria the archaeal chromosome is circular and
comparably small, with high gene density and few noncoding regions. The
10
genes of bacteria and archaea are grouped in operons – cassettes of cotranscribed genes with shared regulatory motifs. The diverse metabolic capacities and life styles of archaea are reminiscent of bacteria. Because of
this, the two groups of organisms were classified together under the term
prokaryotes for a long time, until molecular data revealed their distinct evolutionary histories (Woese and Fox, 1977).
The similarities to eukaryotes are less obvious from morphological and metabolic traits, but the basal cellular machinery of archaea share a larger similarity with eukaryotes than bacteria. Most prominently, the replication machinery of archaea consists of components that are homologous to the corresponding eukaryotic proteins (Grabowski and Kelman, 2003). Translation,
transcription and protein regulation also share similarities with eukaryotes,
(Bell and Jackson, 2001; Maupin-Furlow et al., 2006). For example the archaeal RNA polymerase is closely related to the eukaryotic RNA polymerase II (Werner, 2007).
Unique properties for archaea that separates them from the other two domains include both core cellular and metabolic properties. A feature used for
taxonomic identification is the presence of ether-linked phospholipids of the
membrane as well as monolayer membranes where one single bipolar lipid
spans the entire membrane (De Rosa et al., 1986). This configuration increases thermostability of the membrane and has been speculated to be an
adaptation to hyperthermophilic environments (van de Vossenberg et al.,
1998). The archaeal cell walls lack the polymer murein which is present in
many bacteria (Woese et al., 1978). On the outside of the archaeal cell
membrane, a so called S-layer is formed by a porous, para-crystalline protein
sheet (Albers and Meyer, 2011). There are also morphological traits not seen
in the other domains, such as the square, extremely thin cells of the halophile
Haloquadratum walsbyi (Bolhuis et al., 2004). Metabolically the archaea are
more diverse than eukaryotes. Similar to bacteria there are archaea that make
a living in most habitats in the biosphere. This diversity includes the unique
methanogenesis pathway which makes archaea the sole biological producer
of the powerful greenhouse gas methane. There is also a mode of carbon
dioxide assimilation so far only found in the archaea (Berg et al., 2007).
Evolution
Life is old. During almost the entire 3.9 billion years that the Earth has been
able to host life, life has been present. While the exact dating and nature of
the oldest unambiguous proof of life is under hot debate, an origin of life on
Earth older than 3.5 billion years ago and perhaps as old as 3.8 – 3-9 billion
11
years is not unfeasible (Schopf and Packer 1987; Brasier et al., 2002; van
Zullen et al., 2002). The origination of evolving biomolecules together with
the nature and evolution of this protocellular life remains one of the more
intriguing questions in modern biology. All modern life is thought to stem
from one single, cellular root. This last universal common ancestor – together with accompanying viruses – is known only by what is inferred from
comparisons of now living species. Studies of archaea have always been
connected to models of protocellular evolution and the basal relationship
between the three domains of life.
The view of archaea as viable models for life on early Earth – in the 'archean' geological age, which comprises the first billion years of life on the planet – is as old as that of archaeal research itself (Woese and Fox, 1977; Woese
et al., 1990). The identification of the two main archaeal phyla and the presence of thermophiles in both these groups suggested thermophilic origin for
all of archaea (Woese, 1987), and the image of archaea as remains of primordial life, maintaining a life style reminiscent of the very first protocell,
has never lost its appeal, even as a more complex image of archaea adapted
to virtually all ecosystems – extreme as well as non-extreme – has emerged.
It has been known for a long time that the extremophilic life style of archaea
is not shared by all members of the domain (De Long, 1992), and the continuously emerging evidence of archaeal presence in non-extremophilic ecosystems (Chaban, 2006) challenges an extremophilic archaeal origin. Models
have been proposed of a ‘hot’ archaeal origin where extremophilic archaea
subsequently adapted to and colonized non-extremophilic habitats (Stetter,
2006b), or ‘cold’ where the cell lines adapted to extreme environments stem
from non-extremophilic origins (Boussau, 2008). It should also be noted that
the conditions for life during the archean age are just as debated as the history and nature of the life that lived during that time (Shields and Kasting,
2007).
The archaea as a group was suggested as a result of systematic, phylogenetic
studies where ribosomal RNA of both prokaryotes and eukaryotes was compared (Woese et al., 1977). The organisms were however known before that,
as a group of bacteria being part of the kingdom Monera (Whittaker, 1959).
The group was first named Archaebacteria, and the name Archaea was
coined in order to reflect the fundamental differences between the three primary evolutionary groups of life (Woese, 1990). In the same vein it has been
suggested that the term 'prokaryote' itself is outdated since it comprises bacteria and archaea in the same group in a manner that is not phylogenetically
motivated (Pace, 2006). Although the distinction between archaea and bacte12
ria is generally accepted in the scientific community, the debate has been
long (Mayr, 1998; Woese, 1998).
The origin of eukaryotes is one of the great unanswered questions of modern
biology. Therefore it is no surprise that the nature of the relationship between eukaryotes and archaea is subject to a perennial discussion which
shows no signs of slowing down. The eukaryotic genome contains a mix of
genes which are homologous to genes in both archaea and bacteria. The information processing genes, involved in processes such as replication, transcription and translation, share homology with archaea while the genes with
bacterial homologues tend to have metabolic functions (Rivera et al., 1998).
Most models explaining the origin of eukaryotes involve some kind of merging between a bacterium – either the proteobacterial endosymbiont that
evolved into the mitochondria or an unrelated fusion partner – and an archaeon. This archaeon has been suggested by different models to belong to
virtually every modern archaeal group; a protoarchaeal ancestor; or to an as
yet undiscovered group within the archaeal clade (Forterre, 2011; Gribaldo
et al., 2010; Koonin, 2010; Lake et al., 1984; Martin and Muller, 1998; Martin et al., 2001; Moreira and Lopez-Garcia, 1998; Poole and Penny, 2007).
One recent suggestion places the archaeal fusion partner within the TACK
superphylum, a large clade that includes the cren- thaum- aig- and korarchaea (Guy and Ettema, 2011). This association between TACK and eukaryotes is in part motivated by the increasing number of known shared cellular
systems. These include the cytoskeletal actin homologue crenactin in the
crenarchaeal group thermoproteales and in korarchaea (Ettema et al., 2011;
Makarova et al., 2010; Yutin et al., 2009) and the cren- and thaumarchaeal
cell scission Cdv system, homologous to eukaryotic ESCRT-III components
that is identified and discussed in Paper I and IV in this thesis.
Taxonomy
In the early days of archaeal studies, only a handful of species were known,
but even so one of the very first papers that recognized them as a separate
group was able to distinguish between two main groups, represented by
methanogens and hyperthermophiles (Woese, 1978). The two phyla were
eventually named Eury- and Crenarchaeota (Woese et al., 1990). With very
few exceptions all cultivated archaea belong to either of these groups, and a
substantial part of what we know about archaea is gained from studies of
eury- and crenarchaea. During three decades of studies of archaea a large
number of new species have been described, discovered from both culture
and sequencing based studies. New groups have been added to the old phyla,
and an increasing number of new species do not have an obvious place in the
13
old categories. Motivated by differences in properties and genome sequence
phylogenetics four additional phyla have been suggested, and while there is
no consensus of the phylogenetic status of these groups, the discussion highlights the diversity within archaea. The new phyla that have been suggested
are the Korachaeota, the Nanoarchaeota, the Thaumarchaeota and the Aigarchaeota.
Euryarchaeota is the very first described archaeal phyla since it was ribosomal RNA from organisms of this group that were first distinguished from
bacteria (Woese and Fox, 1977). This phylum classically includes methanogens, halophiles, sulfate reducers and hyperthermophiles. Crenarchaeota is
best known for hyperthermophiles, including thermoacidophiles that in addition to high temperatures also endure low pH values (Woese et al., 1990).
Korarchaea were first identified as a ribosomal RNA sequence which did not
fit into the established groups (Barns et al., 1996). Genome characterization
of the only described species within the phylum, Korarchaeum cryptofilum,
reveal many similarities to euryarchaea, even if ribosomal RNA sequence
analysis places it close to the root of the archaeal tree (Elkins et al., 2008).
The phylum Nanoarchaeota is also known from only one cultured organism,
the highly specialized parasite Nanoarchaeum equitans found in close association with the crenarchaeon Ignicoccus. Interestingly, the ribosomal RNA
sequence is significantly divergent from that of other archaea, to the extent
that it is not targeted by standard archaeal primers. The organism was originally identified with microscopy rather than the more commonly used PCR
based approaches. Due to this feature and the highly specialized biology of
the nanoarchaeal cell, this organism has been assigned its own phylum (Huber et al., 2002). However the close association between N. equitans and its
host suggests that its specialized biology and ribosomal RNA sequence are a
result of reductive evolution – as is not uncommon for parasites – and the
organism has also been described as belonging to the phylum Euryarchaeota
rather than a group of its own (Makarova and Koonin, 2005).
The phylum Thaumarchaeota comprises a vast group of organisms that, although abundant in many habitats and over a large environmental gradient,
are almost exclusively known from environmental sequencing. The presence
of an archaeal clade in non-extremophilic environments that cluster with
crenarchaea has been known for a long time (De Long, 1992), but it was
only after full genome sequencing of the first member of the group – the
sponge symbiont Cenarchaeum symbiosum (Hallam et al., 2006; Preston et
al., 1996) – that the thaumarchaea were proposed to belong to a phylum of
their own (Brochier-Armanet et al., 2008). The Aigarchaeota is yet another
14
deep rooting group, suggested to be a distinct phylum after genome sequencing of the subsurface geothermal archaeon Caldiarchaeum subterraneum
(Nunoura et al., 2011).
Environmental sequencing has greatly expanded the knowledge about the
diversity of archaea. Not only have targeted and random sequencing displayed a large variation within the archaea, with an ever expanding array of
clusters and subclusters. The environments in which the samples are collected have also revealed a much wider range of archaeal habitats than expected,
and it is clear that the early models for habitats accessible to archaea were
too narrow. It is also clear that even while the taxonomical discussion continues, the whole picture is still only emerging. Apart from ever increasing
numbers of sequences belonging to the established and suggested archaeal
phyla, there are also sequences that do not fit any known cluster, suggesting
the existence of even more, as yet unknown, archaeal phyla (Takai and Horikoshi 1999; Vetriani et al., 1999). It can be expected that the full diversity
of archaea and their phylogenetic relationship remain to be charted.
Ecology
The archaea inhabit virtually all viable habitats in the world, from the most
extreme to what we are accustomed to think of as normal, from niches exclusive to themselves to some of the most rich and diverse environments in
the biosphere (Chaban, 2006). The archaea are able to endure and even proliferate in the highest temperature known to host life, with 122ºC being the
current record (Kashefi and Lovley, 2001; Takai et al., 2008; Zeng et al.,
2009). While the archaea might be the best known extremophiles, they are
not the only ones. There are bacteria living side by side with archaea in all
but the most harsh conditions, and there are organisms of all three domains
able to make a living in high salinity and low pH (Rotschild and Manicelli,
2001). With few exceptions (e.g. Könneke et al., 2005) no cultivated archaea
belong to the groups inhabiting non-extremophilic environments. Thus, a lot
of current knowledge of the fundamental environmental limitations of cellular life as we know it has been gained from study of extremophilic archaea.
This adaptation of archaea to several environmental extremes has made them
a viable model for studies of the conditions of life on other planets. Study of
the archaea have time and again forced us to expand our list of environmental conditions able to support life, and thus increased the number of extraterrestrial habitats that can theoretically be host to it. The perhaps most dramatic example is the characterization of hydrothermal vents where complex
ecosystems are centered on primary producers utilizing energy and nutrients
15
from the vents themselves (Takai and Nakamura, 2011). This perspective is
of interest not only for astrobiological study of the conditions for extraterrestrial life, but also for life on early Earth, when both the biosphere and conditions for the first protocells differed considerably from later eras.
Methanogens live in anoxic environments where they convert carbohydrates
to methane. They exist in many environments, from ocean floor and wetlands to the digestive tracts of animals, including ruminants (Vogels et al.,
1980), insects and humans. Methane is an important energy source for many
organisms, and methanogens are therefore often essential components of
their ecosystems (Chaban, 2006). Biologically produced methane used as
biogas has important applications as fuel source, and the significance of methane as a greenhouse gas makes the methanogens a focus for understanding
of the currently occurring global warming.
