Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Science and Technology 572
_____________________________
_____________________________
Cell Cycle Analysis of Archaea
BY
ANDRZEJ POPLAWSKI
ACTA UNIVERSITATIS UPSALIENSIS
UPPSALA 2000
Dissertation for the Degree of Doctor of Philosophy in Microbiology presented at Uppsala
University in 2000
Abstract
Poplawski, A., 2000. Cell Cycle Analysis of Archaea. Acta Univ. Ups., Comprehensive
Summaries of Uppsala Dissertations from the Faculty of Science and Technology 572. 64 pp.
Uppsala. ISBN 91-554-4825-9.
In my thesis, the cell cycle analysis of archaea and hyperthermophilic organisms is presented
for the first time. Crenarchaea from the genus Sulfolobus were used as a model system. Flow
cytometry and light microscopy were applied to investigate the timing and coordination of
different cell cycle events. Furthermore, DNA content, nucleoid structure, and nucleoid
distribution at different stages during the cell cycle were studied. The Sulfolobus cell cycle
was characterized as having a short pre-replication and a long post-replication period. The
presence of a low proportion of cells with segregated genomes in the exponentially growing
population suggested a considerable time delay between termination of DNA replication and
full nucleoid segregation, reminiscent of the G2 period in eukaryotic cells.
The first available collection of conditional-lethal mutants of any archaeon or
hyperthermophile was used to elucidate the coordination of cell cycle events. The studies
showed that chromosome replication, nucleoid partition and cell division in Sulfolobus
acidocaldarius, which are normally tightly coordinated during cellular growth, could be
separately inhibited or uncoupled by mutation.
The ftsZ gene, which is involved in cell division in bacteria and euryarchaea, was isolated
from the halophilic archaeon Haloferax mediterranei. Transcriptional start sites were
mapped, and putative translation initiation elements were identified. In both the upstream and
downstream regions of the ftsZ gene, open reading frames were found to be conserved within
the genus Haloferax. Furthermore, at the 3’ end of the ftsZ gene, the homologs of the
bacterial secE and nusG genes are conserved in almost all euryarchaea analyzed so far. The
studies also demonstrated the functional conservation of the FtsZ protein in different archaeal
species, as well as between euryarchaea and bacteria.
Andrzej Poplawski, Department of Cell and Molecular Biology, Biomedical Centre, Uppsala
University, Box 596, SE-751 24, Uppsala, Sweden
 Andrzej Poplawski 2000
ISSN 1104-232X
ISBN 91-554-4825-9
Printed in Sweden by University Printers, Uppsala 2000
1. Introduction
1.1. Archaea and the concept of three domains of life. Application of
molecular tools in taxonomy has led to the division of life on Earth into
three evolutionarily distinct domains (Fig. 1); Archaea, Bacteria and
Eukarya (188).
Fig. 1. The tree of life based on SSU rRNA analysis. Underlined archaea from genus
Sulfolobus and Haloferax were analyzed in this study. Figure was adapted from the
references (5, 141).
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The phylogenetic uniqueness of archaea was initially inferred from the
comparison of SSU rRNA sequences (187), upon which Archaea was
subsequently subdivided into Euryarchaeota, Crenarchaeota (188) and
Korarchaeota (5).
Delineation of archaea from bacteria, initially a controversial idea,
gradually brought about new understanding of the relationships between
prokaryotes and eukaryotes, and raised the importance of Archaea in the
evolution of life on Earth (35, 186). The recognition of Archaea as a
separate domain of life, and their extremophilic properties attracted the
scientific interest to investigate the biological characteristics of this group
of organisms. Studies of different cellular and molecular properties of
archaeal cells, including lipid composition (89, 178), chemical composition
of the cell wall (87, 88), DNA dependent RNA polymerases, translation
factors and ribosomal proteins (3, 18, 192, 193), have supported the idea of
archaea being distinct from bacteria and constituting a separate branch on
the tripartite tree of life.
The archaeal uniqueness was further supported by analysis of the first
completely sequenced genome of the archaeon Methanococcus jannaschii,
which revealed that more than half of the open reading frames had no
homologs within the two other domains of life (19, 99). In addition, the
mosaic structure of the M. jannaschii genome was reflected by the
presence of either bacterial or eukaryal homologs. The bacterial homologs
were represented mostly by genes involved in metabolism, while the
eukaryotic homologs were found mostly among the genes responsible for
the replication, transcription and translation machineries (19, 42, 99, 139).
Subsequent analyses of genomes from other archaeal species belonging to
either Euryarchaeota or Crenarchaeota (50, 57, 90, 91, 96, 163) led to
similar conclusions, as those based on the analysis of the M. jannaschii
genome. The presence of either bacterial or eukaryal homologs within
archaeal genomes, suggests that by studying archaeal properties insights
into corresponding features in either Bacteria or Eukarya could be
obtained. Furthermore, the discovery of the mosaic structure of the
archaeal genomes resulted in a hypothesis about a chimeric origin of both
archaea (99) and eukaryotes (71, 110).
Comparison of cellular features including RNA processing between the
three domains of life and development of new methods for studying
evolutionary relationships prompted some to favor a eukaryotic-like nature
of the last common universal ancestor (LUCA). According to this
hypothesis, prokaryotes including archaea could appear by a reductive
evolution from an eukaryotic-like ancestor (56, 142, 145).
8
The structure of the tree of life was, furthermore, influenced by the
choice of proteins used for phylogenetic analyses (18, 52, 56). Conclusions
based on those analyses were sometimes contradictory to that inferred from
the SSU rRNA sequences (18, 52, 68, 69, 70) because of one possibility
such as the lateral transfer of genes among species (83, 123, 132).
The uniqueness of archaea could still be supported however, by
phylogenetic analysis using whole-genome comparisons (51).
Thus, a new view of the organization of the tree of life was proposed
with Archaea, Bacteria and Eukarya interconnected through the network of
a horizontal traffic of genes (39).
1.2 Diversity of the Archaea. Archaea constitute a group of organisms
found in many environments. Initially, the crenarchaea and euryarchaea
were found in extreme environments with either high temperature, low or
high pH, or high salinity (168, 170). Later, the application of PCR
combined with SSU rDNA analysis revealed the ubiquity of archaea all
over the planet: from hydrothermal vents, ocean and coastal waters,
through marine and lake sediments, hot springs and soil (141, 168, 173), to
the environment at the doorstep of our department in Uppsala.
1.3 Cellular and molecular features of the Archaea. Since my thesis
concerns the properties of the archaeal cell cycle, as well as the structure
and sequence of regulatory elements for such processes as transcription and
translation, I have included a section describing some features of these
mechanisms in the different domains of life.
Note that the comparison does not reflect the presence of such features
in all of the organisms within each domain, and that the picture may
change as soon as more data becomes available.
Table 1. Major cellular features in the three domains of life
Characteristic
Cell wall
Bacteria
Variable
composition
(murein present)
Ester linked
Absent
Membrane lipids
Eukaryotic-type
cytoskeleton
Nucleus
Absent
Organelles (mitochondria, Absent
chloroplasts, Golgi
apparatus)
9
Eukarya
Variable
composition
(never murein)
Ester linked
Present
Archaea
Variable
composition
(never murein)
Ether linked
Absent
Present
Present
Absent
Absent
1.3.1 Transcription. The basal transcription machinery in archaea has
been suggested to be a simplified version of the eukaryotic RNA
polymerase II machinery (11, 148, 165, 179, 193). Both archaeal and
eukaryal RNA polymerase II are multisubunit complexes. For example,
both Sulfolobus acidocaldarius and yeast RNA polymerase consists of 12
subunits, in contrast to E. coli where the holoenzyme consists of only 4
subunits (Table 2).
Table 2. Transcription in the three domains of life
Characteristic
Bacteria
Eukarya
RNA
Single
Three types
polymerase
4 subunits (2α, β, β´) Multisubunit (12
subunits in yeast)
Mode of RNA Recognition and
Recognition and
polymerase
binding dependent binding dependent on
binding to
TATA-binding protein
on σ factor
promoter
(TBP)
Promoter core -35 box; -10 box
RNA polymerase II:
elements
TATA box,
Initiator element (INR),
Downstream promoter
element (DPE),
TFIIB recognition
element
Transcription Usually not
Transcription factor IIB
initiation
required
(TFIIB) and other
factor
factors
Other
Multiple
Multiple
transcription
factors
m7Gppp cap
Absent
Present
Polyadenyla- Long polyA tail
Long polyA tail
present
tion
absent
mRNA
Absent
Present
splicing
Archaea
Single
Multisubunit (12 subunits in Sulfolobus)
Recognition and
binding dependent on
TBP
TATA box,
TFB recognition
element (similar to
promoter elements for
RNA polymerase II)
TFIIB homologue
(TFB)
Eukaryotic and
bacterial-like factors
Absent
Long polyA tail
absent
Absent
As in most eukaryotic genes, the archaeal TATA box is localized about
30 bp from the transcription initiation start (11, 148, 165). Archaea contain
a homolog of eukaryotic TATA binding protein (TBP), which specifically
recognizes and binds to the TATA box in the promoter region before
loading the RNA polymerase (11, 165, 179). In eukaryotes, TBP is
essential for initiation of transcription in all three RNA polymerase systems
10
(185). All archaeal species with the exception of halophilic ones contain a
single TBP (165).
The TFB recognition element (BRE) is a binding site for transcription
factor B (TFB). The archaeal BRE consists of a minimum of two adenines
located around position -34 to -33 with respect to the transcription
initiation site (164, 165). Upon binding to the BRE, the TFB forms a
complex with the TBP that is necessary for the initiation of transcription.
In some euryarchaea there are multiple TFB proteins (165, 174).
In addition to TBP and TFB, archaea contain many other eukaryotic and
bacterial-like transcription factors (10, 11, 103) that are required for the
efficient regulation of archaeal transcription.
1.3.2 Translation. The translation process in archaea appears to have traits
from both bacteria and eukaryotes (Table 3).