Thermophiles, organisms able to sustain growth at temperatures above 45 C,
and hyperthermophiles (80 C) exist in hydrothermal ecosystems the world
over. These habitats are associated with geothermal activity and exist both as
terrestrial and oceanic systems, including the famous deep sea hydrothermal
vents. These systems are usually rich in potential nutrients made available
from the surrounding rock by heat and abiotic processes. The availability of
metabolites in a specific hot spring is dependent on the surrounding mineral
composition, which is why closely related hot springs fed by the same hydrothermal system well may have dramatically different chemistry, and thus
biological composition. The habitats inhabited by thermophiles are naturally
isolated, immediately associated with a source of geothermal heat and typically subjected to abrupt temperature gradients, to the extent that the optimal
growth temperatures for a lot of these organisms are limited to narrow spatial areas. In ecological terms this makes the hot spring ecosystems island
habitats, i.e. small, habitable patches surrounded by vast inhospitable areas.
The consequence of this is that the biogeographical pattern of thermophiles
differs from what is regarded as the norm for prokaryotes. The rule of thumb
is that for prokaryotes, small numbers and ease of dispersal makes distance
irrelevant, and only environmental selection decides the presence or absence
of a particular species. For the hyperthermophiles Sulfolobus it has been
demonstrated that distance between habitats is a relevant factor in how
strains are related to each other between different hot springs – that the cells
have spread from spring to spring during their colonization history (Grogan
et al., 2008; Reno et al., 2009; Whitaker et al., 2003). It has been suggested
that the viruses that infect these cells do not show a biogeographical pattern,
at least over a regional scale, and thus are efficient enough in their dispersal
16
that the distances between the springs become negligible (Snyder et al.,
2007).
Acidophiles, organisms able to endure extremely low pH values, and halophiles that inhabit hypersaline habitats, often found in connection to marine
systems, are two other commonly studied extremophilic archaea. It can also
be argued that psychrophilic archaea, found in water close to the freezing
point, can be counted to the extremophiles (De Long et al., 1994).
Archaea inhabit vast non-extremophilic environments, such as soil, fresh
water and oceanic water (De Long, 1992; Karner et al., 2001) where they are
important contributors to biogeochemical processes. Archaea in sediments
and bogs produce methane (Chaban, et al., 2006). Archaea in soil and water
have been shown to oxidize ammonium with an activity that rivals bacteria,
and studies of cultivated oceanic archaea have demonstrated an affinity for
ammonium at much lower concentrations than bacteria (Martens-Habbena et
al., 2009).
Archaea can also be found in habitats created by other organisms, such as in
association with other microorganisms or internally in multicellular organisms. They are part of the gut flora of both ruminants, such as cow or sheep,
and of termites, where they produce methane. Even in humans archaea have
been found as a seemingly non-obligate part of the microflora of the digestive tract. Intriguingly enough archaea have never been identified as pathogens, despite their close association with humans and animals. The closest
indication of a disease being caused by archaea is observations of elevated
numbers of methanogens in the oral tract during certain dental diseases
(Cavicchioli et al., 2003). However, it is most probable that this correlation
is a case of opportunistic archaea taking advantage of an already existing
disease, rather than causing it.
Archaeal models
The tree of life is far too large to study in its entirety. The complexity of
even a single cell of a single species is daunting, and the number of species
is beyond counting. In order to study this diversity, necessity dictates extrapolation from well-known study systems to less well studied species. Species
that share habitats can be assumed to share adaptive traits, such as thermophiles using similar mechanisms for keeping the cell structures intact at high
temperatures. Related species can be assumed to share fundamental cellular
traits, such as the similarities in cell division that will be discussed later in
this thesis. Homologous genes can be assumed to share the same function in
17
different species, as in the case of the genes encoding the cell lysis system
VAP which is used in similar manner in the two different viruses SIRV2 and
STIV as discussed in Paper II. An example of homologous genes that do not
share functions is seen with the gene ftsZ which is involved in cell division
in euryarchaea, but seemingly has a different function in thaumarchaea, as
discussed in paper IV.
Model organisms are study systems for which a large body of research is
collected and research tools are readily available. There are archaeal models
for the classical functional and taxonomical groups such as methanogens,
halophiles and hyperthermophiles (Leigh et al., 2011). In these studies, species of the crenarchaeon Sulfolobus as well as the thaumarchaeon Nitrosopumilus maritimus are used as models.
S. acidocaldarius is, together with the related species Sulfolobus solfataricus, the most well studied crenarchaeon. The cells are coccoid and about one
micrometer in diameter (Figure 1). They can be grown in liquid culture and
on plate at a temperature optimum around 80 C, at the remarkably low pH of
2 – 3 (Huber and Stetter, 2000). The strain was isolated from a hydrothermal
hot spring in the national park Yellowstone in the USA (Brock et al., 1972).
The genome has the comparably low GC content of 37%. It is 2.3 Mbp large
and contains 2292 predicted open reading frames (Chen et al., 2005), which
is smaller than the genomes of the related species S. solfataricus, Sulfolobus
tokodaii and Sulfolobus islandicus (Guo et al., 2011; Kawarabayasi et al.,
2001; Reno et al., 2009; She et al., 2001). S. acidocaldarius has been used in
a number of cell cycle studies (Lundgren and Bernander, 2005), and there
are several molecular tools developed for study of it, including protocols for
synchronization of the cell cycle (Duggin et al., 2007; Hjort and Bernander,
1999; Lundgren et al., 2004), microarrays (Andersson et al., 2005) and genetic systems (Berkner et al., 2008).
N. maritimus is the first thaumarchaeon to be cultivated as a pure culture.
The rod shaped cells are tiny, often less than one micrometer in length, and
they grow at room temperature by ammonium oxidization (Könneke et al.,
2005). The 1.6 Mbp genome contains 1997 predicted open reading frames
and has a GC content of 34%, which interestingly is more reminiscent of
Sulfolobus than other sequenced thaumarchaea, where the GC content is
closer to 50% (Walker et al., 2010). There are few molecular tools developed for N. maritimus. However, the ability to cultivate the organism has
18
Figure 1. Cells of S. acidocaldarius seen in transmission electron microscope
made important functional studies possible, as exemplified by the demonstration of its remarkably efficient mode of ammonium uptake (MartensHabbena et al., 2009) as well as the cell division machinery and cell cycle
characterization presented as a part of this thesis.
19
Studies of the archaeal cell
The cell is a dauntingly complex system. Function and survival is dependent
on a tightly regulated network of metabolites, environmental factors and the
cell’s own information processing machinery. Although our understanding
of the cell is increasing there are still fundamental questions yet to be answered.
In this thesis I will discuss four features of the archaeal cell and their implication for both the environment in which the archaea live, but also the shared
evolutionary history between archaea and eukaryotes. In Papers I and IV cell
division in cren- and thaumarchaea is discussed, and a novel system for cytokinesis presented. In Paper IV we also discuss cell cycle organization in
cren- and thaumarchaea. Paper III describes the organization of the chromosome and how multiple origins of replication affect gene localization. In
Paper II we reveal an unknown, lytic life style for the crenarchaeal virus
SIRV2 and describe the highly specialized process in which the virus lyses
the cell. These studies highlight unique features of archaea, but also the similarities that exist between organisms of the three domains.
Cell division in the archaea
Cell division allows the cell to split its biomass and genetic material into two
independent daughter cells, free to grow, migrate and eventually produce
more daughter cells of their own. This is a fundamental aspect of life as we
know it. The cellular mechanisms involved in division are remarkably conserved during the evolutionary history. There are three independent main
mechanisms described – the FtsZ system of bacteria, kor- and euryarchaea
(Makarova et al., 2010; Margolin 2005), the myosin and actin based systems
used by eukaryotes (Morgan, 2007) and the newly described Cdv system
utilized by thaumarchaea and the majority of crenarchaea (Paper I; Paper IV;
Samson et al., 2008).
The FtsZ protein used by bacteria is structurally related to the cytoskeleton
protein tubulin (Margolin, 2005). It forms a ring on the inside of the mem20
brane in the middle of the cell (Bi and Lutkenhaus, 1991). In rod-shaped
bacteria the plane of division is modeled to be identified by cooperating regulatory systems that inhibits FtsZ assembly at cellular poles and at the sites
occupied by the segregated nucleoids (Barak and Wilkinson, 2007). The
eukaryotes display a large variety in cell morphology and structure. By necessity their modes of cell division are varied, but all follow the common
theme of actin and myosin filaments forming a division apparatus (Morgan,
2007). In metazoa a contractive ring is formed by a core of around 20 proteins, directed to the division plane by a complex network of cell cycle regulated factors (Glotzer, 2005).
In contrast to the other domains, cytokinesis in archaea is not homogeneously carried out by one system. There are at least three unrelated apparatuses
for dividing the archaeal cell. Similarly to bacteria, euryarchaea and korarchaea use the FtsZ system (Makarova and Koonin, 2010; Poplawski et al.,
2000; Wang and Lutkenhaus, 1996). Characterization of FtsZ reveals structural similarities to eukaryotic tubulin (Löwe and Amos, 1998) and the protein assembles into a ring in the middle of the cell, which constricts to separate the daughter cells. This assembly and disassembly is associated with
guanosine-5'-triphosphate (GTP) hydrolysis (Margolin et al., 2005). The
crenarchaea contain no gene for FtsZ (Makarova et al., 2010) and until the
study reported in Paper I, their mechanism for cell division was unknown.
Beside these two systems, organisms of the crenarchaeal order Thermoproteales lack genes for both Cdv and FtsZ, and it is not known how they divide
the cell. There are homologues to eukaryotic actin in their genomes that is
shown to have cytoskeletal functions, and although there are indications of
an involvement in cell division this still remains to be demonstrated (Ettema
et al., 2011). Interestingly, Thermoproteus tenax divides in a way that seems
mechanistically different from the cytokinesis utilized by organisms with the
Cdv or FtsZ systems. Just prior to division the cells are rigid with no indication of invagination in the middle, and separation of daughter cells is rapid
and proceeded by a fast vibrating movement. It is proposed that this ‘snapping’ mode of division is made possible by cell wall formation across the
middle of the cell before the split takes place (Horn et al., 1999; Sonobe et
al., 2010; Wildhaber and Baumeister, 1987). Whether or not this mechanism
is related to the cell division of other thermoproteales, and what – if any –
role is played by the actin homologues in cell division remains an open question.
Besides the thermoproteales, there are still a number of organisms where cell
division falls outside the three main systems (Bernander and Ettema, 2010;
21
Erickson and Osawa, 2010). A number of strains of bacteria of the PVC
superphylum (that includes planctomycetes and chlamydia) have seemingly
lost their ancestral FtsZ gene. Some parasites lack a known cell division
systems, presumably as a result of severe genome reduction. In some cases
the missing genes have been incorporated in the host genome, and it is plausible that cell division in most of these organisms is dependent on factors
from the host. Organisms without cell wall of both archaea and bacteria have
been shown to lack known cell division system. This so called ‘plasma
anomaly’ suggests alternative ways to produce daughter cells. Interestingly,
mutants of the bacterium Bacillus subtilis able to grow without cell wall
have been shown to divide even after the gene for FtsZ is knocked out. This
division implements a mechanism where the cell produces membrane enclosed protrusions that cleaves into separate bodies. Another example of
cells that divide without apparent use of a specific mechanism is Mycoplasma genitalium that has been seen utilizing the cell motion apparatus to drag
the daughter cells apart.
These examples show that there are unknown, unrelated mechanisms for
dividing the cell and fundamental discoveries still remain to be made. However, understanding of cell division as a concept has taken a large step forward with the discovery of the cren- and thaumarchaeal cell division system
Cdv, described in Paper I and IV.
Cell division in crenarchaea (Paper I)
In Paper I we identified, and coined the name for, the cell division machinery Cdv. In earlier studies (Lundgren and Bernander, 2007) cultures of S.
acidocaldarius were synchronized in G2 phase. Upon release to continued
growth total RNA was sampled in intervals and the global transcriptome
measured with microarrays. Several genes were identified with expression
patterns displaying distinct cyclic behavior, with gene expression increasing
and then soon again dropping, suggesting a narrow time span of induced
gene activity. This peak in transcription coincided with the postreplicative
stages of the cell cycle, during which the duplicated chromosomes align
(Robinson et al., 2007) and separate to opposite poles of the cell which finally divides to form two new daughter cells. Three of the identified genes –
Saci_1374, Saci_1373 and Saci_1372, named cdvA, cdvB and cdvC – are
organized in a cluster with short intergenic spacers. They have very similar
expression profiles, and we decided to study them further.
The function, phylogeny and cellular location of the three Cdv proteins were
explored. The genes were amplified with PCR and cloned into E. coli ex22
pression vectors. The expressed proteins were purified with metal ion columns that target added histidine tags. Antibodies were produced against the
proteins and used to identify structures formed by the target protein in fixed
exponential phase cells stained with fluorophore labeled secondary antibodies as well as the DNA targeting stain DAPI. The cells were studied with
fluorescence microscopy.
Microscopy is one of the oldest and most fundamental tools available to the
microbiologists – arguable this entire field of science was formed when Antonie van Leeuwenhoek used microscopes of his own invention to perform
some of the first studies of microorganisms in the seventeenth century.
While microscopy studies have been indispensable to characterize the anatomy of the cell, the function of a certain structure is usually not obvious, and
several cellular features are simply not visible under the light microscope. In
fluorescence microscopy, specific stains are deployed to distinguish between
choice structures. Various stains target features such as DNA, cell walls,
membranes and cytoskeleton components. The molecular era of microbiology allows the sensitivity of stains to increase by design of carrier molecules
directly for the desired target. Two common carrier molecules are oligonucleotides and antibodies. The carriers bind to a signal molecule, such as fluorescent groups (as we used in this study), detectable enzymes or binding for
further antibody targeting. Signal molecules with distinct signals make it
possible to stain the same sample for several different targets, as we do in
this study, by using fluorescent groups that are excited by light of different
wavelengths. For organisms with an available genetic system an elegant
method of protein visualization is the recombinant addition of a fluorescent
fusion tag, such as green fluorescent protein (GFP), directly to the target
protein (Ludin and Matus, 1998). These and similar methods make a detailed
study of subcellular anatomy possible.
S. acidocaldarius cells are roughly spherical with a diameter in the range of
one micrometer. The general cell morphology was seen using phase contrast
microscopy while DNA and protein localization were visualized with excitation of specifically bound fluorophores. When the Cdv proteins were targeted with specific antibodies we saw all three proteins form band structures in
the center of the cell, at a right angle to the plane of nucleoid segregation
(Figure 2, 3 and 4). The bands were only seen in a subset of the total cell
population, which matched the gene expression pattern where only cells in a
narrow stage of the cell cycle displayed gene activity. The bands were
overrepresented in cells with segregated nucleoids but also found in a small
number of cells where the nucleoids were not segregated. This suggests that
the structure was formed before the replicated nucleoids were separated from
23
Figure 2. Fluorescence microscopy images of S. acidocaldarius (photo: Erik Pelve)
and N. maritimus (photo: Ann-Christin Lindås) stained with DNA targeting dye and
fluorescent antibodies targeting CdvA. The first panel shows phase contrast images
of the cell. The second panel shows the DAPI-stained DNA (blue) with the two
nucleoids segregated. The third panel shows CdvA structures (green). The fourth
panel is an overlay of DNA and CdvA-images where the CdvA bands are positioned
between the segregated nucleoids.
Figure 3. Fluorescence microscopy images of CdvB structures
in S. acidocaldarius. The first
panel shows phase contrast images of the cells. The second
panel shows the DAPI-stained
DNA (blue). The third panel
shows CdvB structures (green).
The fourth panel is an overlay of
DNA and CdvB-images. Both a
ring and band is seen, which
suggests that the cells are differently aligned. The structures
have formed prior to nucleoid
segregation. Photo: Ann-Christin
Lindås.
24
each other. The bands covered the entire width of the cell and when the cell
constricted in the late phase of cell division the bands shrunk in size with the
mid-section, following the width of the shrinking cell envelope. Thus, all
three Cdv proteins formed bands in the middle of dividing cells. They
formed before nucleoid segregation and remained during constriction until
the daughter cells were separated. The same bands were seen for CdvB and
CdvC in an independent study, where interaction between the proteins also
was demonstrated (Samson et al., 2008).
Transcription studies confirm the cell cycle specific function of the cdv
genes. UV irradiation of exponentially growing S. acidocaldarius cultures
caused downregulation of the genes (Fröls et al., 2007; Götz et al., 2007).
The same was seen in comparison of transcription patterns of cells in stationary and exponential phase. These observations are consistent with a
function of the genes in basal cellular processes, since cells under stress are
less likely to produce new daughter cells. Yet another indication of essential
gene function comes from production of gene knockout mutated cell lines.
Attempts to delete the cdvC gene have not been successful (Hobel et al.,
2008). While negative results like these are unsuitable as direct evidence, the
difficulties observed for cdvC knockout production still serves as an indication of an essential function for the genes, especially when viewed in the
light of the full body of gathered observations.
A classical road to study the inner workings of the cell is disruption of specific cellular processes with antibiotics. Exponentially growing cultures of S.
acidocaldarius were treated with antibiotics and analyzed in microscope.
Tunicamycin has been shown to inhibit cell division in S. acidocaldarius
(Hjort and Bernander, 2001). Cells treated with this antibiotic were arrested
in the cell division stage, and correspondingly the number of cells with Cdv
bands was increased threefold. Cells treated with the antibiotic daunomycin,
which inhibits the cell after replication (Hjort and Bernander, 2001), displayed disturbed nucleoid morphology and no Cdv bands were seen, consistent with cell cycle arrest. Radicicol treated cells produced Cdv bands in
normal proportions, but also displayed DNA segregation and cell division
with no apparent arrest of the cell cycle. Mutational study is another method
for study of protein function by means of their disruption. S. acidocaldarius
mutants DG132 and DG134 (Bernander et al., 2000) showed deficiencies in
genome segregation and cell division respectively. No mutations were found
in the cdv genes, and Cdv bands were seen in microscopy, although with
altered morphologies. The phenotypes of the mutants are thus not caused
directly by damages to the Cdv proteins and may have been an effect of gen-
25
Figure 4. Cdv structures in S. acidocaldarius. The first panel shows phase
contrast images of the cells. The second panel shows DAPI stained DNA
(blue) and the third panel fluorescent antibodies targeting CdvB (green). The
fourth panel is an overlay of the DNA and CdvB images. Cdv structures are
seen in cells with segregated nucleoids.
26
eral cellular stress, or possibly direct damage of yet unidentified components
of the division system or its regulation.
It has been said that nothing makes sense in biology except in the light of
evolution (Dobzhansky, 1964). In the context of identifying the function of
the Cdv proteins, the evolutionary perspective gave a last and highly convincing piece to the puzzle. The CdvB and C proteins are both homologous
to components related to the eukaryotic ESCRT-III system, a system used
for cell membrane reorganization during multivesicular body formation,
viral release and cytokinesis (Hurley and Hanson 2010; Morita et al., 2010).
CdvB is homologous to the ESCRT-III components Snf7, while CdvC is
homologues to the associated factor VPS4. Thus, an archaeal function for
Cdv in membrane rearrangement – such as during cytokinesis – is analogous
to the function of the eukaryotic homologues. Interestingly, CdvA is not
homologous to any eukaryotic proteins and is specific to archaea.
The Cdv components are not universally present in the crenarchaea – as we
have seen they are missing entirely from the thermoproteales. Despite the
presence of FtsZ in euryarchaea, there are proteins with a possible relation to
CdvA in a number of phylogenetically dispersed euryarchaea. There are no
Cdv components in the only sequenced korarchaeon (Makarova et al., 2010).
In S. acidocaldarius there are three paralogues to cdvB – Saci_0451,
Saci_1601 and Saci_1416 – and in other sequenced crenarchaea there are at
least two extra cdvB paralogues in the genome. The functions of these proteins are not fully determined. Saci_1601 has a similar expression pattern to
the cdv genes but forms no band structure in the cell. These components
have been associated with virus release (Ettema et al., 2009; Maaty et al.,
2006) and vesicle formation (Ellen et al., 2009). Though no mechanistic
details have yet been provided, these associations are highly interesting since
they mirror analogous processes in eukaryotes.
There are several parts of the ESCRT systems in eukaryotes that lack homologues in archaea. Models suggest that ESCRT-III is recruited by the other
systems to detach budding vesicles after targets for vesicle transport are
identified and a budding membrane is initiated (Hurley and Hanson, 2010).
The system is flexible with several different paths all leading up to the same
mode of membrane manipulation, which explains the many processes in
which ESCRT-III is involved. Interestingly, in the genomes of euryarchaea
potential homologues to both cdvB and C are always found in pairs
(Makarova et al., 2010). It has been noted that the Vps4/ESCRT-III interaction is present in both eukaryotes and archaea, which together with the
27
known involvement in cytokinesis suggest an ancient conserved function as
well as sequence homology.
The phylogenetic distribution of the cdv genes gives yet another indication
of involvement in cell division. The Cdv system shares a functional analogy
with the bacterial FtsZ system, forming a contracting ring in the middle of
the cell. Even if there is no homology between the components, the Cdv
system forms an analogous structure. Euryarchaea divide with the FtsZ system known from bacteria. The ftsZ gene is present in most known euryarchaeal genomes as well as the genomes of kor- and thaumarchaea. However, the gene is not present in the crenarchaea (with the exception of a distantly related homolog in S. solfataricus (Makarova et al., 2010)). The cdv
genes however are present in all sequenced genomes of crenarchaea of the
orders Desulfurococcales and Sulfolobales. This pattern of complementary
presence/absence suggests functional analogies. Cells with FtsZ function
without Cdv, and cells with Cdv have no need for FtsZ.
Intriguingly as an exception to the pattern noticed above the thaumarchaea
have genes for both FtsZ and Cdv which raises the question of their mode of
cell division. This question is further addressed in Paper IV.
Cell division in thaumarchaea (Paper IV)
The thaumarchaea are found in soil and ocean environments which make
them a substantial part of the biosphere (Chaban et al., 2006). In the genomes of sequenced thaumarchaea – to date N. maritimus (Walker et al.,
2010), Cenarchaeum symbiosum (Hallam et al., 2006), Nitrosoarchaeum
limnia (Blainey et al., 2011) and Nitrososphaera gargensis (Spang et al.,
2010), and also Caldiarchaeum subterraneum (suggested to belong to the
phylum Aigarchaeota) (Nunoura et al., 2011) – there are homologues to both
the crenarchaeal Cdv and the euryarchaeal FtsZ cell division systems. In
Paper IV we demonstrate that Cdv is used for cell division in N. maritimus.
In a similar experimental procedure as the one described for the identification of crenarchaeal Cdv, N. maritimus homologues of cdv and ftsZ were
cloned into E. coli expression vectors and antibodies were produced from
purified proteins. These were used to stain and analyze ethanol fixed cells
from exponentially growing N. maritimus cultures in fluorescence microscopy.
The N. maritimus cell is rod shaped, with a diameter less than half a micrometer. The nucleoids were seen in DAPI-staining as oblong patches, fol28
lowing the cell shape as it grows. In a substantial proportion of cells two
clearly distinct nucleoids could be seen at opposite poles of the cell. CdvA
and C formed bands in the center of the cell, between the segregated nucleoids (Figure 2). These bands were only seen in cells with segregated nucleoids, and a large fraction of these cells showed an elongated morphology
compared to cells without Cdv bands. The structures were similar to the Cdv
bands observed in S. acidocaldarius, but since N. maritimus is rod shaped,
rather than coccoid like S. acidocaldarius, the centered position of the Cdv
bands between the nucleoids was easier to discern in N. maritimus.
The CdvB homologues lacked band structures, but two of the three studied
homologues displayed a temporal pattern similar to the CdvA and C proteins, with an indistinct, uniform cell signal in the same cells as those with
CdvA and C bands. This means that the proteins were present only in a subset of the cells in the culture, which matched the presence of the bandforming Cdv components, suggesting a function for the proteins in the same
process. This observation is analogous with the S. acidocaldarius Cdv system where the transcription pattern of cdvB is similar to cdvA and C. In contrast to Cdv, FtsZ formed no band structure in N. maritimus. The FtsZ signal
was uniformly present in the cells, and unlike CdvB there was no distinguishable temporal correlation with the Cdv bands or segregated nucleoids.
In the light of these observations, we conclude that the Cdv machinery is
used for cell division in thaumarchaea, just as in many crenarchaea. The
function of FtsZ in these cells is not known, but it has been observed that the
thaumarchaeal FtsZ homologues displays sequence divergence from the
euryarchaeal and bacterial FtsZ in the GTP binding T4 loop (Busiek and
Margolin, 2011), which makes a shift in function from cell division plausible. Our study gives no indication as to what this alternative function is.
Cell division – discussion
The observations reported in Papers I and IV demonstrate that the Cdv system is used for cell division, and is the system of use in most cren- and
thaumarchaea. The mechanistic and functional properties of the system have
only begun to be explored, although there are a number of important clues
already discovered.
Protein structure analysis demonstrates an interaction between the proteins
CdvB and CdvC in S. solfataricus (Obita et al., 2007). The same was seen in
S. acidocaldarius using yeast-two-hybrid experiments (Samson et al., 2008).
Similarly, pull-down studies indicated an interaction between CdvA and B
29
(Samson et al., 2011). An 18 residue C-terminal peptide of CdvA, named
E3B after ESCRT-III binding, was shown to bind to a winged-helix motif of
CdvB. Co-crystallization of the interacting domains revealed a variation of
winged-helix architecture with unique topological features. There was no
interaction seen between CdvA and CdvC. Thus, with this observation of
binding between CdvA and CdvB, the chain of interaction between all three
Cdv proteins was confirmed. This is reminiscent of the eukaryotic binding
between Vps4 (CdvC homologue) and ESCRT-III (CdvB homologue),
which is interesting given the sequence divergence between the binding domains of CdvB and ESCRT-III proteins.