Table 3. Translation in the three domains of life
Characteristic
rRNA
Ribosomes
(subunits)
Shine-Dalgarno
(SD)
Translation
initiation codons
Translation
factors
Initiator tRNA
m7Gppp cap
Bacteria
5S, 16S, 23S
70S (30S + 50S)
Present
Eukarya
5S, 5.8S, 18S, 28S
80S (40S + 60S),
70S in organelles
Absent
Archaea
5S, 16S, 23S
70S (30S + 50S)
Mainly AUG,
Other initiation
codons (GUG, UUG
etc.) also used to
minor extent
Multiple initiation,
elongation and
release factors
Formyl-methionine
Absent
Initiation essentially Mainly AUG,
restricted to AUG
Other initiation
codons (GUG, UUG
etc.) also used to
minor extent
Multiple initiation, Both eukaryal and
elongation and
bacterial-like factors
present.
release factors
Methionine
Methionine
Present
Absent
Present
SD-like sequences are present in many archaeal genes and are localized
3 - 10 nucleotides in front of the initiation codon (2, 11, 36). Recent
experimental studies with Sulfolobus solfataricus demonstrated that the
Shine-Dalgarno is essential for the initiation of translation, at least in
crenarchaea (27). A SD-independent initiation of translation could proceed,
but only after the artificial removal of the 5´ untranslated mRNA leader
region in front of the initiation codon (27). In archaeal genes that lack SD,
translation was suggested to start from the first initiation codon located
downstream of the transcription initiation start (36). The process of
11
translation initiation of such genes in Sulfolobus might be enhanced by yet
an unidentified sequence elements, located downstream of the initiation
codon, inside a coding sequence (175).
As in bacteria, archaeal ribosomes are composed of 30S and 50S
subunits. Archaeal rRNA further parallels bacterial rRNA since it also
consists of 16S, 23S, and 5S RNAs. Furthermore, archaea possess both
bacterial-like and eukaryotic-like factors (104) required for different stages
of the translation process.
1.3.3 Genome organization. The size range of an archaeal chromosome is
on average similar to that of bacteria: about 1.7 – 5 Mb. Mesophilic
archaea often contain megaplasmids (54). A 100 kb megaplasmid was
detected in Halobacterium sp. NRC-1 (133), and in species from the genus
Haloferax, the sizes of megaplasmids range from 130 kb to 620 kb (127).
In addition to different types of plasmids, archaea contain a spectrum of
different kinds of other genetic elements such as viruses, transposons and
insertion sequences (191, 194).
The DNA in mesophilic archaea is negatively supercoiled, and
hyperthermophilic archaea contain relaxed to positively supercoiled DNA
(23, 53, 73, 74, 111). The positive supercoiling was proposed to be created
by reverse gyrase, which introduces positive supercoiling into covalently
closed circular DNA (34, 53, 54, 111, 176). Several proteins including
histone-like proteins and histones (found only in euryarchaea) (60, 108,
143, 154, 195), were implicated to actively participate in structuring the
archaeal chromosome. Smc proteins, which are required for chromosome
condensation, segregation and variety of other aspects of chromosomal
maintenance within bacteria and eukaryotes (26, 75, 76, 79, 84, 85, 171)
together with topoisomerases, have been detected in archaea as well (12,
44, 53, 54, 111).
1.4 Cell cycle. All cells grow, duplicate their genomic DNA, and divide in
order to survive. The cell cycle has been divided into distinct stages called
G1, S, G2, and M in eukaryotes, and B, C, and D in bacteria (137). The
periods remain distinct and separate in eukaryotic cells, whereas in bacteria
the boundaries overlap when the growth rate is so fast that the generation
time is shorter than the time required to duplicate and segregate the
genomes (i.e. when C+D is longer than the doubling time) (137).
An enormous amount of data has been acquired for both the eukaryotic
and bacterial cell cycles. Yet, almost nothing was known about the cell
cycle features of archaea by the time I started my research. This was
mostly due to limitations in the genetic systems, lack of conditional-lethal
12
mutants and technical problems which made it difficult to apply flow
cytometry and microscopy to study these organisms.
Below, I present the major known features about archaeal replication,
partition and division before proceeding to the Present investigation and
Discussion describing the archaeal cell cycle.
1.4.1 DNA replication. In eukaryotes and bacteria, a large number of
replication genes have been identified and their products isolated and
characterized biochemically to provide a detailed description of this
process (4, 100, 181). In archaea, proteins essential in DNA replication
(Table 4) were mostly found to be homologs of eukaryotic equivalents (12,
13, 18, 22, 41, 92, 106).
Table 4. Replication in the three domains of life.
Function
Bacteria
Eukarya
Archaea
Origin
recognition
DnaA
Origin recognition
complex (ORC)
Orc1/ Cdc6
Replicative
Helicase
DnaB
Mcm
Single strand
DNA binding
protein
Clamp loader
Ssb
Minichromosome
maintenance proteins
(MCM)
Replication protein A
(RPA)
Rfc (2 subunits)
Sliding clamp
(processivity
factor)
Main replicative
DNA polymerase
DnaN
Replication factor C
(5 subunits)
Proliferating cell
nuclear antigen
(PCNA)
Family B
γ complex
Family C
Rpa
Pcna
Family B or D
(euryarchaea)
Family B
(crenarchaea)
DNA ligase,
ATP-dependent
DNA strand
DNA ligase,
DNA ligase,
ligation on
NAD-dependent
ATP-dependent
lagging strand
Processing and
DNA Pol I,
FEN 1,
FEN 1,
removal of
RNaseH
RNaseH
RNaseH
primers
The table includes proteins with either identified or predicted functions.
Very little is known about the number of origins of chromosomal
replication in different archaeal species. The GC skew analysis based on
the asymmetrical distribution of nucleotides or short oligomers on leading
13
and lagging DNA strands, and on the abrupt changes of such distributions
in regions close to the origin and terminus of replication, were applied to
search for origin of replication in archaeal chromosomes (62, 109, 150,
151, 153).
This analysis failed to demonstrate the presence of skew distribution in
M. jannaschii and Archaeoglobus fulgidus, suggesting that these species
may initiate replication of chromosome from a multiple origins (153).
Skew analysis suggested a presence of a putative single origin of
replication in euryarchaea such as Methanobacterium termoautotrophicum
and Pyrococcus horikoshii (109, 153). Using a combination of skew
analysis and the identification of an early replicating chromosomal segment
in synchronous cells, a single origin of replication was identified in
Pyrococcus abyssi (131).
In crenarchaea, the location and number of origins of replication remains
to be identified.
In all organisms, the initiation of chromosomal replication requires
binding of an initiation factor at the origin. Such function is performed in
bacteria by DnaA and in eukaryotes by proteins assembled as an origin
recognition complex (ORC) (4). Eukaryotic ORC consists of six subunits.
In archaea the presence of a homolog of the largest subunit of an eukaryotic
ORC, ORC1, was identified, in the chromosome of Pyrococcus furiosus
(92).
A homolog of an eukaryotic Cdc6 protein was detected in several
archaeal species. In eukaryotes CDC6 binds to ORC complex and
participate in loading helicases (MCM proteins), required to unwind the
double-stranded DNA. Both DNA binding and helicase activities were
experimentally demonstrated for archaeal Mcm protein (25, 94, 177).
Cdc6 protein also shows similarity at amino acid level to ORC1, what
may suggest similarity in functions between both proteins (92). Archaeal
Cdc6 might be involved in loading of helicases as well as function as an
initiator protein (13).
All DNA polymerases are grouped into a distinct families, referred as A,
B, C and X according to their sequence similarity and drug sensitivity (18,
22). Members of family B DNA polymerases are found both within
crenarchaea and euryarchaea (22, 92). In contrast to crenarchaea, which
contain multiple family B polymerases, euryarchaea possess only one
equivalent from the same group (21, 22, 43, 92). Similar to their eukaryotic
counterparts several members of archaeal family B polymerases are
sensitive to aphidicolin (22, 92). Some members of archaeal family B
14
polymerases contain protein introns, referred to as inteins (19, 22, 78, 92,
134, 144, 190).
Recently a new class of polymerases, denoted as family D, was
discovered in euryarchaea (22). In P. furiosus, the DNA polymerase from
this family referred to as Pol D, was found to be a heterodimeric enzyme
constituted of two subunits: large DP 2 subunit with catalytic activity, and
small DP 1 subunit which shows significant similarity to the eukaryotic
polα, polδ, polε polymerases (22, 92, 117).
Usually, different types of DNA polymerases are assigned to replicative
and/or repair synthesis and recombination. The exact role of archaeal DNA
polymerases is not entirely understood (13, 18, 22, 92) and needs to be
investigated further.
A processivity factor stimulates the replicase activity of DNA
polymerase (4, 92, 138, 181). It encircles the DNA strands and through
interaction with DNA polymerase ensures the connection of the replisome
with DNA template during replication (4, 138). Archaeal processivity
factor was found to be a homolog of an eukaryotic protein called
proliferating cell nuclear antigen (PCNA) (12, 13, 22, 41, 92). In all
completely sequenced euryarchaea only one homolog have been identified
(19, 22, 91, 92, 96, 163), while in crenarchaea multiple Pcna were detected
(22, 32, 90, 92). In both Sulfolobus solfataricus and Pyrococcus furiosus,
Pcna was experimentally shown to stimulate DNA polymerase activity (20,
32).
Pcna is loaded onto a DNA template by a clamp loader, which in archaea
is a homolog of the eukaryotic replication factor C (RFC) (12, 13, 19, 41,
90, 91, 92, 96, 163). In contrast to eukaryotic RFC, which consists of five
subunits, archaeal Rfc is composed of only of two subunits (22, 41, 92, 93).
The homologies found between eukaryotic and archaeal replication
proteins strongly suggest that replication machinery is more reminiscent
of its eukaryotic counterpart than the bacterial one.
1.4.2 Chromosome partitioning. The sequences reminiscent of
centromere-like elements and homologs of ParA and ParB proteins,
involved in plasmid and chromosome partitioning in bacterial cells (59,
126, 128, 158, 159, 184), were also identified in archaea (12, 24, 59, 126).
Archaeal centromere-like elements have been implicated to be essential for
plasmid stability (13, 127, 160).
1.4.3 Cell division. The FtsZ protein is present in almost all bacteria,
archaea, and chloroplasts studied so far (115, 152). Recently, FtsZ was also
identified in mitochondria (9, 48, 124). FtsZ polymerizes into a ring
15
localized at the septum during the division process, and was suggested to
provide an infrastructure for other cell division proteins (15, 16, 46, 116,
120, 152). FtsZ was also proposed to participate in the cell constriction
process through conformational changes of its protofilaments (114).
The FtsZ protein of the euryarchaeon M. jannaschii (19) showed a high
level of similarity in tertiary structure to eukaryotic tubulin (47, 113, 136).
Tubulin and FtsZ constitute a separate GTPase family with a distinct GTP
binding motif: GGGTG(T/S)G (33, 135). The similarities between tubulin
and FtsZ suggest that FtsZ might be an ancestor of eukaryotic tubulin (45,
46, 49, 120).
In addition to the FtsZ, euryarchaea contain the homolog of bacterial
MinD protein (12, 58, 152). In bacteria MinD is required for proper
positioning of the septum during the process of cell division (152). Both
MinD and ParA belong to a superfamily of ATPases, which consists of
many proteins, including those essential for plasmid and chromosome
partitioning (98). Database searches using archaeal homologs to bacterial
Soj protein, which is required during the sporulation and for proper plasmid
and chromosome partitioning (172), recognized MinD proteins and vice
versa (12), what may suggest partially overlapping functions of MinD and
Soj proteins in archaeal cells (12).
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2. Present investigation
Part I: Cell cycle characterization of Sulfolobus
(papers I and II)
1.1 The use of Sulfolobus in cell cycle studies. The idea of archaea
constituting a separate domain of life generated significant interest in
studying the cellular and molecular properties of this group of organisms.
However, the archaeal cell cycle remained uncharacterized when I focused
my research interests on these organisms (see Introduction).
The hypothesis of crenarchaea being relatives of eukaryotic organisms
(149) had an influence on my decision to leave my studies of E. coli in
order to analyze the archaeal cell cycle. I was interested in understanding
to what extent the archaeal cell cycle displayed unique characteristics, and
which properties were reminiscent of those found in bacterial and
eukaryotic cell cycles.
By testing a variety of archaeal species, I found that crenarchaea from
the genus Sulfolobus were relatively easy to grow, since they did not
require strict anaerobic conditions and high pressure for cultivation as
many other archaea do. Moreover, and contrary to halophilic archaea,
Sulfolobus cells were relatively easy to keep intact during the experiments.
For these reasons, I chose the crenarchaeal species Sulfolobus
acidocaldarius and Sulfolobus solfataricus as my primary subjects for the
first cell cycle analyses of archaea and hyperthermophilic organisms.
Before describing my experimental work, I will briefly outline the major
genetic and physiological features of the genus Sulfolobus.
1.2 Properties of genus Sulfolobus. A characteristic feature of the
members of the genus Sulfolobus (17) is that they are thermoacidophiles
(169, 170). The optimum growth temperature and pH are about 80°C and
pH 3, respectively (64, 169). The doubling time varies between 4 - 8 hours.
The cells are surrounded with a glycoprotein S layer (64, 88, 147, 183).
The sizes of the Sulfolobus genomes are about 3.0 Mb (8), and the
genomic base composition is around 37% GC (63, 157). Sulfolobus cells
possess reverse gyrase (95), which introduces positive supercoiling into
covalently closed circular DNA (53, 54, 55, 95), and is suggested to be
essential for stable maintenance of the chromosome at high temperatures
(53).
1.3 Introduction to flow cytometry. The flow cytometry technique has
been successfully applied in cell cycle studies of both eukaryotic and
17
bacterial organisms (31, 161, 162, 167). In flow cytometry, cells fixed and
stained with DNA-specific fluorescent dyes pass through a measuring
window at high speed. The light scatter (a measure of cell size) and the
fluorescence (reflecting DNA content) for each cell is recorded, registered
and analyzed by the computer software. DNA content and cell size in a
population can then be represented as a two- or three-dimensional plot
(Fig. 5, paper I). From DNA content distribution, the proportion of cells in
different stages of the cell cycle can be measured and the length of the
different cell cycle periods can be calculated.
1.4 Control experiments. We used flow cytometry to analyze the DNA
content and cell size distributions of Sulfolobus species. In parallel, I
studied the structure and distribution of their chromosomes within archaeal
cells, using phase-fluorescence microscopy, after staining with DNAspecific dyes.
Since this was the first time such analyses were performed with archaea,
we designed control experiments to ensure the accuracy and reliability of
our studies (Fig. 2, paper I). Treatment of cells with DNase resulted in total
loss of fluorescence signal, without any affect on the light scatter
distribution. Treatment with proteinase K did not significantly influence
the DNA content distribution of the population, though there was a
reduction in the light scatter signal. Microscopy revealed that ethanol fixed
cells followed by proteinase K treatment became transparent (not shown).
The fluorescence signals, however, were still maintained by the cells (not
shown). Hence, proteinase treatment did not significantly alter the integrity
of the S layer but most probably had an influence on the content of internal
cellular proteins. There was almost no effect on DNA content and cell size
distributions after treatment with RNase. Combined treatment with RNase
A and proteinase K affected the light scatter distribution, but not the shape
of the DNA content distribution. The fluorescence signals were slightly
reduced upon such treatment. When DNA content and nucleoid structure
in cells treated by Dnase, Rnase A, and proteinase K were analyzed by
microscopy (Fig 1, paper II), the results were essentially the same as those
obtained by flow cytometry.
In conclusion, these experiments demonstrated that the fluorescence
signals corresponded to DNA. Hence these techniques could be used for
the visualization of DNA and determination of DNA content.
1.5 Genome sizes and numbers. Estimates of genome sizes of Sulfolobus
species have been published previously. The genome size of S. solfataricus
(DSM 1617) was estimated to be 3.1 Mb (157), while S. acidocaldarius 7
18
had a genome size of 2.7 Mb (97). We used flow cytometry to compare
with the available data, genome sizes of Sulfolobus species analyzed in this
study.
In agreement with published data, the genome size of S. solfataricus
(DSM 1616) was found to be significantly larger than that of S.
acidocaldarius (DSM 639) both in the stationary and exponential phases.
This difference was irrespective of the stains: 4-6-Diamidino-2phenylindole (DAPI), Hoechst (Fig. 3, paper I) or mixture of mithramycin
and ethidium bromide (MEB) used in the analysis (paper I).
To determine the DNA content of Sulfolobus with regard to the size and
number of chromosomes, E. coli cells with known integral number of
chromosomes were used as a reference scale (not shown). Using this scale,
the DNA content from exponential phase cells, corresponding to the first
and second peaks (Fig. 2 and paper I) were found to reflect one and two
genome equivalents, respectively. Cells from the stationary phase were
found to contain almost exclusively two genome equivalents (Fig. 2 and
paper I).
When non-intercalating drugs such as DAPI (28) were used, the genome
sizes of both S. acidocaldarius and S. solfataricus were estimated as less
than 15% from the predicted values. The flow cytometry analysis using
MEB resulted in an underestimation of the Sulfolobus genome size.
Furthermore, flow histograms of MEB-stained DNA showed a slightly
displaced location of the 2-genome peak in stationary phase (Fig. 6, paper
I), which was not apparent when non-intercalating drugs were used (Fig.
2). Since the GC content is 37% in Sulfolobus and 50% in E. coli,
underestimation of the size of Sulfolobus genome using the MEB stain
could be explained by the preferential binding of mithramycin to GC-rich
regions (28). In addition, as DNA is negatively supercoiled in bacteria, and
in a relaxed to positively supercoiled state in thermophilic archaea (53, 73,
74, 111), superhelicity could as well affect the DNA-binding properties of
different drugs.
In conclusion, the number of Sulfolobus genomes alternates between one
and two in the exponential phase, and upon entering stationary phase
Sulfolobus cells contain two genome equivalents.
Furthermore, this experiment provided an easy way to estimate relative
sizes of archaeal genomes. Exact determinations of genome size require
comparisons between species with the same, or closely similar, GC content
and chromosomal structure.
1.6 The cell cycle. The DNA content distributions obtained from the
flow cytometry analyses (Fig. 2) were used to calculate the lengths of the
19
different cell cycle periods (Fig. 9, Discussion) with the help of computer
simulations. The amount of cells in the B, C and D periods was estimated
and converted to relative lengths of the cell cycle periods as described in
paper I. The DNA content distribution in an exponentially growing culture
of S. acidocaldarius was found to be slightly dependent on the optical
density at the time of sampling. Therefore a range of values is given below
for the length of the cell cycle periods in this species. The B period in both
Sulfolobus species occupied not more that 5% of the cell cycle. The length
of the C period was calculated to be 37% (157 min) for S. solfataricus, and
ranged between 26 - 40% (55 to 85 min) for S. acidocaldarius. The
postreplication period was estimated to be 60% in S. solfataricus and
between 58 - 72% (124 - 153 min) for S. acidocaldarius.
The flow cytometry analysis indicated that most of the cells contained
two genome equivalents, while microscopy demonstrated that in a majority
of the cells a single fluorescence focus was present (Fig. 2).
Fig. 2. DNA content distributions and nucleoid visualization in S. acidocaldarius cells
from exponentially growing (upper panels) and stationary phase (lower panels)
cultures. The cells were fixed in ethanol and stained with DAPI before analysis. Arrows
indicate cells with two separated nucleoids, as well as dividing cells. The bar is equal
to 2 µm.
20
The number of cells in the population with two fully separated nucleoids
was estimated by microscopy to be about 4% for S. solfataricus and 5% for
S. acidocaldarius.
Thus, most of the fluorescence foci should contain two unseparated
genomes. This feature indicated that G2 stage (see section 1.4,
Introduction) was a part of the Sulfolobus cell cycle. This period was
estimated to occupy about 50% of the cell cycle.