It has been demonstrated that CdvA is able to bind to liposomes constructed
from archaeal lipids, and also to recruit CdvB to the liposomes (Samson et
al., 2011). The liposomes were deformed by the presence of CdvA, but even
further when both CdvA and B were allowed to interact, which is reminiscent of the eukaryotic ESCRT-III machinery’s ability to bend cell membranes. Protein detection studies indicate that cellular presence of the CdvA
protein does not follow the cyclic abundance of mRNA transcript but displays a more uniform presence, despite the band-like protein structure only
being seen in a small subset of cells. There is also an indication of differences in transcription regulation between CdvA and CdvB which corresponds to expression studies in S. solfataricus where cdvA is transcribed
separately from cdvB and C (Wurtzel et al., 2010).
Sucrose gradient centrifugation has demonstrated interaction between protein CdvA and CdvB, as well as CdvB and CdvC in Metallosphera sedula, a
hyperthermophilic crenarchaeon of the order Sulfolobales, related to S. acidocaldarius and S. solfataricus (Moriscot et al., 2011). While there was no
direct interaction between CdvA and C, the binding between CdvB and
CdvA and C, respectively, does not inhibit each other. This suggests that
CdvB binds to both CdvA and C in the cell, and that these interactions build
up the Cdv apparatus. All three proteins formed structures when purified
from M. sedula (Moriscot et al., 2011). CdvA formed large structures, which
in SEM images were revealed to consist of large helices, formed by 2 - 3
CdvA filaments each. Interestingly, it was shown that these structures were
only formed in the presence of DNA and that interaction with CdvB did not
break up the filaments. CdvC formed ring structures which were reminiscent
of the eukaryotic structure. Together with CdvA, CdvC formed large structures consisting of both proteins in equal proportions. Also CdvB formed
multimers.
30
The cdvA gene is present in only a single copy in the genomes where it is
found, and the cdvA phylogeny follows established cren- and thaumarchaeal
ribosomal phylogeny closely. This suggests an evolutionary history where
horizontal gene transfer has played a minor role, consistent with a complex
role for CdvA in the cell cycle (Moriscot et al., 2011).
Our studies of Cdv as well as these observations suggest a model where
CdvA interacts with DNA, possibly as part of a structure facilitating segregation of the nucleoids, and then locates to the cell membrane in the middle
of the cell. It recruits CdvB which in turn recruits CdvC and together builds
the cell division apparatus. The mechanistic function of the cell invagination
and eventual cytokinesis is probably analogous to the eukaryotic ESCRT-III
and Vps4 system. In this model CdvA recruits CdvB and C in a role analogous to the eukaryotic ESCRT components not present in archaea.
The Cdv machinery represents an ancient mode of membrane reorganization,
shared between cren- and thaumarchaea and eukaryotes. The system has
proved to be versatile and have a function in different cellular processes.
Learning more about these structures will teach us more about the fundamental properties of the archaeal and eukaryotic cell, and thereby give valuable insight into the relationship of these two domains.
The archaeal cell cycle
The cell cycle is divided into three basic parts – the prereplicative or G1 (gap
1) stage, the replication or S stage (DNA synthesis) and the postreplicative
stage which includes the G2, M and cytokinesis stages (gap 2, mitosis and
division). Newly divided cells enter into the G1 stage with a single copy of
the genome. Upon entry into the S stage the DNA replicates and the cell then
contains two chromosomes in the G2 stage until the nucleoids segregate and
the cell divides again.
The crenarchaea are related over long evolutionary distances and adapted to
a range of different habitats and cell growth conditions. Despite this the organization of their cell cycles displays remarkable similarities (Lundgren et
al., 2008). Flow cytometric studies of exponentially growing cultures of
species from all three orders of crenarchaea reveal common patterns in the
cell cycle, the organizational path the cell takes through reproduction. In all
species of crenarchaea studied to date, the basic cell cycle organization is
similar. After division the cell quickly enters S phase and copies the chromosome. It then rests in postreplicative stage for the majority of its cell cycle,
31
until the cell divides and the two resulting daughter cells quickly move
throughout G1 phase and S phase to reach the ‘resting stage' in G2 phase
(Lundgren et al., 2008). This common trait is intriguing as it speaks of either
conservation through the entire crenarchaeal evolutionary history, or an active selection. The cell cycle organization of euryarchaea is less homogenous, displaying different patterns in chromosome numbers, replication timing and resting stage (Maisnier-Patin et al., 2002; Majernik et al., 2005;
Malandrin et al., 1999; Soppa, 2011).
As we have seen, thaumarchaea were until recently considered a deeply
branching clade of crenarchaea (Brochier-Armanet et al., 2008). Most species of this group are non-extremophilic and their cellular features combine
traits known from crenarchaea and euryarchaea. Thus, it is of interest to contrast our knowledge of the unity of the crenarchaeal cell cycle organization
to corresponding analyses of the thaumarchaea. Also, the presence of thaumarchaea in the large ecosystems of soil and ocean and significant involvement in global cycling of carbon and nitrogen means that understanding of
their life cycle has environmental significance. This study was conducted
and reported in paper IV.
Cell cycle organization of thaumarchaea (Paper IV)
Cultures of N. maritimus were grown at exponential phase into stationary
phase and samples were collected and fixed in ethanol. To quantify the DNA
content and thereby give an estimation of each cell’s progress through the
cell cycle, the cells were analyzed by flow cytometry.
Flow cytometry separates cells in a stream of fluid and passes them by a
laser and optical detector system in single file. In this way data is collected
and quantified for individual cells, and trends within the population can be
detected. The cell scatters light in a manner that depends on size, shape and
surface composition. In a sample containing only cells from a single species,
light scatter can be used as an estimate for cell size. Fluorescence labeling
shows presence of target molecules, most commonly DNA. In this study
DNA is stained with ethidium bromide and mithramycin, alternatively with
SYTO green. This method allows very precise monitoring of culture growth
parameters such as cell size, DNA content, replication progress and relative
time spent in different parts of the cell cycle.
Analyses of N. maritimus demonstrated that the majority of the cell population in exponential growth phase contained one single chromosome, a smaller part contains two chromosomes and surprisingly large part contains be32
Figure 5. Flow cytometry histograms of DNA content in exponential phase cultures
of N. maritimus and S. acidocaldarius. The central distribution in each graph represents the cells of the population. For both species two main peaks can be seen – one
large and one small – that correspond to cells with one respectively two chromosomes (marked by vertical lines). The area between them corresponds to cells undergoing replication. In N. maritimus the culture is dominated by cells with one
rather than two chromosomes, and in S. acidocaldarius it is the other way around,
reflecting different organization of the cell cycle. Of note is the population of cells
undergoing replication. Given the long generation time in N. maritimus the relatively large group of cells in the process of replicating their DNA suggests a low replication speed. The shift in location of the DNA distributions on the X-axis between the
species represents differences in genome size.
tween one and two chromosomes, residing in S phase (Figure 5). The relative amount of replicating cells is large given the long generation time of N.
maritimus, 33 hours under the growth conditions used.
The exponential age distribution of the culture means that measured cell
cycle lengths will be biased towards newborn rather than dividing cells. In
33
order to compensate for this, simulations were used to calculate the length of
each cell cycle phase (Michelsen et al., 2003). The prereplicative period of
the cell was found to be 19 - 29%, the replicative period to be 45 - 53% and
the postreplicative period to be 24 - 30%. The span in numbers represents
variation between different time points during the exponential phase.
When cell cultures approached stationary phase there were fewer cells with
two chromosomes, and the G1 peak increased. However, even long after the
cell cultures had reached stationary phase, the two chromosome peak was
not completely gone, and the S-phase population still remained.
Replication speed was estimated to be 13–15 bp/s if two replication forks are
postulated, i.e. one origin of replication rather than multiple as in several
species of crenarchaea. This replication speed is low compared to the extremophilic crenarchaea (Lundgren et al., 2004), and more similar to eukaryotic replication (Conti et al., 2007; de Moura et al., 2010; Jackson and
Pombo, 1998).
Cell cycle organization – discussion
The crenarchaeal uniformity in cell cycle organization is intriguing when the
evolutionary distances and differences in habitat and life style that separate
the different crenarchaeal lineages is taken into consideration. It is equally
intriguing that the cell cycle of N. maritimus does not display the same pattern. The thaumarchaeal cell cycle is dominated by the prereplicative and
replicative stage, in contrast to the crenarchaeal cell cycle where the cell
cycle is dominated by the postreplicative stage.
There are several possible reasons for the cell cycle organization of N. maritimus. It might be an adaptation to extremely nutrient poor environments,
where a slow replication and a majority of the cell cycle spent with less than
two full chromosomes rations the available nutrients and energy. This explanation ties into the high affinity to ammonium and thus adaptation to nutrient
poor conditions displayed by N. maritimus (Martens-Habbena et al., 2009).
The slow replication speed compared to crenarchaea might also be a result of
the low temperature in the ocean.
The thaumarchaea inhabit a very diverse range of habitats and are likely
adapted to very different life styles. It remains to be seen if the thaumarchaeal cell cycle displays the same uniformity as the crenarchaeal, and ultimately
if these various modes of cell cycle organization better reflect an ancestral or
adaptive trait.
34
Chromosome organization of crenarchaea
It is of crucial importance for all living organisms that the genetic information encoded in the chromosome is used correctly. Therefore it is not
surprising that the chromatin is subject to several orders of organization. The
DNA sequence itself, presence of neighboring elements, gene order and density, spatial location in the cell as well as presence and interaction of associated proteins all play important roles in determining expression and regulation of genes (Parada et al., 2004).
The archaeal chromosome shares many features with the bacterial, but while
bacteria consequently have one single origin of replication there are archaea
where replication initiates at multiple distinct sites of the chromosome. This
means that several pairs of replication forks start independently from different sites to replicate the genome. In the studied species of Sulfolobus three
origins of replication, unevenly distributed over the chromosome, have been
identified experimentally (Lundgren et al., 2004; Robinson et al., 2004) or
suggested from bioinformatic analysis (Chen et al., 2005; Flynn et al.,
2010). At least two origins of replication are found in the related crenarchaeon Aeropyrum (Robinson and Bell, 2007), and there are also multiple origins
of replication in Halobacterium (Bergquist and DasSarma, 2003; Coker et
al., 2009), Methanocaldococcus jannaschii (Maisner-Patin et al., 2002) and
Haloferax volcanii (Norais et al., 2007). One origin of replication has been
identified in Pyrococcus abyssi (Myllykallio et al., 2000) and Archaeoglobus
fulgidus (Maisner-Patin et al., 2002).
The genome of S. solfataricus has undergone extensive re-arrangement
(Chen et al., 2005; She et al., 2001), and there are indications that one of the
origins in S. acidocaldarius has been incorporated into the genome from
external sources, probably an independently replicating plasmid (Robinson
and Bell, 2007). These observations suggest that the exact number of origins,
as well as their spatial localization on the chromosome, is not essential for
the archaeal cell.
Genes are not regularly spaced on the chromosome. Several evolutionary
forces cluster genes with similar function, similar expression and similar
importance. The perhaps most fundamental mode of gene clustering is the
formation of operons in bacteria and archaea where several genes are coexpressed as a single transcript from a single promoter. Clustering genes in
an operon can be advantageous from a gene regulation perspective since the
gene expression will be under common control. Also, horizontal gene transfer is facilitated by genes of the same function sharing the same transcrip35
tional cassette (Lawrence and Roth, 1996). There is a tendency for highly
expressed genes to cluster to origins of replication in fast growing bacteria
(Couturier and Rocha, 2006). This is thought to be caused by a selection for
utilization of the gene dosage effect, in which genes close to the origins are
present with an extra copy in the cell during chromosome replication. Genes
in high demand – for example ribosomal genes – are by being located close
to an origin of replication thus copied as early as possible in this part of the
cell cycle.
Highly conserved genes often have important functions for the cell. In bacteria these genes tend to cluster together on the chromosome (Fang et al.,
2008; Martin et al., 2003). Genes with functions in the same cellular process,
as well as genes with similar expression profiles are also generally clustered
in bacterial genomes (Dandekar et al., 1998; Overbeek et al., 1999). The
same clustering of genes with similar function and expression pattern is seen
in eukaryotes (Lercher et al., 2002). In addition, conserved genes and housekeeping genes tend to cluster in regions of high gene density. Smaller clusters of genes with similar transcription pattern are also seen, probably regulated by chromatin organization and localization within the nucleus (Hurst
2004). In both humans and Drosophila melanogaster gene expression can be
correlated to replication timing (Schübeler et al., 2002; Woodfine et al.,
2004). Possibly, this correlation between transcription and replication is a
consequence of chromosome topography, and that the protein machinery
needed for one process also facilitates the other by opening up the chromosome.