The T period (the time from the initiation of visible septum constriction
to cell separation (72)) was estimated by calculating the percentage of cells
in the population with visible constriction. This period was found to be 4%
(19 min) and 5% (13 min) for S. solfataricus and S. acidocaldarius,
respectively.
In conclusion, our analysis showed, that a short B and a long
postreplication period characterizes the cell cycles of both S.
acidocaldarius and S. solfataricus. In addition, the G2-like period, which
constitutes a part of the postreplication stage, was found to occupy a large
fraction of the Sulfolobus cell cycle (paper I and II).
1.7 Nucleoids of Sulfolobus cells. Studies of chromosome distribution and
structure have not been performed previously for any archaeon or
hyperthermophile. Therefore, nucleoid structure from both the exponential
and stationary phases of Sulfolobus cells was analyzed either in ethanolfixed or living cells (Fig. 3).
A
B
C
D
Fig. 3. Nucleoid structure in the exponential and stationary phases of S. acidocaldarius
cells. A and B, staining with DAPI of living cells from exponential (A) or stationary
phase (B). C, DAPI staining of ethanol-fixed mixed population containing exponential
and stationary phase cells. D, phase contrast illumination of C.
21
Both approaches showed that the structure of the Sulfolobus nucleoid
differs between cells analyzed in the exponential and stationary phases
(paper II).
1.7.1 Nucleoid structure in exponential and stationary phases. The
stationary phase nucleoid was represented by a single fluorescence focus,
which covered most of the cell interior and had few distinguishable
features as compared to those from the exponential phase (Figs. 2 and 3).
The structure of the nucleoid was not affected by the type of treatment and
was similar in both ethanol-fixed and living cells (Fig. 3).
Furthermore, the transparent appearance of the cells from the stationary
phase (Fig. 3), after ethanol fixation, suggested differences in cellular
properties as compared to exponentially growing cells.
1.7.2 Nucleoid distribution in exponential phase. In order to unravel the
pattern of chromosome partitioning, a search for cells in different stages of
nucleoid processing was performed for both Sulfolobus species (Fig. 4).
A
B
C
D
E
SA
SS
Fig. 4. Nucleoid structure and distribution in Sulfolobus cells. SA, S. acidocaldarius,
SS, S. solfataricus. Column A, cells with a single nucleoid. B, cells with two nucleoids
aligned in parallel. C, cells with separated nucleoids. D, cells with separated nucleoids
and visible constriction. E, complex nucleoid structures.
The majority of the cells in both S. acidocaldarius and S. solfataricus
contained a single fluorescence focus, localized in the middle or at the side
of the cell (column A). A small proportion of the population contained two
nucleoids, displayed as clear foci or arcs within the cells. Column B shows
cells in which the two nucleoids were aligned in parallel. Column C
indicates cells containing fully separated nucleoids located at opposite
sides of the cell. This nucleoid localization pattern was preserved during
22
cell division, as shown in column D. Examples of aberrant nucleoid
structures are demonstrated in column E.
1.8 Morphological features. An unusual morphological feature which we
called budding was observed in S. solfataricus and S. acidocaldarius cells
under particular conditions. Budding was sporadically present in cells from
the exponential phase, analyzed either directly under the microscope or
after spreading on agar containing 10 mM Tris-HCl. A high number of
buds was observed when cells from a liquid culture were spread onto 1%
gelrite or agar in water (Fig. 5). Pronounced budding was also detected in
S. acidocaldarius after prolonged incubation with DAPI (not shown).
A
B
A
S. acidocaldarius
B
S. solfataricus
Fig. 5. Examples of budding Sulfolobus cells. Cells from exponentially growing
cultures were observed by microscopy after spreading on 1% gelrite in water (column
A) or 1% agar in water (column B). The upper row represents phase contrast
illumination, while the middle and bottom rows show nucleoids in budding cells after
the live cells were stained with DAPI for 60 min (followed by the same treatment as in
row 1).
23
Part II: Conditional-lethal mutants of S. acidocaldarius
(paper III)
2.1 Isolation of mutants. A major step towards further analysis of the
Sulfolobus cell cycle was achieved with the first isolation and
characterization of conditional-lethal mutants of S. acidocaldarius.
Conditional-lethal mutants are required to study the function of essential
genes (86, 119). In a conditional-lethal mutant, substitutions in the amino
acid sequence of a protein result from the treatment of the cells with a
mutagen. This affects proper folding of the mutant protein, and alters its
activity under conditions which have no influence on the corresponding
properties of the wild-type protein (86, 119). During exposure to the nonpermissive condition, the mutated protein can lose its activity completely
or partially, depending upon the substitution.
The isolation of conditional-lethal mutants of S. acidocaldarius was
accomplished by chemically mutagenizing the cells of strain DG64 with
nitrosoguanidine. Nitrosoguanidine is a mutagen known cause transitions,
transversions, and -1 frameshift mutations (166).
Mutants of S. acidocaldarius, which failed to grow on solid media after
a shift from 70°C to 83°C, were isolated. The non-permissive temperature
was close to the optimal growth temperature for wild-type Sulfolobus cells.
At permissive temperature, the DNA content distributions, nucleoid
structure, cell morphology and growth properties of the mutants were
found to be similar to those observed for the DG64 parental strain (paper
III, and Fig. 6). After the shift to the non-permissive temperature, growth
and viability of the parent strain DG 64 was not affected (Fig. 3, paper III).
Flow cytometry experiments showed that the percentage of cells in the B
and C periods in this strain decreased slightly after the shift, but the shape
of the DNA content distribution was similar to that at the permissive
temperature (Fig. 2, paper III). Furthermore, nucleoid structure of DG64
after the shift was similar to that observed at 70°C (Fig. 2, paper III).
Since the flow cytometry and microscopy parameters of the DG64 parent
strain remained similar at both temperatures, differences in the phenotypes
of the various mutants were likely to have resulted from mutations.
2.2 Phenotypic reversion. The observed phenotypes of the conditionallethal mutants of S. acidocaldarius could be due to mutations in several
genes.
Reversion of the mutations, which completely or partially restore the
wild-type phenotype of the cell, can occur through a suppressor mutation
24
introduced either outside the gene (extragenic suppression) or in the same
gene (intragenic suppression). Alternatively, mutations can revert by a true
reversion which restores the wild-type sequence of the gene (86, 119).
Single mutations will revert at much higher frequencies compared with
multiple mutations. For example, if the reversion frequency of a point
mutation in gene of E. coli was about 10-8 revertants per generation (119),
than the reversion frequency of a double mutation would be at the level of
10-16.
The values of reversion frequency observed in our collection of
conditional-lethal mutants, were about 10-6 to 10-7 (Table 5), and were
close to the frequencies of spontaneous pyrE and pyrF mutants of S.
acidocaldarius (82). This is much higher than would be predicted by two
or more independent reversions. Hence, we assumed that only one gene
was affected in most of the mutants.
2.3 Classes of mutants. A final collection of 34 conditional-lethal mutants
grown in liquid medium at 70°C were analyzed for alterations in DNA
content, nucleoid structure and cell size distributions, after a shift to the
non-permissive temperature of 81°C. Flow cytometry and microscopy
analysis, together with measurements of growth and viability, were used to
divide the mutants into five classes (Table 5).
2.3.1 Class I. Mutants from this class were characterized by arrest in the
post-replication stage. Class I was further subdivided into subgroups Ia and
Ib (Table 5).
Class Ia was characterized by the uncoupling of cell growth from
replication, as demonstrated by mutant DG132. A continuous increase of
cell size without concomitant cell division, after a shift to the nonpermissive temperature, was observed in both flow cytometry and
microscopy analysis (Fig. 6). There was also a decrease in the growth rate
accompanied by a slight decrease in cell viability (Fig. 3, paper III).
Arrest at the post-replication stage along with the absence of cells with
one genome equivalent observed after the shift (Fig. 6), suggested that
ongoing replication was allowed to finish but that no new initiation of
replication occurred.
Continuous changes in nucleoid structure were evident, especially at late
time points in the experiment. The nucleoids became highly unstructured
and were usually localized to the side of the cells (Fig. 6). In addition, cells
with reduced fluorescence signals, probably representing DNA
degradation, were detected.
25
In mutants from class Ib, represented by DG146, replication was again
blocked about 3 hours after the shift, and the cells were arrested in the
post-replicative period for the rest of the experiment (Fig. 6). In contrast to
DG132, little or no cellular growth occurred in DG146, as indicated by the
largely unaffected light scatter distribution (Fig. 6) and growth
measurements (Fig. 3, paper III). Cells of DG146 were viable for more
than 30 hours after the shift. A dramatic drop in cell viability (Fig. 3, paper
III), and DNA degradation (Fig. 6), was observed 48 hours after the shift to
nonpermissive temperature.
2.3.2 Class II. Mutants from classes II to V (Table 5) were not arrested in
specific cell-cycle periods after the shift to non-permissive temperature.
Mutants from class II were characterized by a progressive decrease in
growth followed by loss of cell viability (Fig. 3, paper III), which
coincided with DNA degradation inside the cells (Fig. 6).
2.3.3 Class III. This class (Table 5) is represented by DG134 and is
characterized by an increase in both DNA content and cell size after the
shift to non-permissive temperature. The growth and viability were
undisturbed for at least 10 hours after the shift and were slightly reduced at
later time points of the experiment (Fig. 3, paper III).
Flow cytometry revealed that there was no arrest in the post-replication
stage after the shift. Instead, the DNA content and cell sizes continued to
increase (Fig. 6). The increase in DNA content was evident at late time
points as peaks corresponding to up to 5 genome equivalents emerged in
the population (Fig. 6). In addition, a large broadening of the light scatter
distribution was observed (Fig. 6) what suggests that the growth of some
cells in the population was inhibited at different time points of the
experiment.
Although some DG134 cells with unstructured nucleoids similar to
those observed in DG132 were detected, the presence of many cells with
clearly separated nucleoids at late time points during the experiment (Fig.
6) provided evidence for an actively operating partitioning system at
nonpermissive temperature in this mutant.