Thus, in both bacteria and eukaryotes genes tend to cluster according to
function, expression, conservation pattern and relative importance for the
cell. In paper III we study the clustering of genes in archaea.
Transcription topology of Sulfolobus (Paper III)
In Paper III, the global transcription pattern of the chromosome was examined. Microarray studies of both S. acidocaldarius and S. solfataricus
showed a clear bias for highly transcribed genes clustering to the origins of
replication.
Microarray technology allows for large scale quantification of nucleic acid.
The arrays consist of a library of DNA probes, matching the genes of the
organism of choice, attached to a glass slide (Andersson et al., 2005). Sample nucleic acid is labeled with fluorescent dyes (Cy3 and Cy5 dye in this
study) and hybridized to the slides together with a reference sample. The
36
relative amount of fluorescent dye is quantified for each probe with laser
detection. Thus, while microarray allows measurement of the global RNA
expression of the cell, only relative quantification is possible. Microarray
technology is most commonly used for transcriptome analysis by RNA
quantification, but quantification of DNA is equally possible and has been
instrumental in the detection of the three origins of replication in Sulfolobus
(Lundgren, et al., 2004). The microarray experiments described in Paper III
were designed to show the expression of each gene in a comparable fashion.
Therefore genomic DNA – rather than RNA – from stationary phase cultures
was used as a standard since all genetic markers in that sample are present in
equal copy numbers. For the gene expression, global RNA from both exponential and stationary phase cultures were used.
When the signal ratios were calculated for each gene, a clear bias was seen
(Figure 6). Highly expressed genes clustered around the sites of chromosome
replication with the expression levels declining with distance. Three peaks of
elevated transcription were seen, specifically at the sites of origin initiation.
The pattern obtained was reminiscent of a marker frequency plot where
DNA markers from growing cultures were hybridized against DNA from
stationary cultures and which thereby, when plotted over the chromosome,
gave a visualization of active origins of replication. However, in the transcription characterization plot the slopes of the peaks were steeper than in
the DNA marker frequency plot. This observation rules out the possibility
that the pattern is caused by gene dosage effect, where the simple increase in
number of gene copies after replication also increase the level of transcription at a corresponding level. In this experiment we measured a more than 4fold average expression ratio for S. acidocaldarius between genes located
adjacent to or distant from origins of replication, in contrast to the 1.3-fold
average gene copy ratio measured in growing cultures (Lundgren et al.,
2004).
Similar bias was seen for both S. acidocaldarius and S. solfataricus that,
although related, have radically different genetic layout due to extensive
chromosome rearrangement in the evolutionary history of S. solfataricus
(She et al., 2001). Although three origins are present in both species, their
locations differ (Lundgren et al., 2004). Even so the correlation between
highly expressed genes and origins of replication is maintained, although to
37
Figure 6. Relative transcription over the chromosome in S. acidocaldarius and S.
solfataricus. Each dot represents the logarithmic relative expression of a single gene.
The data comes from one hybridization. The arrows indicate the origins of replication (from Lundgren et al., 2004) where highly transcribed genes cluster.
a weaker extent in S. solfataricus. This indicates an active selection for the
pattern and suggests adaptive advantages for the organism.
Inspired by these observations the underlying structure of the clustering was
examined further and a consistent pattern of gene localization being biased
by proximity to origins of replication emerged. The gene density is higher in
regions close to origins of replication than in intermediate areas of the chromosome. Intermediate DNA sequences are shorter, between genes of both
the same and opposite strands. There are no transposons in the chromosome
of S. acidocaldarius, but S. solfataricus is one of the most transposon dense
chromosomes of all sequenced organisms (She et al., 2001). These transposons are mainly located in late-replicating areas of the chromosome, leaving
the area surrounding the origins of replication free from transposons. This
38
observation goes hand in hand with the elevated gene density and suggests
an active selection against transposons in these regions.
Housekeeping genes code for basal cellular functions such as replication,
transcription and translation. Due to their fundamental role in the cell,
housekeeping genes tend to be highly conserved. There are between 100 and
200 genes identified in most of the sequenced archaeal genomes (Makarova
et al., 2007) that can be considered an archaeal ‘pan-genome’. These genes,
that constitute most of the house keeping genes, follow the by now familiar
pattern of elevated density in transcriptionally active regions.
These observations form a cohesive image of an actively selected adaptive
maintenance of the regions surrounding origins of replication with a bias for
the presence of genes of importance for the organism; highly transcribed
genes, housekeeping genes and panarchaeal genes. In both S. acidocaldarius
and S. solfataricus this pattern is seen for all three origins of replication.
Chromosome organization – discussion
Extremophiles live a hard life. The highly mutagenic nature of the habitats of
these organisms necessitates adaptations to reduce genetic damage, and transcription studies have shown a constant activity of DNA repair systems
(Fröls et al., 2007; Götz et al., 2007). During the majority of the cell cycle of
Sulfolobus, the cell has two copies of the chromosome (Lundgren, 2004).
This might conceivably be a protection against gene damage by providing
the cell with two functional copies of each gene for repair of damaged sequences by homologous recombination. The same selection pressure might
be a driving force behind the transcriptional bias and clustering of panarchaeal genes revealed here. The genes that cluster near the origins of replication are the most important for the organism – highly transcribed genes that
are extensively used, conserved genes that have an essential function for
phylogenetically dispersed organisms as well as genes with known function
in central cellular processes. Conversely, the genetic regions of least value
for the organisms are located far away from the origins of replication. Thus
it is possible that the observed transcription pattern and associated clustering
of essential genes is another trait to reduce DNA damage by environmentally
induced mutagenesis. This clustering ensures that the most important genes
will be the first copied during DNA replication and thus the first parts of the
chromosome with an extra copy for potential repair.
Another possibility is that the trait is a regulating mechanism for gene activity. By clustering of highly transcribed genes and origins of replication, a
39
chromosomal region is formed with an overall high presence of DNA binding proteins, belonging both to the transcription and replication apparatus. In
this ‘hot-spot’ region DNA access is simplified, which might also have energetic advantages in an extremophilic environment where the DNA by necessity is tightly wound and add a potential measure of structural control.
In both these models, the multiple origins of replication increase the amount
of chromosomal regions in proximity to an origin, and thus increase the potential for both genetic safe keeping and chromosome structure dependent
gene regulation.
A bioinformatic survey of six genomes of the related organism S. islandicus
described a bias in degree of genetic evolution which correlated with distance from origins of replication (Flynn et al., 2010). In this study it was
determined that nucleotide substitution rate of both synonymous (dS) and
non-synonymous (dN) nature – that is, both mutations that do not affect and
that affect the amino acid sequence of the encoded protein – correlate positively with distance from the nearest origin of replication. This means that
genes close to origins have a lower evolutionary rate than more distant
genes. Similar to our observation in S. acidocaldarius and S. solfataricus it
was also noted that conserved genes – in this study conserved between the
sequenced genomes of S. islandicus rather than within the entire archaeal
domain – cluster to the origins of replication. The pattern was especially
pronounced for genes involved in transcription and translation. In contrast,
genes associated with energy metabolism and transport, genes that can be
attributed to the organisms’ habitat and life style rather than the internal
workings of the cell, tended to be more common in areas distant to the origins of replication. In addition, codon there was also a bias in codon usage
by genes close to origins of replication compared with more distant genes.
The observations made in S. islandicus are all in line with our observations
in Paper III, but contradict the authors’ initial hypothesis that the bias in
evolutionary rates, gene order and codon usage seen in bacteria should be
non-significant in archaea with multiple origins of replication in a relative
small genome, such as Sulfolobus. According to this hypothesis the extra
copies of genes close to origins cause a dosage effect in expression levels
that in turn cause the discrepancies in evolutionary rate. Multiple origins of
replication in a small genome should reduce the dosage effect and therefore
also the differences in evolutionary rate. As seen in both of these studies,
however, there is a strongly pronounced bias for evolutionary rate as well as
gene content and transcription, centered around three origins of replication
rather the one origin in bacteria (Flynn et al., 2010).
40
Interestingly the bias is less pronounced for one of the three origins of S.
islandicus, suggesting that it fires later in the cell cycle (Flynn et al., 2010).
Similar suggestions have also been made for one of the origins of S. acidocaldarius (Duggin et al., 2008).
The presence of multiple origins of replication in Sulfolobus and the pronounced bias in transcription and function of the genes that surround them
suggest that these regions of the chromosome are subject to strong evolutionary forces. While the exact forces forming and maintaining the origins of
replication remain to be discovered, these studies clearly demonstrate that
multiple origins of replication in crenarchaea are adaptive traits with a selective value for the cell.
Viral release and infection in extremophilic
environments
Wherever there are living cells, there are viruses that infect them. In every
ecosystem, for every organism, they are an influential part of life. Viruses
have for example been estimated to be as much as ten times more numerous
than cells in sea water (Wommack and Colwell, 2000). Due to their size and
the ease with which they are transferred, virus particles can spread over large
distances to a magnitude large enough to significantly affect local population
structure (Snyder et al., 2007). The effect a virus has on its host is reflected
in the evolution of cellular mechanisms to resist infection. One example is
the CRISPR system (clustered, regularly interspaced short palindromic repeats) which is used by both bacteria and archaea to defend against invading
virus by direct targeting of specific sequences of nucleic acid (Kariginov and
Hannon, 2010).
Despite the comparably small number of archaeal viruses that has been studied they display a remarkable variation in morphology. Among the more
noticeable are the fittingly named bottle-necked virus ABV, the claw-end
virus AFV1 and the two-tailed lemon shaped virus ATV, with the ability to
change its morphology outside the cell (Prangishvili et al., 2006). There are
also more ordinary shaped viruses, such as H1, which is similar in shape to
head-and-tail viruses of bacteria.
There is no single genetic element shared between all viruses, which mean
that their phylogeny cannot easily be determined by using approaches analogous to the trees built by ribosomal genes for cellular life. Also, for distinct
viral groups, rapid genetic evolution makes this approach difficult. One way
41
to study phylogenetic relationships for viruses is by comparing the morphology of the virion capsids. With few exceptions – such as the head-and-tail
viruses mentioned above – this classification of viruses results in three distinct clades, one for each domain of life (Prangishvili et al., 2006). This observation supports the description of viruses as ancient entities, which have
coevolved with their cellular hosts from the time of the last universal common ancestor – or even earlier.
A virus that maintains a stable infection of its host without killing it is described as lysogenic (Prangishvili, 2006). The genetic material of a lysogenic
virus is incorporated in the genome and, while not actively killing the host, it
can under the right circumstances produce and secrete new virus particles.
The alternative viral life style is lytic, where the infecting virus produces a
large number of virions and kills the host cell, thereby releasing the resulting
virions into the surrounding environment for new infections.
In bacteria, lytic viruses break out from the cell by direct enzymatic degradation of the cell wall. There are also indications of inhibition of cell wall synthesis. The degradation system itself is of different complexity in different
bacteriophages, ranging from single proteins to elaborated systems for
transport and synchronization of the degradation apparatus (Bernhardt et al.,
2002). Holins are specialized structures that assemble in the bacterial membrane and, at a coordinated point of the infection cycle, permeabilizes it by
formation of large ring structures (Saava et al., 2008), which gives phage
encoded degrading enzymes access to the cell wall. The exact scheduling
with which cell wall degradation takes place speaks of a tightly regulated
mechanism (Wang et al., 2000).
Archaeal viruses have traditionally been described as lysogenic, and it has
been theorized that the extreme habitats prevent the lytic life style (Prangishvili and Garret, 2005). The virus SIRV2 has been described as lysogenic
(Prangishvili et al., 1999). However, in Paper II we demonstrate that SIRV2
is in fact lytic and utilizes a spectacular mechanism for cell lysis, unrelated
to the bacterial systems.
SIRV2 infection of Sulfolobus islandicus (Paper II)
To understand a virus is to understand its relationship with the host, and
despite the wealth of morphological and genetic diversity that has been seen
for archaeal viruses only a small number of studies of the host-virus interaction dynamics have been conducted (Prangishvili et al., 2006).
42
Sulfolobus rod-shaped virus 2 (SIRV2) infects S. islandicus LAL14/1. The
virus particle is linear without envelope and it belongs to the family Rudiviridae (Prangishvili et al., 2006). The 35.5 kbp genome consists of double
stranded DNA – as is the case for most archaeal viruses. The virus and its
host are adapted to hyperthermophilic, acidophilic habitats (Prangishvili et
al., 1999; Prangishvili et al., 2006; Rice et al., 2001; Vestergaard et al.,
2005; Vestergaard et al., 2008).