In addition, transparent cells, most probably resulting from the loss of
cellular integrity, were present at a low frequency within the population.
2.3.4 Class IV. The shift to non-permissive temperature did not result in
pronounced changes in the growth rate or morphology of the mutants from
this class. This class was characterized by the presence of cells with more
than 2 genome equivalents after overnight incubation at 81°C (Table 5).
26
2.3.5 Class V. Mutants from this category did not show any detectable
phenotype within the time used for our analysis (usually more than 24
hours). However, the conditional-lethal phenotype was evident upon
extended incubation on plates and liquid media (not shown).
Fig. 6. Examples of conditional-lethal mutants with interesting cell cycle phenotypes.
Bar equal to 2 µm.
27
Table 5. Phenotypic effects after the shift to non-permissive temperature*.
Mutant class
Ia
Ib
4
5
Number of isolates
132
146
Representative strain (DG number)
Flow cytometry
1. Cell cycle arrest in D period
3
3
2. Cells with >2 chromosome equivalents
(o/n)
3. Cell size increase
+
4. DNA degradation
o/n
o/n
Mass increase after shift
1.Significant decrease as compared to wt
+
+
+
2. Rapid growth arrest†
1.2 x 10-7
5.8 x 10-7
Reversion frequency
(S.D.)
(1.4 x 10-7;
(5.9 x 10-7;
n = 7)
n = 5)
IIa
5
149
IIb
3
155
III
4
134
IV
3
117
10
(+)
6
6
+
-
(o/n)
-
+
-
+
(+)
1.3 x 10-7
(8 x 10-8;
n = 6)
+
8.2 x 10-6
(1x 10-5;
n = 3)
-
*Designations: +, phenotype present; -, phenotype absent; 3, 6, 10, o/n, hours post-shift at which the phenotype first
became evident. Parentheses indicate an intermediate phenotype.
† OD increase corresponding to less than one mass doubling after the temperature shift to 81°C. Parentheses indicate a
mass increase corresponding to between one and two mass doublings.
Part III: The Haloferax mediterranei ftsZ gene
(paper IV)
3.1 The idea behind the project. The ftsZ gene is important for cell
division in prokaryotic organisms (see Introduction). The interesting
morphological properties of halophilic archaeon H. mediterranei prompted
us to initialize an investigation of the cell division process by analyzing the
structural and functional properties of the ftsZ gene and protein in this
organism.
3.2 The H. mediterranei ftsZ gene. Preliminary experiments demonstrated
that primers directed against the Haloferax volcanii ftsZ gene (182) also
hybridized to H. mediterranei DNA. Hence, about 20 different primers
covering the entire ftsZ gene region of H. volcanii (GenBank Accession No.
U37584) (182) were designed. The ftsZ gene region from H. mediterranei
(1983 bp) was then amplified and sequenced (GenBank Accession No.
AF196833).
The ftsZ gene was localized within the sequenced region, the average GC
content, transcription and translation regulatory elements, and other
properties were determined (Table 6).
Table 6. The main features of the ftsZ gene of H. mediterranei
(GenBank accession No. AF196833)
Gene
Type
Length
Base count
Average GC content
TATA box (positions with reference to the
No. AF196833 nucleotide sequence)
Shine-Dalgarno
Initiation codon
Termination codon
Length of the FtsZ protein
Identity with the entire H. volcanii ftsZ gene
ftsZ
paralog 1
1092 nt
225 A, 326 C, 341 G, 200 T
61%
5´-TAATTA-3´ (410-415)
Identity with the entire H. volcanii FtsZ
protein
GTP binding motif
Z-ring (FtsZ ring) at division site
ftsZ flanking gene at 5'-end
ftsZ flanking genes at 3'-end
97%
5´-GAGG-3´ (475-478)
5´-ATG-3´ (487-489)
5´-TAG-3´ (1576-1578)
363 aa
90%
GGGTGTG (868-888)
present
orf X
secE, nusG
29
Phylogenetic analyses of ftsZ genes revealed that they branch into two
separate clusters, denoted as paralog 1 and paralog 2 (49). The high
sequence identity at both the nucleotide and amino acid levels between the
H. mediterranei and H. volcanii ftsZ genes and proteins automatically
grouped ftsZ of H. mediterranei as a paralog 1 (Table 6).
3.3 Transcription initiation signals. The transcription initiation start site
was mapped using primer extension analysis (Fig. 2, paper IV). The
putative TATA box was found to be localized about 30 nucleotides
upstream of the transcriptional start site for the ftsZ gene (Table 6).
3.4 Shine-Dalgarno sequence. There are at least two potential SD
sequences (SD) for the ftsZ gene in both H. mediterranei and H. volcanii.
In both strains, one SD was located 3 bp and the other one 9 bp upstream of
the 2nd putative translation initiation codon of the ftsZ gene (Fig. 1, paper
IV). In H. volcanii, an additional potential SD sequence was localized 15
bp upstream of the 2nd initiation start codon.
In H. mediterranei, the distance between one of the SD sequences and the
initiation codon was the same as that for the nusG gene in the same region.
This sequence was therefore suggested as the probable recognition site for
translation.
3.5 Frameplot analysis. In species with high chromosomal GC content,
there is an uneven distribution of the GC content within the codons. The
average GC content is the highest at the third position and the lowest at the
second position (14). This non-random GC distribution allows for the
determination of coding regions (14, 81). In addition, translational start and
stop codons are indicated by the program. These are usually located on the
border between the random and non-random GC distribution.
3.5.1 Translation initiation starts. Considering the results from the
examination of the nucleotide sequence, there could be three possible
translation initiation start sites (ATG) for the ftsZ gene in both H.
mediterranei and H. volcanii. The frameplot analysis was used, in order to
differentiate between these alternatives (Fig. 7). The analysis showed that
the 1st codon was located on the ascending part of the GC distribution, the
2nd on the border between random and non-random distribution and the 3rd
well inside the coding sequence (Fig. 7) and hence rejected as a potential
start site. The presence of the SD element in front of the 2nd ATG codon
30
was used as evidence that this codon was the preferred translation initiation
codon for the ftsZ gene in both Haloferax species.
Fig. 7. Frameplot analysis of the ftsZ gene region of H. mediterranei (GenBank
Accession No. AF196833) and H. volcanii (GenBank Accession No. U37584). The
relative GC content in different codon positions is shown in a graphical representation
with open reading frames indicated above the graphs. The possible translation initiation
codons and their orientation are indicated by arrowheads (<, >). Vertical bars indicate
the possible stop codons. The putative translation initiation and stop codons for the ftsZ
31
gene, as well as for other genes from the analyzed region are indicated below each
graph.
3.5.2 GTP binding motif of the FtsZ protein. The first and the second
nucleotide in all glycine and threonine codons are GG and AC, respectively.
Since the GTP binding motif of the FtsZ protein (Table 6), consists of
71.4% glycine and 28.6% threonine, it could be directly identified by
frameplot analysis as a pronounced increase in the average GC content at
the second position within the codons (Fig. 7).
3.5.3 Conserved gene order in the ftsZ gene region. Frameplot and
database analyses revealed the existence of conserved ORFs both upstream
and downstream of the ftsZ gene (Fig. 7 and Table 6). The analyses
demonstrated the presence of the orfX gene upstream of the 5´-end,
conserved only within the genus Haloferax. The same analyses showed a
conserved cluster of secE and nusG genes located downstream of the ftsZ
gene. This cluster, together with downstream ribosomal genes, is preserved
in almost all archaeal and bacterial organisms (49, 180).
3.6 Functional conservation of the FtsZ protein. The extensive
identity at the amino acid level among the Haloferax FtsZ proteins (Table
6) prompted us to perform immunolocalization studies of the FtsZ protein
in paraformaldehyde-fixed H. mediterranei cells, using an antibody
directed against the H. volcanii FtsZ (Fig. 8).
In H. mediterranei, in most of the non-constricting cells and in cells
with asymmetric constrictions, any formation of the FtsZ ring (Z-ring) was
not detected (row F). Some cells contained a fluorescent half-band (row D)
probably a Z-ring under construction, although a poor access of antibody
in such cells cannot be excluded. In most cases, for H. mediterranei (row
A, B and C) as well as for H. volcanii (row G), the FtsZ protein was
visualized as a fluorescent band across the cells. The demonstration of a
clear ring structure was possible for a few cells (row A).
32
Fig. 8. Examples of immunolocalization of the FtsZ protein in H. mediterranei and
H. volcanii. Cells from exponentially growing culture of H. mediterranei and H.
volcanii were chemically fixed in paraformaldehyde, and after the immunolocalization
of the FtsZ protein were performed, as described in paper IV. Bar corresponds to 2 µm.
33
3. Discussion
Part I: Cell cycle analysis of Sulfolobus
(papers I, II and III)
1.1 Introduction. The combination of flow cytometry and microscopy has
been successful in cell cycle studies of bacteria and eukaryotes (see section
1.3, Present investigation), and the work presented in this thesis
demonstrates that this approach could also be used for the first cell cycle
analysis of archaea.
This study revealed interesting characteristics of the Sulfolobus cell
cycle (Fig. 9), including features reminiscent of those observed in both
bacteria and eukaryotes. Below, I will discuss properties of the cell cycle,
nucleoid organization, different morphological features, and the use of
conditional-lethal mutants.
Fig. 9. The cell cycle of Sulfolobus. A, relative lengths of the cell cycle periods is
indicated in the pie graph. Cells are indicated as circles outside the pie graph. Cells with
the striped area are in the replicative period (C period), while cells with filled areas are
either in prereplicative (B period) or postreplicative period. Postreplicative period
consists of G2, Partition and Division (G2, Par and D) stages. B, DNA content
distribution obtained by flow cytometry analysis of exponentially growing culture.
34
1.2 The B period. The small amount of time occupied by the B period
during the cell cycle (about 5%) indicates that the replication machinery is
competent for initiation early after cell division. As the nucleoids are
localized to opposite cell halves during the division period (Fig. 4 column
D), the B period could be devoted to structural changes and physical
movement of the chromosome or origin of replication to a particular place
in the cell. Physical movement of the origin of replication to a place
containing components of the replication machinery has been shown in
bacteria (107, 112), and may be required for triggering the initiation of
replication in Sulfolobus cells.
1.3 The C period. The genome of Sulfolobus species is about 40% smaller
as compared to E. coli, but the length of DNA replication period was found
to be similar to that of E. coli growing within the same range of doubling
times.
The growth temperature of Sulfolobus cells is above the melting point of
their DNA. At high temperature, there is a pronounced deamination of
cytosine compared to mesophilic growth conditions (61). Since the
mutation rate in Sulfolobus living in extreme environments corresponds to
the values observed for E. coli (82), Sulfolobus cells may have developed a
DNA repair system (66) able to cope with the denaturation and
thermodegradation of DNA. Archaeal DNA polymerases were shown to
recognize uracil generated as an effect of the deamination of cytosine and
to stop in front of such lesions until they are repaired (61, 105). Frequent
stops of the replisome caused by the need for removal of DNA lesions by a
DNA repair system could therefore result in an increased length of the C
period.
The differences in the length of the C period could also be due to other
features, such as differences in the replication machineries or differences in
chromosome structure between archaea and bacteria. Very little is known
about the regulation of Sulfolobus DNA replication. By taking into
consideration the difference in genome sizes between E. coli and
Sulfolobus, a reasonable assumption is that there is a single origin of
replication. Multiple origins would decrease the time required for
replication of the Sulfolobus chromosome and shorten the length of DNA
replication period.
1.4 The G2 period. Analysis indicating a low proportion of cells with two
segregated genomes in the population suggests a considerable time delay
between termination of DNA replication and full chromosome separation.
Thus, a period reminiscent of the G2 stage in the eukaryotic cell cycle (129,
35
137) is proposed to be present in Sulfolobus cell cycle. The G2 period
constituted about 90% of the post-replication period.
The low proportion of cells with fully separated nucleoids in Sulfolobus
could suggest that the chromosomes may remained connected after the
completion of replication, as was postulated for the eukaryotic G2 phase
(129). During the long G2 stage, possible DNA damage could be repaired
through recombination between the two copies of Sulfolobus chromosome.
The use of multiple copies of the chromosome, and homologous
recombination as a defense mechanism against lethal effects of the
environment was suggested for the bacterium Deinococcus radiodurans,
which has an extraordinary capacity to withstand high levels of damaging
agents such as ionizing or UV radiation (6, 7, 29, 125). Similar systems
protecting the chromosome from the lethal effects of high temperature
might operate in Sulfolobus, although this must be experimentally proven.
The reason for the presence of two genome equivalents in the G2 period
might also be the maintenance of a second copy without the need for
recombination. Such a strategy might assure survival of the cell in
extremophilic conditions, if damage would occur in one of the copies of the
genome.
1.5 DNA partition. A rearranged structure of the Sulfolobus nucleoid
was observed in cells, which had entered the partition stage (Fig. 4, column
B). Paired nucleoids were highly condensed, and their alignment was
reminiscent of that observed for eukaryotic chromosomes during mitosis.
Such similarities may indicate that certain stages of the partition process in
Sulfolobus could be similar to that observed during eukaryotic mitosis.
In contrast to slowly growing bacteria in which chromosome partitioning
may be a gradual process occurring during replication (189), Sulfolobus
cells appeared to partition their chromosomes during a limited time period
of about 5% of the cell cycle, suggesting rapid movement towards the
opposite halves of the cell.
Recent studies showed the presence of nucleotide repeats in both
Sulfolobus chromosome (24) and Sulfolobus pNOB8 plasmid (160). Such
repeats are implicated in process of chromosome and plasmid partitioning
in prokaryotes (126), and could possibly represent archaeal equivalent of
centromere-like element (13, 24).
The genes encoding putative ParA and ParB proteins were found in the
Sulfolobus pNOB8 plasmid (12, 13, 24, 160). These proteins are homologs
of sop/par genes involved in chromosome and plasmid partitioning in
bacterial cells (59, 128). Par B protein binds to centromere-like element
36
and participates in partitioning by utilizing the energy from the ATP
hydrolysis, which is provided by ParA (59, 128, 159).
Nucleotide repeats identified in the Sulfolobus chromosome may
constitute binding sites for protein(s) actively participating in process of
chromosome partitioning, in a manner similar to that, described for a
bacterial Par A and ParB proteins.
Under physiological conditions, initiation of chromosomal replication in
Sulfolobus is dependent upon the completion of both chromosome
partitioning and cell division (paper I and (77)). Such coupling can be
destroyed in the conditional-lethal mutants of S. acidocaldarius (e.g.
DG134), in such a way that both chromosome replication and partitioning
remain active in the absence of cell division (Fig. 6 and paper III). Results
from experiments with another mutant DG132 showed that in the absence
of chromosome partitioning, DNA replication is inhibited as well (Fig. 6
and paper III). Hence, these results together indicate that, in Sulfolobus
cells chromosome partitioning but not division is the process essential for
the initiation of chromosomal replication (paper III).
1.6 Cell Division. The similarity between the T period (the time from the
initiation of visible septum constriction to separation of the cells (72)) in
Sulfolobus and slowly growing bacteria (paper I) might indicate that the
septation process operates through a bacterial-type mechanism. However,
there is no FtsZ protein in crenarchaea and so far there is no information
about the presence of any other cell division proteins in Sulfolobus.
Furthermore, the only experimental evidence so far, for the mechanism
of crenarchaeal cell division, was a demonstration of snapping division in
vivo in Thermoproteus tenax (80). Whether the same or similar mechanism
for cell division operates within Sulfolobus remains an open question.
The active replication in the absence of cell division observed after the
shift to the non-permissive temperature of the DG134 cell division mutant
(Fig. 6) indicates that cell division is not essential for the initiation of
replication (see above). Under physiological conditions, the cell division
period could be necessary to assure a competent state of the replication
machinery before the relatively quick initiation (of chromosome
replication) event.
1.7 Nucleoid structure. There was a clear difference in nucleoid structure
between exponential and stationary phase cells (Figs. 2 and 3). In
exponential phase, the nucleoids were more structured and did not fill the
entire cell, while upon entrance into stationary phase, they became more
relaxed and occupied a larger part of the cell interior.
37
In E. coli, coupling between transcription, translation and translocation
of membrane proteins has been proposed to affect the structure of
nucleoids. In this model, external loops of chromosomes containing genes
coding for membrane proteins are thought to be connected with the cell
membrane (189). Therefore, changes in the expression of the membrane
proteins upon entrance into the stationary phase would affect the anchoring
and result in differences in chromosome structure. Similar mechanism
could operate in Sulfolobus cells and result in the relaxation of the DNA
structure in stationary phase.
Alternatively, the structure of the Sulfolobus chromosome may be
influenced by level of the expression of proteins involved in the regulation
of DNA supercoiling and condensation, which could be dependent on
changes in growth phase. The structure of the Sulfolobus chromosome was
suggested to be dependent on the balance between reverse gyrase activity,
creating positive supercoiling, and the relaxing activity of type II
topoisomerases (111) as well as other proteins. The S. solfataricus 7 (Sso7)
protein was shown to facilitate the hybridization of complementary strands
of DNA (67), and was suggested to participate in maintaining the dynamic
structure of DNA at high temperature (67). Changes in the amounts of
proteins related to the Smc family, topoisomerases, and Sso7 levels, can be
a consequence of differences in the expression of genes and turnover of
proteins upon the entry into the stationary phase and could be a reason for
relaxed nucleoid structure. Similar to Sulfolobus the relaxed structure of the
nucleoids was also observed in stationary phase cells of E. coli (1).
In contrast to Sulfolobus cells, the structure of the nucleoids in stationary
phase cells of the euryarchaeon M. jannaschii was much more compact
compared to nucleoids from exponential-phase cells (118). Contrary to
euryarchaea, Sulfolobus and E. coli do not contain histones, proteins that
tightly compact the chromosomes. Thus, the nucleoid structure in such
organisms might depend on the superhelical properties of the chromosome.
A decreased level of reverse gyrase expression in Sulfolobus upon entrance
into stationary phase could result in decrease of the linking number of the
chromosome, which could lead to the relaxation of the nucleoid structure.
In contrast, decreased level of expression of topoisomerases in stationary
phase of euryarchaeal cells may result in a more compact structure of the
nucleoids, since the topoisomerases would no longer prevent condensation
of the DNA by histones.
1.8 Budding. The appearance of bud-like extrusions in some S.
acidocaldarius and S. solfataricus cells under special condition (Fig. 5), is
reminiscent of the corresponding process observed in wall-less L-forms of
38
E. coli growing in the presence of calcium. In wall-less L-forms of E. coli
budding was speculated to be an unusual division pattern (140).
A presence of only a single bud in each of the analyzed cell, suggest that
budding could be explained by taking into consideration differences in the
structure of the cell envelope at one particular place of the cell. This could
be due to proceeding, but still yet unfinished synthesis of the S layers, after
the completion of the division process. Alternatively, changes in the
structure of the cell envelope at one particular place of the cell may result
from the initiation of a new cell division event. Therefore, the place for bud
emergence could be used as a marker indicating either the localization of
division in the mother cell or the position of the next division.
The thinner cell wall could also be an advantage during the mating
described for Sulfolobus cells (65, 146, 155, 156) because it would make it
easier for donor and recipient cells to establish an intercellular connection
between each other.
1.9 Use of conditional mutant in studies of Sulfolobus physiology.
Conditional-lethal mutants offer insights into the metabolic and molecular
processes involved in biosynthesis, polymerization and assembly of
molecules within the cell. They are also important for understanding the
function of a variety of proteins involved in DNA replication, transcription,
translation, energy metabolism, biosynthesis of building blocks,
catabolism, transport of macromolecules, and other processes essential for
the growth and viability of cells. Below, I will briefly discuss selected
examples for the use of conditional-lethal mutants of S. acidocaldarius.
1.9.1 Mutations affecting growth, protein synthesis and metabolic
pathways. The group of mutants from class Ib (Table 5) did not show any
effects on cell integrity. Growth and replication were rapidly halted after
the shift to nonpermissive temperature, as exemplified using DG146 (Fig.