In Paper II the infection cycle of S. islandicus and SIRV2 was explored. Cell
cultures were grown to exponential growth phase and inoculated with viruses at different multiplicity of infection (MOI, a measurement of the ratio of
infecting virus per potential host). A range of methods were deployed to
monitor cell growth response and cellular DNA degradation as well as viral
DNA accumulation and virion production, and the viral release system was
described.
Optical density (OD) is the standard measurement of cell culture growth and
was deployed to follow the kinetics of infected culture. While the optical
density of uninfected cultures steadily increased, the curve flattened out during the course of virus infection. However, since empty cells scatter light
just as well as living, the opacity of the culture is not a direct measurement
of cell vitality and might therefore be a misleading indicator of infection
dynamics. Counts of colony forming units (CFU) measures the number of
viable cells in a culture by plating, and demonstrated a dramatic reduction of
viability in infected cultures compared to controls. The CFU counts also
revealed growth of a small subpopulation of S. islandicus cells from early
infection that was revealed to be immune to further SIRV2 infection. A rigorous examination of growth conditions revealed that the stagnation of OD
and decrease of CFU values remained true also for different MOIs (~7 and
~7 x 10-3), different temperatures (70 C, 75 C and 78 C), different pH values (3.0 and 3.5), and different medium richness (1:1 and 1:5 dilution). Also,
three different S. islandicus strains were used.
As we have seen, flow cytometry measures the DNA content of each individual cell which gives an estimate of total cell viability in the culture. In
cultures infected by viruses, cellular DNA – clearly visible prior to infection
in the distribution of two genome peaks typical of crenarchaea (Lundgren¸ et
al., 2008) – was degraded over time and empty cells where all DNA is degraded accumulated instead (Figure 7). We also saw an intermediate accumulation of viral DNA. This visualization of internal viral DNA was only
seen at high MOIs, which demonstrates how the viral replication takes over
the cellular machinery.
43
Figure 7. Flow cytometry histograms of cell size and DNA content for cultures of S.
islandicus strain LAL14/1 infected with SIRV2 at three time points after infection.
At the time of infection the culture is healthy, but after 11 hours the majority of the
cells are DNA-less. Although the cellular DNA becomes highly degraded during the
course of infection the light scatter signal from the surface remains rather undisturbed, due to empty cells still being present in the growth medium. Because of this,
the lytic properties of SIRV2 are not readily observed with optical density based
methods.
CFU counts and flow cytometry suggested that a subset of the infected cells
had acquired immunity against SIRV2 infection. In order to confirm this,
great pains were taken to confirm the identity of resistant subpopulation as
being the same S. islandicus LAL14/1 strain that was initially infected, rather than a contaminating SIRV2-insensitive strain. 16S ribosomal DNA
sequence and EcoRI restriction patterns of the whole genome were compared with uninfected LAL14/1 as well as related Sulfolobus strains, and
they showed identical patterns to the uninfected cells. In addition, four different, five times re-streaked clones were infected with SIRV2, and eventually growth was observed after several days of non-increasing OD values.
44
This confirmed the initial susceptibility of LAL14/1 cultures to SIRV2, followed by the eventual gain of resistance.
As an additional test of cell viability, DNA coding for host specific 16S ribosomal RNA was visualized with dot blot hybridization. This technique
uses radioactive markers to quantify DNA of a specific sequence. The same
method was used to estimate the amount of internal cellular virus DNA, and
it was revealed that while cell specific DNA was lost, viral DNA accumulated.
The exact kinetics of virus infection time and burst size was confirmed with
1-step growth study where an S. islandicus strain was infected with SIRV2 at
a low MOI. The medium is replaced after initial incubation to ensure that all
infecting virus particles had been adsorbed within a defined amount of time
and quantification of released virus particles was performed in a time series
by counting plaque forming units (PFU). This method enabled us to monitor
the infection progress from one single time point of infection.
Transmission electron microscopy (TEM) and scanning electron microscopy
(SEM) images of infected cells showed a remarkable sight. Approximately
ten hours after infection, wedges were seen sticking out of the cell membrane, apparently rupturing it. These pyramidal structures had seven-sided
bases and left holes in the membrane and cell wall. In the tip of the pyramid
was a darker dot. During the course of infection, the virus associated pyramids (VAPs) were located in the membranes of both living and empty cells.
Also, virus particles were seen inside the cells, mainly in bundles taking up a
large part of the cellular space.
When considered together, these experiments give a model of the infection
cycle of SIRV2. During the first 8 hours after virion infection the virus DNA
is replicated and the host DNA is degraded. After 10 hours fully assembled
virus particles are formed and arranged in bundles, and at the same time the
pyramidal VAPs emerge from the S-layer. During the next four hours the
VAPs open up to puncture the cell envelope and release the virus. Empty
cells remain with punctured envelope and free from both host DNA and viral
structures. The virus particles are free to infect new cells and the infection
cycle starts again.
Virus of the archaea – discussion
The results clearly demonstrate a lytic life style of SIRV2. The CFU counts
and flow cytometry independently show a decrease in amounts of viable
45
cells during the course of viral infection. The flow cytometry together with
the dot-blot assay also demonstrate a degradation of the host specific DNA.
Finally the electron microscopy images show cells being ruptured by specialized structures, proving that SIRV2 is adapted to lyse the host rather than
remain in the living cell in a lysogenic state of ongoing infection. At the
same time, the lack of drop in OD value shows how the earlier description of
SIRV2 as a lysogenic virus was formed. The stable OD value despite cell
rupture and death is most likely due to the remaining presence of empty cells
in the media. Also, the emerging subpopulation of resistant cells will contribute to the turbidity of the culture, and will eventually grow into a detectable growing cell population in its own right. This proves the value of detailed examination of the relationship between virus and host, using several
sources of information. The VAPs used for cell rupture are structures unique
to archaeal viruses and do not correspond to any other known viral release
mechanism.
In analogy to the SIRV2 life cycle study, it has been demonstrated that the
Sulfolobus Turreted Isocahedral Virus (STIV) also has a lytic life cycle
(Ortmann et al., 2008) and even shares the same release mechanism as
SIRV2, forming pyramidal structures that rupture the cell envelope (Brumfield et al., 2009). In this study the lytic life cycle was demonstrated and
monitored directly with TEM/SEM imaging.
Follow-up studies have given valuable insight into the nature and mechanism of the VAPs in both SIRV2 and STIV (Quax et al., 2010, 2011; Snyder
et al., 2011). The VAPs of both viruses consisted of seven triangular structures forming a heptahedrical pyramid. In SIRV2 each pyramid was modeled
to be symmetric with two 74º and one 34º angles. Open VAPs formed apertures that let virus particles out of the cell. At this point, seven triangular
sides of the VAP were still connected at the base, and the triangles had acquired a more rounded form, resembling shark fins in their morphology. The
pyramids were found in different sizes but with identical proportions, which
means that the structure grows gradually but forms its basic shape at initial
assembly. Only large pyramids were seen in open form.
Proteomics studies revealed that the VAPs of SIRV2 consisted of the 10 kDa
protein p98 (Quax et al., 2010). Available in large number in SIRV2 infected
cells this protein was found in the cytoplasm as soon as 3 hours after infection, and at all times when VAPs were visible, p98 was found in the membrane. The protein was exposed on the cell surface, consistent with its role in
piercing the cell envelope.
46
The native host of SIRV2 is S. islandicus. When the SIRV2 p98 protein was
expressed in the related species S. acidocaldarius, which is immune to
SIRV2 infection, the pyramidal structures were assembled and found in the
membrane (Quax et al., 2011). The pyramids were of similar size range as
the native VAPs of infected S. islandicus but no open structures were found.
Remarkably, the same was true for p98 expressed in E. coli. Pyramids assembled and gathered in the cell wall, just as in S. acidocaldarius and in the
native host. The VAPs found in E. coli did not reach the same size as in Sulfolobus and did not open, but were nevertheless capable of self assembly
without any other archaeal or viral factor.
The VAPs of STIV displayed very similar properties to the VAPs of SIRV2
(Snyder et al., 2011). The pyramidal structure was formed by only one protein, c92, which was present in the cell at early stages of the infection and
which concentrated to the cell membrane to form the pyramids. The structure was autonomous from the virus particle, it was seven-sided and opened
up in a similar fashion to the SIRV2 VAPs. Also when expressed heterogeneously in another organism – S. solfataricus – the pyramids were capable of
assembly, although not of opening. They were found in various sizes in the
cell. The ability of VAPs from both SIRV2 and STIV to assemble from expression of only one gene in systems as different as Sulfolobus and Escherichia demonstrates a remarkable versatility in the temperature range, pH
range and variation in internal cellular chemistry. It is also of interest to note
that the seven-fold structure is rare in nature (Al-Khayat et al., 2009; Förster,
et al., 2003).
There are very few genes shared between SIRV2 and STIV, but the genes
encoding the proteins identified in both viruses as being a major component
of the VAPs are homologous. SIRV2-p98 and STIV-c92 share 55.4% amino
acid identity, which includes a transmembrane domain. This is yet another
indication that the VAPs consist of very few factors (Quax et al., 2010;
Snyder et al., 2011). Homologues of this gene are present in a number of
viruses of both the Rudiviridae family to which SIRV2 belongs and to the
Stygiolobus family of STIV (Quax et al., 2010). Interestingly, there are
closely related viruses of both families that lack the gene – such as the Acidianus rod-shaped virus 1, ARV (Vestergaard et al., 2005) and the close STIV
relative STIV2 (Happonen et al., 2010). Presumably these viruses are either
in possession of a different cell disruption mechanism or simply lack the
lytic life style altogether. The similarity between the SIRV2 and STIV VAP
proteins is higher than between SIRV2 and the more closely related virus
Stygiolobus rod-shaped virus (SRV), which suggests that the gene has been
horizontally transferred (Quax et al., 2010).
47
The VAPs constitute a remarkable virus-encoded structure, capable of assembly and function autonomously from the capsid. The term 'virodome' has
been coined for this kind of structure (Prangishvili and Quax 2011). Apart
from the VAPs, it is suggested that the term could be used for the cell wall
damaging holin structures of bacteriophages.
Since the first identification of these remarkable cell lysis structures in
SIRV2 and STIV several fundamental questions about their nature and
mechanism have been answered, but as is always the case in descriptive
science, every answer give rise to more questions. As intriguing as it is to
see the ease with which the VAPs assemble in different systems, it is just as
interesting to note that the final stage of the VAP maturation, where the pyramids open and release the viruses, does not seem to be functional in any of
the transgenic systems. It is tempting to speculate that either the virus particle itself or another virus encoded factor is needed for VAP opening, or perhaps that the effects of viral infection of the cell itself serves as a trigger for
the opening mechanism.
Another point of interest is the apparent narrow phylogenetic spread of this
cell disruption mechanism, only being present in a two archaeal virus families despite the ease with which the trait should be able to be horizontally
transferred. The VAPs are encoded by only one gene, the formation and
localization of the pyramidal structure in the membrane has proven highly
resistant to environmental and cellular variation, and – not least important –
the encoded trait can be expected to have a high adaptive value for lytic viruses of all domains. VAP is a school example of a trait that should be horizontally transferred, but yet it is only described in two viruses. This observation requires an explanation.
One possibility is that the lytic life style is uncommon among hyperthermophiles and that the two described viruses are exceptions. From this follows
that VAPs are not common since cell lysis is not a highly adaptive trait
among archaeal virus. This fits with the earlier description of crenarchaeal
viruses as mainly non-lytic. However, both SIRV2 and STIV are well
adapted to their host and habitat, and there is no suggestion that they are
uncommon in nature which calls this line of reasoning into question. Another possible explanation is that other release systems, such as the bacterial
holin/endolysin system (Wang et al., 2000) has a higher adaptive value for
many organisms and therefore outcompete the VAPs. The holin/endolysin
system is widespread among lytic bacteriophages. Similarly, other undiscovered cell rupture mechanisms might exist among archaeal viruses and constitute a similar competition barrier. Supporting this idea, the crenarchaeal vi48
rus ATV has also been shown to be lytic, although the mechanism for cell
rupture is not clear (Häring, 2005). The extremophilic life style of SIRV2
and STIV might prevent gene flow to viruses inhabiting less extreme habitats. The undersampling of archaeal viruses (Prangishvili, 2006) might even
cause the apparent rarity of VAP to be an artifact that will be proven wrong
by further sampling. As a last possibility, the VAPs might be a young trait in
evolutionary terms, which has so far lacked the time to spread wider than it
already has.
It has been theoreticized that the harsh conditions of the hydrothermal environments where these viruses and their hosts live are inimical to the virions,
which is why extremophilic viruses are selected for a lysogenic life style
where direct contact with the environment outside the cell is minimized
(Prangishvili and Garrett, 2005). The growing number of crenarchaeal viruses described as lytic, as well as the description the specific cell rupture
mechanism VAP, shows that the viral particle is not intrinsically susceptible
to the harsh environments of the hot springs, and that the infecting viruses
just as well as the host cell is to be regarded as extremophiles.
49
Conclusions
Archaea is the least studied of the three domains of life. Much is still unknown about their fundamental functions and relationships. The four papers
included in this thesis discuss different aspects of the biology of the archaeal
cell. We have discovered a previously unknown mode of cell division shared
between cren- and thaumarchaea. This Cdv system is homologous to the cell
membrane reorganizing ESCRT-III system of eukaryotes, which suggest an
ancient evolutionary history of this system. We have seen a thaumarchaeal
cell cycle organization that differs from that of crenarchaea, possibly reflecting adaptation to an extremely nutrient poor environment. Global transcription patterns as well as gene localization of Sulfolobus is correlated with
early replicating regions which indicate active selection for multiple origins
of replication. This might serve as protection from DNA damage caused by
harmful environments or as a regulatory feature. Lastly we have described a
previously unknown lytic life style for the extremophilic virus SIRV2 and
shown a highly specialized mechanism for cell rupture. These studies have
given new important insight into the inner workings of the archaeal cell.
50
Perspectives
Studies of the archaea have again and again changed our fundamental biological understanding and forced us to re-write the framework in which we
place all biological research. The constraints of cellular and viral life, the
basal evolutionary history on Earth, the fundamental organization of the cell,
the origins and nature of eukaryotes, the principles of ecological dynamics –
all have been reshaped thanks to increased understanding of archaea. If for
no other reason, we owe it to ourselves to keep studying an organism group
so full of surprises.
Methodology in microbiology
The research presented in this thesis focus on four different topics – cell
division, cell cycle strategies, chromosome organization and viral release.
Four different archaeal model organisms are used for the study – S. acidocaldarius, S. solfataricus, S. islandicus and N. maritimus. The studies are
united by their significance for cells throughout the tree of life, and also in a
shared methodological approach where all levels of information transactions
within the cell are taken into account.
The information dynamics of the cell follows a straight path, from the intrinsic potential encoded in the genome through the utilized capacity of the transcriptome, followed by the functions realized by the proteome and last the
actual phenotype of the cell in interaction with the environment. All these
levels of information are discussed in this thesis. Cell phenotype is studied
with growth assays and flow cytometry. Protein dynamics is studied with
fluorescence microscopy. Global transcription is measured with microarray
technology and genome content and dynamics is studied using dot-blot technology. Also, previous studies of these features – especially genome sequencing and transcription mapping – have been invaluable resources for the
model systems used and for the setup of these experiments.
In this age of large-scale genomics and bioinformatics, an increasingly large
part of the body of scientific knowledge is derived from homology based
51
predictions where the function of a genome or a protein is estimated from
similarities with known genes from related organisms. While an indispensable tool, this line of methodology has its pitfalls which one needs to be aware
of, mainly the larger uncertainty that follows prediction based on homology
over large evolutionary distances. That is why experimentation is still crucial, even despite the overwhelming capacity of bioinformatics. While large
scale bioinformatics is used to draw a more and more detailed map of the
diversity of life, experimentation puts physiological markers on the phylogenetic map.
The identification of the thaumarchaeal cell division machinery reported in
Paper IV gives a good example of this dualism. Prior to the study presented
in this paper, it was known from genomic sequencing that the thaumarchaeal
genome contained genes for two distinct cell division machineries – the FtsZ
machinery and the Cdv machinery. Based on the indicated involvement of
Cdv paralogues in other processes than cell division, FtsZ was predicted to
be the most likely candidate for cell division in thaumarchaea (Makarova et
al., 2010; Samson and Bell, 2011). However, when experimentally tested,
our study proved that Cdv is the primary cell division apparatus. No suggestion of an FtsZ involvement was seen, and the role of FtsZ is not yet known.
Thus the simplest solution predicted from sequence homology was not correct. This conclusion is also reinforced by the observation that the GTP binding domain of FtsZ of thaumarchaea is significantly divergent from the corresponding domain of euryarchaea (Busiek and Margolin, 2011) This
demonstrates how inference made from gene homology in some cases need
to be subject to experimental testing, especially when conclusions are extrapolated between distantly related groups.
A sound principle in experimental science is that extraordinary claims needs
extraordinary proof. An observation that fits seamlessly in generally recognized models needs less support than the observation that radically changes
earlier known theories. Our studies provide an interesting example of this
principle. Claims that are bold and new – such as the discovery of the Cdv
genes as a cell division system – do not necessarily contradict other models.
The lack of FtsZ in crenarchaea was well known, and thus the existence of
an unknown system for cell division was easily inferred. The identity of the
system needed rigorous proof, not because previous claims needed to be
addressed but simply because the novelty of the study means there is less
previous knowledge to relate it to. Another example is found in Paper II. The
claim that the virus SIRV2 has a lytic life cycle is a direct contradiction of
earlier models, and careful experimental work was needed to provide support
for the new discovery. A range of methods were deployed to carefully moni52
tor and control the infection dynamics. Similar studies have been conducted
earlier, using optical density measurements as the primary method for estimation of infection progress. In order to disprove the resulting description of
SIRV2 as a lysogenic virus, our study monitored the infection on several
levels. Apart from optical density we also quantified cellular and viral DNA
together with cell integrity, viability and immunity, resulting in a detailed
map of the virus-host infection dynamics. Thus, the novel claim we make is
supported by rigorous evidence, and our new model consistent with previous
observations.
The scientific method depends on reproducibility. The reliability of an experiment is ensured by its ability to be independently tested. However, all
too often the practical application of this principle is lacking, due to limitations in resources and simply the amount of labor that would be required for
systematic, independent testing of all reported claims. In that aspect the research presented in this thesis is in privileged minority, since the findings of
three of the articles have been corroborated by similar studies. The identification of Cdv as a crenarchaeal cell division system (Samson et al., 2008),
the characterization of VAPs as a viral lysis system (Brumfield et al., 2009)
as well as a bias in gene clustering to the three origins of replication in Sulfolobus (Flynn et al., 2010) have been corroborated in independent labs, in
the Cdv case with the same and in the VAP and gene clustering cases with
different study systems than in our studies.
Microbiological science is an ever growing body of knowledge, but it is also
a craft. Every experiment yielding usable data is preceded by countless hours
in the laboratory preparing material, sounding potential methods and fine
tuning the analytical data yielding step that can be rather short and uncomplicated compared to the entire laboratory procedure. Scientific progress is in
no small part dependent on the hands that hold the pipette and the accumulated skill in the laboratory. Experimental success depends on minor details
that are not always included in official descriptions of a method, such as the
humidity in the incubator or the correct way to stabilize the microarray slide
with small pieces of pipette tips.
Just as important as the ability to perform the experiment is the ability to ask
the right questions and design the right experiment to begin with. There are
concerns that the rapid advancements of methods in microbiology shifts
focus from an analytical perspective to a descriptive one (Prosser, 2007).
The revolution in DNA sequencing technology makes production of very
large dataset feasible and there is a risk of the collected amount of data being
too unstructured and unconnected to theoretical frameworks to be useful in
53
comparisons. The more subtle risk is that the eagerness to fill the ever so
large white spots on the map makes us forget to connect the dots between
our new findings and what is already established. Microbiology has always
been driven by methodological advances, and studies of the archaea are no
exception. The very first discovery of archaea as a separate group (Woese
and Fox, 1977) was a result of utilizing brand new methods of ribosomal
RNA comparisons, and many milestones reached since then are also the
results of new methods or technologies. Examples include PCR amplification of DNA from uncultured organisms which identified archaea in oceanic
environments (De Long, 1992) or the genome sequencing technology which
as one of the first microorganisms gave the full genome of Methanococcus
jannaschii (Bult et al., 1996). Together with progress in methodology, research is driven by the right questions being asked. One such example from
archaeal research is the discovery and cultivation of organisms living at a
higher temperature than 100 C, which utilized established technology to
address novel questions (Stetter, 2006a).
As is usually the case with dichotomies, most research lies between the two
poles – made possible by new technique but motivated by ideas. The discovery of the crenarchaeal cell division machinery presented here is a good example. The transcription map that made the study possible was dependent on
modern microarray technology (Lundgren and Bernander, 2007), but identification of the cell division machinery needed focused empirical analysis of
the candidate genes obtained from the transcription map.
Archaea by name and nature
Archaea as a separate group of organisms has been under hot debate, naturally so, since the concept of three domains and a fundamental division between two prokaryotic groups has forced rethinking of earlier models of the
tree of life (Woese, 1998). While fundamental questions remain unanswered
– primarily the relationship between archaea and eukaryotes (e.g. Guy and
Ettema, 2011), the archaea as one distinct group is now part of established
biological theory. Interestingly the old word ‘prokaryote’ is still widely used
even if it has been criticized for lumping archaea and bacteria together in the
same group, despite taxonomic differences (Pace, 2006). Given the many
shared metabolic and ecological properties of bacteria and archaea it is hard
to argue against such a useful shorthand for “non-eukaryotic microorganisms”, but the debate is interesting since it highlights the importance of labels.
54
One of the cornerstones of biology is classification. With the overwhelming
quantity and variation of living organisms we need labels to sort the information. Shared names imply shared properties, and in the evolutionary perspective of modern biology, also a shared genetic history. The separation of
archaea from bacteria is motivated by just this, their lack of shared ancestry
earlier than the last common ancestor of all life. The fundamental difference
between the three domains of life is highlighted by archaea and an evolutionary perspective motivates the name and separation from bacteria. In a
three-domain worldview there are always three perspectives on an evolutionary question, which increases the accuracy. This perspective would not
necessarily be present if the archaea were regarded as yet another subgroup
to bacteria.
The same is true for the current discussion about thaumarchaea which up to
recently were seen as a subgroup of crenarchaea (Brochier-Armanet et al.,
2008). In different versions of the archaeal phylogenetic tree there are a
number of deep-rooting groups with slightly different positions. While the
exact similarities and differences that motivate the nomination of new phyla
can and should be discussed, the division between cren- and thaumarchaea
serves to highlight important differences. Most obvious is the question of
habitat – most crenarchaea are extremophilic and most thaumarchaea live in
habitats with no environmental extremes. There are also differences in cellular features, such as the differences in cell cycle organization and the thaumarchaeal presence of the ftsZ gene discussed in this thesis. The genomes of
thaumarchaea have been shown genes thought to be exclusive to either crenor euryarchaea (Brochier-Armanet et al., 2008). These differences are accentuated by the nomination of a thaumarchaeal phylum.
It is interesting to note that while the concept of archaea has become well
established, the name of the archaeal domain is misleading. The name archaea itself means ‘ancient’ and alludes to the geological age Archea which
is the age when life first appeared on Earth (Woese and Fox, 1997). However, archaeal organisms are not any more – or less – ancient than those of the
other domains, nor do they have a specific connection to the archeal geological age. Archaea are – just as all other living organisms – the modern descendants of ancient cell lineages. When the name was coined it was suggested that the extremophilic archaea were closer in phenotype and life style
to the cells living on the young Earth, but as we know from the large groups
of non-extremophilic archaea those habitats are not typical for all archaea,
and the properties of the ancestral archaea as well as the habitats and life
styles of the first living cells remain under debate.
55
The label we assign the organism groups we study influence the way we
think about them. A quick glance through this thesis is enough to demonstrate the importance attached to terms such as ‘Archaea’, ‘Crenarchaea’,
and ‘Thaumarchaea’. One reason for this is that a cell or a species is not
studied just for its own sake. It is also studied as a model for a group of organisms, for a life style or for fundamental traits equally valid for all life.
The labels we attach help us decide how extensively a study can be extrapolated from.
The universal through the particular
Through roots and shoots, the tree of life is interconnected. Archaea is one of
the three domains of life and thus holds important clues to the function of all
cellular life. As a model, studies of archaea have been used to gain insight in
the function of the eukaryotic cell (Bernander, 2007), the origin of eukaryotes (Guy and Ettema, 2011), life under extreme conditions (Daniel and
Cowan, 2000), search for life in space (Kendrick and Kral, 2006) and for the
conditions for the first life on an early Earth (Müller et al., 2005). To study
archaea is to study all these perspectives and more. It is interesting, given the
relatively few research groups that focus on archaea, how many fields of
biology that stand crowded around the water bath, trying to pry loose its
secrets.
The diversity of archaea is remarkable. In habitat and life style they range
from the very coldest to the hottest, they live alone in the most extreme environments and are ubiquitous in the largest ecosystems of the biosphere. Evolutionally they are one of the three fundamental domains of life, with roots in
the first living cell on the planet and with a cell structure that holds the key
to understanding our own inner secrets. The archaea are a unique group of
organisms but studies of them have yielded fundamental understanding of
the whole tree of life and all cells on Earth.