6). However, the cells remained viable for a long period, more than 30
hours after the shift (Fig. 3, paper III). Since the initiation of replication
requires de novo protein synthesis, the observed arrest at the postreplication
stage with two genome equivalents and the inability to initiate replication
(Fig. 6) might imply damage of any component of the protein synthesis
machinery.
Furthermore, the observed phenotype could be a consequence of
blocking particular metabolic pathways participating in the synthesis of
amino acids or ATP, which is required for biosynthesis, polymerization and
assembly reactions, and affect the replication of DNA and other growth
properties.
39
1.9.2 Mutations affecting cellular integrity. The DNA degradation in
mutants from class II (Table 5) coincides with the relatively rapid drop in
viability (Fig. 3, paper III) and ghost-like appearance of the cells, after the
shift to nonpermissive conditions (Fig. 6).
The phenotype of the class of conditional-lethal mutants of S.
acidocaldarius discussed in this section suggests that they may be useful
for searching for genes that are essential for proper functioning of the cell
envelope.
Membrane stability of archaeal cells is essential for protection against
lethal effects of extreme temperature in the living environment (40, 87, 88,
89, 178). This stability is due to the presence of ether links and isoprenoids
in the membrane lipids (40, 89, 178). Mutations in the gene(s) participating
in isoprenoid synthesis could allow penetration of external acid into the
cells. This might be followed by acidification of the cytoplasm, leading to a
quick loss in cellular viability.
A proper structure of the membrane is important for electron transport,
which is dependent upon proteins located in the membrane (102). As the
pH of the medium is about 3, an efficient respiratory system for expelling
protons out of the cytoplasm of Sulfolobus cells is essential for preserving
the internal cellular pH, which is close to neutrality. Furthermore, the
electron transport is required for the synthesis of ATP. Mutation in the
protein components of such a system might lead to problems with ATP
synthesis and the effectiveness of the proton motif force. This would affect
metabolism and significantly change the internal cellular pH, leading to
acidification of the cytoplasm, degradation of DNA and cell death.
The S layer of Sulfolobus consists of glycoproteins (64, 88, 147, 183).
Mutation(s) affecting the glycosylation pathway or proper function of the
components of the S layer may alter its structure and/or integrity. This may
result in damage of the integrity of the cell wall, as well as denaturation of
cellular proteins, leading to a cell death.
The processes described above can be studied in Sulfolobus using
mutants from class I, II, and V, which constitute more than 60% of our
collection. This large fraction of conditional-lethal mutants provides
possibilities for discovering essential genes involved in the regulation of
various cellular processes in the archaeal cell.
40
Part II: The ftsZ gene and cell division of
H. mediterranei
(paper IV)
2.1 Introduction. In this section, I will discuss cell division in halophilic
archaeon H. mediterranei, as well as transcription and translation of its ftsZ
gene. The gene organization around ftsZ in all euryarchaea was analyzed
using the information provided by frameplot and database analyses. The
extent of functional conservation between archaeal and bacterial FtsZ
proteins was investigated. Finally, the relationship between euryarchaeal
FtsZ and eukaryotic tubulin will be discussed.
2.2 Transcription initiation. Analyses of several promoter sequences of
the euryarchaeal ftsZ paralog 1 suggested the presence of a putative TATAbox motif which matched the general archaeal TATA box consensus
sequence, which is 5´-YTTAWA-3´ (where Y= C or T; W=A or T) (174).
In contrast, the TATA box of Haloferax ftsZ (Fig. 1, paper IV)
resembled neither the above consensus nor the recently proposed consensus
for genes of halophilic archaea, that is 5´-TTTWWW-3´ (164). The
sequence of the TATA box of Haloferax ftsZ fit with the core consensus
sequence for protein coding genes 5´-TWWWWR-3´ (where W= A or T;
R=A or G) (30).
In halophilic archaea, there are multiple TATA binding proteins (TBPs)
(133, 165, 174). Thus, the TATA box of Haloferax ftsZ that do not fit the
consensus sequences might instead constitute a binding site for an
alternative TBP. However, it is necessary that more genes from H.
mediterranei be sequenced and analyzed in order to support such
speculation.
2.3 Translation initiation signals. Two ATG translation initiation starts
were proposed, the first located immediately downstream of the
transcription initiation start and the second positioned downstream of the
putative SD sequence (Fig. 1, paper IV and Fig. 7).
The presence of SD motifs in front of the 2nd initiation codon implies a
bacterial-like SD-dependent mechanism for translation initiation of the
Haloferax ftsZ gene. A similar mechanism for translation initiation could
also be suggested for the ftsZ paralog 1 of M. jannaschii, which also have a
putative SD in front of the initiation codon.
However, the second possibility that translation could be initiated by an
unknown mechanism from the 1st ATG codon located close to the 5´end of
the ftsZ mRNA cannot be excluded. The short (3 bp) distance between the
41
transcription initiation start of the ftsZ gene and the 1st ATG initiation
codon make the possibility for the presence of SD-like sequence or any
regulatory elements upstream to this codon, unlikely.
In bacteria and in S. solfataricus, in genes without a SD sequence in
front of the initiation codon, other regulatory sequences located instead
downstream of the initiation codon might constitute a functional equivalent
of the absent SD sequence (101, 175). In S. solfataricus, possibility for a
presence of such regulatory sequence elements located even at the 3´-end
of the mRNA, was also considered (175).
Haloferax may use a similar to Sulfolobus strategy in order to initiate
translation of the ftsZ gene from the 1st ATG codon. Sequence elements
with partial identity to SD such as 5´-GGA-3´ which is located downstream
of the 1st ATG initiation codon and partially overlaps it, or other yet
undetected signal elements located downstream of the 1st ATG codon,
might be necessary for initiation of translation of the ftsZ gene from this
codon.
2.4 Gene organization around ftsZ. The gene cluster identified in the
genus Haloferax, containing ftsZ, secE and nusG, (Fig. 5, paper IV and Fig.
7) is a part of a larger cluster coding for proteins involved in transcription
translation (ribosomal proteins), and protein secretion. Even though gene
order may not be preserved in general in evolution (130), this gene cluster
is most likely important since it was found to be conserved in many
bacteria and archaea (180), and hence proposed to be present already in the
last common universal ancestor of all organisms (LUCA) (180).
The clustering of the ftsZ gene with secE, nusG and other genes is
present in euryarchaea (paper IV and (180)), but not in bacteria. For
example, in E.coli the ftsZ is grouped with downstream genes such as the
envA required in early stage of lipopolisaccharide biosynthesis and cell
separation, gene with unknown function, secA required for proteins
translocation across membrane (37), and the mutator gene, mutT.
Therefore, linkage of the ftsZ in archaea could be explained as an
accidental event, resulting from the horizontal transfer of the ftsZ gene to
an archaeal ancestor, and integration of the ftsZ gene close to the cassette of
genes, including secE and nusG.
The one base pair spacer between secE and nusG, and the presence of an
SD in front of the nusG gene strongly suggests that the genes are cotranscribed and co-translationally coupled. The clustering of secE and nusG
could facilitate the physical interaction between their gene products.
However, the absence of secE in the euryarchaeon Pyrococcus implies that
physical interaction between the NusG and SecE proteins is not an absolute
42
requirement. Clustering might rather provide a way to coordinate gene
expression, but this remains to be proven.
2.5 FtsZ and tubulin. The structural similarities between tubulin and FtsZ
proteins led to the hypothesis of FtsZ being an ancestor of tubulin (see
Introduction).
Analysis of several archaeal genomes revealed the presence of multiple
ftsZ genes, clustered separately on the phylogenetic tree and referred as
paralog 1 and paralog 2 (49). Phylogenetic analysis suggested that ftsZ may
diverge into multiple copies already in the archaeal ancestor (49). Thus,
tubulin might have evolved from a non-essential copy of the duplicated ftsZ
gene (49, 120).
The FtsZ paralog 1 from Haloferax was demonstrated to be functionally
conserved in both bacteria and archaea. Considering its high identity at the
amino acid level to other euryarchaeal FtsZ paralog 1 proteins (paper IV),
this might imply that the euryarchaeal FtsZ paralog 1 proteins are devoted
to the formation of the Z-ring and all are essential for cell viability. Hence,
this would exclude the paralog 1 from being an ancestor of tubulin. Instead,
FtsZ paralog 2 could be a potential candidate for a tubulin precursor.
2.6 Morphological features. Some cells in the H. mediterranei population
were found to be asymmetrically invaginated. This type of morphology
was reminiscent of the cell division pattern observed in spherical rodA
mutants of E. coli affected in peptydoglycan synthesis (38).
A Z-ring could only be observed in a few of the asymmetrically
invaginated H. mediterranei cells, whereas a majority of such cells did not
exhibit any fluorescence (Fig. 8, row E and F). The absence of a
fluorescent signal could be explained in different ways, including technical
problems (e.g. poor staining and/or fixation conditions) or problems with
the stability of the Z-ring. It would be interesting to target this problem
using a FtsZ-GFP fusion, which eliminate the problem of harsh treatment
of the cell during chemical fixation and immunolocalization studies. A
GFP fusion was preferred rather than the immunolocalization for
localization studies of other bacterial proteins such as Soj, and MinD (121,
122).
To conclude, the lack of fluorescent signal in a majority of the cells with
asymmetric invaginations suggests that the morphology of H. mediterranei
cells is not enough to draw conclusions about the exact pattern of cell
division.
43
2.7 Cell division. Since morphology was insufficient criterion to predict
the division pattern of Haloferax cells, localization of the Z-ring was used
to target this problem. The bacterial-like pattern of cell division in
exponentially growing cells of H. mediterranei was reflected in the
structure and presence of a Z-ring. The localization of the Z-ring to the cell
septum at different stages of cell division in H. mediterranei (Fig. 8),
suggests that the Haloferax cells divide by medial fission as observed in
many bacteria, including E.coli.