Archaeal research has come a long way. First thought to be a fringe group of
bacteria clinging to life in a next to barren environment, the archaea are now
known to be an ubiquitous, diverse, ancient and ecologically essential group
of organisms. And while our understanding of them has exploded these last
decades we have still only scratched the surface. In 1978 Carl Woese wrote
‘The full extent of diversity in the archaebacteria needs to be appreciated’.
Three decades later, I can only agree.
56
Final reflections
It has been a long time – a long time since the first archaeal cell tentatively
unfolded its flagella to smell the sulfur of a new biosphere, a long time since
Carl Woese and George Fox opened the ribosomal Pandora's box that broke
the pro- and eukaryotic taxonomy dichotomy and a long time since I, in my
master’s thesis, wrote that the crenarchaeal cell division system had yet to be
identified. Since then much water has flown through the water bath. It has
been a time filled by the arts and crafts of labwork; of microarray spots, flow
cytometry cell counts, of late evenings staring at DAPI dots in the microscope that look more and more similar to stars for each passing hour. There
have been a lot of dots, these years. It has been a lot of fun connecting them.
The research presented in this thesis demonstrates fundamental properties of
the archaeal cell and adds contours to some of the blank spots on the map of
our biological understanding. However, as is always the case with discoveries, each answer we have provided has given rise to even more intriguing
questions. While the ongoing sequencing revolution has given us a glimpse
of the true diversity of archaea, this, most of all, serves as a reminder of the
daunting task in front of us, to name and categorize, to study and understand.
This is indeed an exciting time to be a microbiologist.
57
Svensk sammanfattning
Archaea är en av livets tre domäner – den mest fundamentala indelningen av
levande organismer. Arkéerna är encelliga mikroorganismer som morfologiskt är mycket svåra att skilja från bakterier. De är båda relativt små, saknar
organeller och cellkärna och är oerhört varierade i livsstil och metabolisk
kapacitet. Arkéer är dock närmare släkt med eukaryoter än bakterier. Cellens
basala maskinerier för informationshantering – replikation, transkription och
translation, jämte andra basala cellulära processer som proteinreglering –
består av liknande maskineri i arkéer och eukaryoter. De arkéella proteinkomplexen är generellt enklare och består av färre komponenter. Det gör
dem till tacksamma modeller för att förstå den eukaryota cellen eftersom de
arkéella maskinerierna är nedskalade till sina mest nödvändiga delar. De
mest välstuderade arkéerna är anpassade till extrema livsmiljöer; till heta
källor, saltbassänger, undervattensvulkaner, syrasjöar och andra förhållanden
som utmanar gränserna för var liv överhuvudtaget kan existera. Det har dock
visats att arkéer finns i stora antal även i icke-extrema miljöer, i de vidsträckta havs- och jord-ekosystemen men också associerade med djur och
människor. De har en nyckelroll i viktiga geokemiska cykler, som kretsloppen för kväve och kol, och de är de enda kända organismerna som producerar den viktiga växthusgasen metan.
I den här avhandlingen presenteras fyra studier av arkéers cellbiologi. Som
modellsystem används arter av släktet Sulfolobus, anpassade till ett liv i heta
källor och släktet Nitrosopumilus, anpassade till kalla och näringsfattiga
miljöer. Sulfolobus är ett av de mest välstuderande arkéella modellsystemen
vilket innebär att det finns flera skräddarsydda molekylära tekniker för arbete med dem, och att nya studier kan sättas i sammanhang av en stor mängd
tidigare forskning. Nitrosopumilus är den mest välstuderade arkén från sin
miljö, och även om den är ny som modellsystem så har studier av den gett
viktig förståelse om kallevande arkéer. Sulfolobus och Nitrosopumilus räknas till de båda fylumen Cren- respektive Thaumarchaeota.
Celldelning är ett kritiskt steg i livscykeln för varje levande organism. Det
finns tre huvudsakliga mekanismer för att dela cellen. Det bakteriella FtsZmaskineriet som även används av organismer i fylumet Euryarchaeota, be58
står av en proteinring längs insidan av cellmembranet som snörper av cellen
på mitten. Det eukaryota systemet är varierat i sin form och funktion för att
tillgodose behoven för de olika celltyper som finns hos till exempel djur och
växter, men det bygger på en gemensam bas i proteinerna aktin och myosin.
Cren- och thaumarkéer använder inte något av de tidigare kända celldelningssystemen utan visas i denna avhandling dela sig med ett nytt system
med tre proteiner som vi namnger Cdv (cell division). Cdv-systemet bygger
liksom FtsZ upp en proteinring som snörper av cellen, och tycks också interagera med DNA, kanske för separation av de blivande dottercellernas kromosomer, men mekanismen är inte känd. Cdv-systemet är besläktat med det
eukaryota systemet ESCRT-III som används till att bilda membranblåsor för
bland annat proteinsortering. Systemet har också visat sig användas av virus
för att lämna värdcellen, och intressant nog nyligen också för celldelning.
Tillsammans med indikationer på att arkéella protein som är närbesläktade
med Cdv-komponenterna också har en funktion vid virusinfektion och
bildande av membranblåsor antyder släktskapet mellan det arkéella och eukaryota systemet att Cdv som celldelningssystem är en uråldrig cellulär
funktion som föregår den tidpunkt då arkéers och eukaryoters utvecklingslinjer skildes åt. Det finns dock en grupp crenarkéer, av ordningen Thermoproteales som inte delar sig med Cdv, och vars celldelningsmekanism förblir
okänd. Intressant nog har thaumarkéer gener för båda celldelningssystemen
Cdv och FtsZ. I den här avhandlingen visar vi att Cdv används till celldelning, men vad FtsZ har för funktion hos thaumarkéer är än så länge okänt.
Cellcykeln hos crenarkéer har visats vara oväntat likriktad. Trots att crenarkéerna uppvisar stor spridning i livsstil och evolutionärt släktskap så tillbringar de större delen av sin livscykel med två kopior av kromosomen. Efter delning går de båda dottercellerna omedelbart in i replikationsfas och kan
därefter spendera lång tid med dubbla genom innan nästa celldelning. Thaumarkéer å andra sidan visar vi i den här avhandlingen har en cellcykel dominerad av prereplikativ och replikativ fas. Efter celldelning så dröjer det innan
DNA-replikation, och när den väl påbörjas går replikationen långsamt, med
hastigheter som påminner mer om eukaryoter än andra arkéers. Vi tolkar det
som en anpassning till de näringsfattiga miljöer cellerna lever i, vilket stämmer med observationen att Nitrosopumilus är anpassade till upptagning av
energikällan ammoniak vid mycket lägre koncentration än bakterier i samma
miljö.
Sulfolobus och andra crenarkéer påbörjar replikation av genomet på flera
olika distinkta positioner på kromosomen, vilket står i skarp kontrast mot
bakterier där det bara finns en startpunkt för replikation av varje kromosom,
trots att den bakteriella och arkéella kromosomen i övrigt har många gemen59
samma drag. Vi visar i den här avhandlingen att genuttrycket är ojämnt över
kromosomen. De högst uttryckta generna är konsekvent lokaliserade i tidigt
replikerade regioner medan lägre uttryckta gener befinner sig längre bort
från replikationsstarten. Samma mönster återfinns om man tittar på essentiella gener vars funktion är viktig för cellen. Gentätheten är överlag hög i
tidigt replikerande regioner, och genelement som inte har någon funktion för
cellen, som transposoner, är lokaliserade långt från replikationsstarten. Våra
observationer visar ett selektionstryck för att gener av stor betydelse för cellen placeras i tidigt replikerande regioner och noterar att multipla replikationsstarter ökar den tillgängliga mängden tidigt replikerande regioner.
Crenarkéer lever generellt i utsatta miljöer där risken för mutationsskador är
stor. Tidig replikation av viktiga gener innebär att de tidigt i cellcykeln finns
i en extra kopia som kan användas för reparation vid DNA-skada. Det stämmer med observationen att crenarkéer replikerar sin kromosom tidigt och
därmed har en extra kopia av hela genomet under större delen av sin livscykel. En alternativ förklaring är att klustren av högt uttryckta gener har en
reglerande funktion där lokala regioner av hög aktivitet underlättar tillgång
till generna för transkriptionsmaskineriet.
Virus som infekterar arkéer har en uppsjö olika utseenden och egenskaper,
av vilka många aldrig har setts för virus från någon av de andra domänerna.
Virus utvecklas i intim samklang med sina värdceller, och förståelse av ett
virus funktion innebär förståelse av deras interaktion. I den här avhandlingen
visar vi att viruset SIRV2 som infekterar Sulfolobus lyserar sin värdcell,
något som tidigare endast var känt från ett fåtal fall av virus som infekterar
crenarkéer. Viruset har en högst specialiserad mekanism för att öppna värdcellen där pyramidformade strukturer genomborrar cellmembranet inifrån
och öppnar sig utåt som flikar för att släppa ut de färdiga viruspartiklarna.
Strukturerna bildas och agerar oberoende av viruspartikeln, och vi ger dem
benämningen VAP (virus associated pyramids).
Vi bekräftar med fyra studier av arkéers cellbiologi gemensamma drag med
både bakterier och eukaryoter, men också helt unika egenskaper, som anstår
en av de mest fundamentala organismgrupperna. Vi diskuterar skillnader och
likheter mellan cren- och thaumarkéer och deras släktskap med eukaryoter.
Vi demonstrerar okända system för celldelning och virusinducerad celllysering och organisation av kromosom och cellcykel som tidigare inte setts i
våra modellsystem. Forskningen som presenteras i den här avhandlingen har
ökat vår kunskap om fundamentala aspekter av cellens funktion och samtidigt visat på nya, intressanta vita fläckar på den biologiska kartan.
60
Acknowledgements
I wish to thank my supervisor Rolf for freedom when possible, for support
when needed and for sharing your enthusiasm for dots in microscopes and
water baths. Thanks for inspiration, for perspective and for giving me the
chance to work with this fascinating group of organisms. Thanks, AnnChristin for invaluable help and amazing work. You really are one of the
best I ever seen in the lab. Thanks Anna, Sara and Ester for enthusiasm,
good work and plenty of interesting fika conversations. Thanks to the previous archaeologists, Maria, Anders, Magnus and Karin E. for introducing
me to the lab and the field.
I am thankful for the funding agencies, FORMAS, the EBC research
school, Helge Ax:son Johnsons stiftelse and the Agouron institutue, for
financing my research.
Thanks Stefan B. for inspiration and for a really interesting course. Thanks,
the people of the UMC network for fun discussions and the Geobiology
crowd for showing me the stromatolites and for those long, wonderful days
with hot sun and hot springs. Thanks to Karin T., Emma and the rest of the
Giardia group for diving into the mysteries of microarray together with me.
Ariane and the rest of the Pasteur institute virus group for a nice collaboration and for your amazingly rigorous experimental setup. Willm, José and
the rest of the Nitrosopumilus people for letting us work with your amazing
organism.
My co-workers – past and present – of Molecular Evolution and the rest of
EBC, for creating a fun atmosphere and for the endless fikaroom discussions. I especially want to thank Jonas for the stories, Johan for the ties,
Kirsten for sharing the gains and pains of teaching, Kashia for dinosaurs
and Neanderthals, Björn B. for raising the quality of our Journal Clubs,
Björn N. for opening my eyes for the sequencing revolution, Eva for sharing my pain about the micro array scanner, Fredrik for the fencing, Minna
for teaching my son Finish, Fei-Fei for always being happy, Hillevi for the
time I got to know you, Lionel for endless enthusiasm and for introducing
me to the secrets of genome sequencing. Thijs for sharing my fascination
61
with the most interesting of bugs. Jennifer for pumpkins and anecdotes,
Lisa for finding the fun in everything, Håkan for being such a good sport
about my computer’s frail personality, Jan, Siv and Otto for ideas and inspiration. Robert for the historical perspective and for the beauty and rhythm of
labwork, Karin O for always looking out for us all, Kristina and Ann-Sofie
for taking care of the lab and helping us new hands out. Thanks to Oliver for
your happy, upbeat nature and for keeping the good flow going.
I also want to thank my fellow students and teachers, from Uppsala University, SLU and everywhere, for walking the bumpy roads of science together
with me. A large thanks to my own students, who have always kept me on
my toes and spurred me towards greater understanding. Thanks to the organizers and participants of my popular science presentations – especially
Marie Öhrn and associates for the squeker toy concert at our scientific salon, and the Young Scientists for really interesting evolutionary debates.
Thanks to all my friends, old and new, lost and kept. You are mad. You are
wonderful. Thanks for all the fun we have had and the stories we have
shared.
I wish to thank my relatives and my family – Mamma, Pappa and Tomas
– for never failing faith and support, and for always sharing my enthusiasm.
Thanks for teaching me to look at the world and enjoy what I see. Thank
you, my beloved Maja for everything, as always, and Simon because you
are truly Extraordinary.
62
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