However, and in contrast to E. coli, the final stages of cell division in H.
mediterranei were characterized by the presence of a morphological
structure reminiscent of a cellular bridge, to which the FtsZ protein was
localized (Fig 8, row B and C). These morphological structures could result
from contraction of the cell by the Z ring. Such process of contraction of
the cell may be driven by changes in the curvature of FtsZ filaments (114).
Although the presence of the FtsZ protein and formation of the Z-ring
indicate conservation of at least initial stages of cell division between
bacteria and archaea, only few cell division proteins (see Introduction)
were identified in completely sequenced euryarchaeal genomes (12, 152).
Hence, more studies are required to reveal the complete mechanism of cell
division process in the genus Haloferax.
44
4. Future projects
4.1 Correlation between growth rate and length of cell cycle periods.
The correlation between the length of particular cell cycle periods and the
growth rate can be studied by the flow cytometry analysis of cells growing
in different media. If a decrease in growth rate is followed by an increase in
the length of the post-replication period, this would support the model in
which the initiation of replication is coupled to the division of the cell.
However, if the pre-replication period increases in parallel within the
generation time of the culture, then replication would not be coupled to the
division process.
4.2 Cell division. After the development of a genetic system, the cell
division mutants should be complemented using a DNA library from the
parent strain DG64, in order to find a functional equivalent of the FtsZ
protein in Sulfolobus.
Furthermore, I want to understand how the cell division process
proceeds in the cell. Since, DG134 is affected in cell division and contains
up to 5 properly segregated genome equivalents, the cell division process
could be followed using microscopy and flow cytometry after the cells are
returned to the permissive temperature.
4.3 The ftsZ genes in halophilic archaea. In almost all completely
sequenced euryarchaeal genomes multiple copies of the ftsZ gene have
been detected. In contrast, only a single copy of ftsZ gene has been reported
in halophilic archaea so far. Therefore, it would be interesting to
investigate how many copies of the ftsZ gene may exist in halophilic
archaea.
A PCR fragment constituting a probe against either paralog 1 or 2 of the
ftsZ gene may be used to screen a genomic library. Positive clones would
be isolated, sequenced, subcloned into a shuttle vector and used for further
analysis.
After isolation of the ftsZ gene(s), an effort could be made towards
construction of a vector with a FtsZ-GFP fusion to study the localization of
different FtsZ proteins in living cells. The FtsZ-GFP fusion could be used
to elucidate whether formation of the Z-ring is common to both FtsZ
paralogs or primarily restricted to FtsZ paralog 1.
45
In archaea containing multiple copies of the ftsZ gene, different
approaches such as mutagenesis or antisense regulation, may be used to
knock out a particular copy. This will help to elucidate the function of
particular paralogs and to reveal which archaeal ftsZ genes are essential for
cell division and viability.
4.4 Cell cycle studies of halophilic prokaryotes. These studies should be
done to get an overview, and to be able to generalize about cell cycle
properties in crenarchaea and euryarchaea, as well as to compare cell cycle
properties between deeply branching extremophilic archaea and bacteria.
Thus, cell cycle analyses should be extended to halophilic archaea as well
as halophilic and thermophilic bacteria, and I have already initiated such
studies.
Preliminary results revealed that in halophilic archaea, the growth
conditions affect cellular morphology and nucleoid structure. In rod-shaped
halophilic archaea, the nucleoid structure resembles that of E. coli, but in
those with pleomorphic morphology the nucleoids have rather a complex
network-like structure. Flow cytometry analysis shows that the halophilic
archaea contain up to three genome equivalents in both exponential and
stationary phases. In addition, anisomycin, which inhibits protein synthesis
in eukaryotic cells, dramatically affected the nucleoid structure and cellular
morphology of halophilic archaea.
I have also initiated cell cycle studies of halophilic bacteria, such as
Halomonas elongata. In the near future, the same approach will be used to
study the cell cycle properties of the thermophilic bacterium Thermus
thermophilus.
46
ACKNOWLEDGMENTS
I would like to express my gratitude to everyone, who contributed in many different
ways, to my thesis:
To all of the former and present members of the Departments of Microbiology
and Medical Genetics in Uppsala for providing a pleasant and stimulating atmosphere
to work in.
Rolf Bernander, my supervisor, for his strong support, stimulating discussions,
understanding and patience. I am very grateful for the freedom I had during
development of the procedures for studying the archaeal cell cycle and microbial
diversity in Uppsala.
Professor Kurt Nordström my first boss in Sweden, who provided me with the
opportunity to come and study in Uppsala. I appreciate your determination in
broadening our knowledge by pushing us to read books outside of our subject area.
Santanu Dasgupta, for sharing your knowledge about different aspects of life,
including science, art, religion, and Indian food. I am also grateful for your incredible
input into the correcting of my Ph.D. thesis.
Karin Carlson, for her understanding of my requirements for more time to finalized
writing of my thesis, and discussions about microbial diversity which help me to
improve the procedures for my course with the students.
Klas Flärdh, for endless explanations of frameplot analysis and interesting
comments about my work. Without your support with Endnote program, I would
probably never have finished typing the references into my thesis.
Dennis Grogan, from the University of Cincinnati (USA) for a nice and fruitful
collaboration. I am very grateful for the email discussions and the tricks you suggested
to help with growth, plating and preservation of the Sulfolobus strains.
Diarmaid Hughes, you always impressed me with the very pedagogic and organized
way you prepared presentations and answered question during Monday seminars.
Leif Kirsebom who as a new Head of MIKRO brought to our department a powerful
spirit.
Christina Friberger, for her excellent and irreplaceable help in so many different
things.
Professor Alina Taylor, Krzysztof Kucharczyk and Ewa Laskowska from the
Department of Biochemistry, University of Gdansk who supervised my first steps into
science.
Anna Podhajska, and Karol Taylor, professors of the Department of Microbiology
and Molecular Biology at the University of Gdansk, for creating an enthusiastic interest
in all students, including myself, in microbiology and molecular biology.
Jean-Pierre Bouche and Kayemuang Cam, from the CNRS, Toulouse in France,
where my interest in the ftsZ gene and various aspects of cell cycle began. I had a nice
time and gained a lot of experience while being in your lab.
Thomas Åkerlund, for introducing me into the world of light microscopy. The
haloarchaeal group from the lab. of professor Shiladitya DasSarma: Nitin B., Fazeela
M., Stacy C., Eric G., and Sean K., for their help and nice time in Amherst. The
members of Dr. Harald Huber lab. from the University of Regensburg for their help
at the beginning of my "Sulfolobus career".
47
To old and new archaea people Ulrika, Laurence, Karin H., Henrik K. (Blotte)
and Cecilia, for help, being understanding of my (dis-) organization and most
importantly for reminding me almost every day to eat.
Joanna Kufel, who from my perspective kept me alive, although Leif Kirsebom,
would say she has controlled my life.
Emilia, one of the best friends I have ever had and my supplier of chorizo.
Sophie, for being my Favorite French Star, and her two inseparable friends, our
Greek Goddess Christina and their Latino Amigo Javier.
Lina, with her amazing eyes, Betina and Emma S., all of whom I will remember
until the end of my life. The beautiful girls Johanna B., Lotta I., Mira, Pernilla,
Patricia and Solveig R., who were all absolutely required for keeping MIKRO alive in
my heart. Staffan, Agnetha R., Annika, Tord, Maggan, Åsa E., Helena S., Hilde E,
Magnus L., Fredrik S., Thomas Å., Per and Dan, for nice times at old MIKRO.
Janne, the Ju-jutsu master; Bjorn, the (Z) Ring man; Tessi, the lucky man. Helen
W., for support and non-stop discussions in the flow room and over the phone. Doro,
for making very good cakes. Christina Ninalga, for the English proofreading of my
thesis. Lotta T. and Helena Ö., for being nice persons. Esteban B., for nightshift
discussions. Hywel, Nils, and Mathias, for the help with computers and other things
that I probably forgot to mention. Fredrik P., for having fun watching pictures of your
home made from a satellite. Tao, for your determination in getting things done, in
whatever conditions, and for your original personality. All the ladies from Disken, for
nice Swedish chats, cookies, and access to the autoclave whenever I needed it.
Anna-Chey, who constantly supplies loading buffer for agarose electrophoresis and
buffers for the preparation of competent cells. Lena M., for being my "swedish
mother.”Solan, without you I would probably never have realized what was wrong with
saying “KEBO catalog.” Eva B., for improving my Swedish. Eva F., I spent most of
my 5 years time at the BMC at your place trying 10,000 different combinations in order
to make the perfect "archaeal" medium. Without your high quality components and
clean lab, it would have been hard to succeed. Stig, Eddie, and Hamid, for technical
support whenever I needed it and for making fun of me while working in Eva F.’s lab.
Anders A., for letting me live in his apartment.
DOA, Veronica M., Lotta O, Hai-Tao, Johan B, Cecilia, and Ylva for years of
running the course together. Jan Zabielski, for random discussions about life in Great
Britain. The PCR/ABI "club", Lucia C., Ludmila, Paula, Delal, Inger, Anh-Nhi and
Kiki, for their discussions and help necessary for improving my PCR and sequencing
skills.
Dima, Stanislav, Edward, Gala, Annika, for rakija, mushrooms, parties, russian
lessons, and nice time throughout the years. Jolka, Przemek, Betito, Albert, Jan,
Tomek, Maciej, Mikolaj, Patryk, Sebastian, Martin, Cici, GosiaK, and Gosia W.,
Helena H., Amilcar (Katracho), David (Chapin), Dick (King of Salsa), Susan,
Mike, Anders, and Annika, for their presence in Sweden. All people from the "old
times" at Lasse Maja and Sernanders Krog for nice time. The Bulgarian Mafia; Nina
(number 1), Natalia, Elli, Petrana, Svetlio, and Timour, for perfect company and
salsa lessons. I also would like to thank to Aneta and Witek. Edyta, Prezes, Tow.
Trzeciak, Tomek, Jacek, Marek and Ania K., Zbyszek R., Zbiszek SZ, and Joasia
Cz. for everything they did to support my work and life in Uppsala .
All of the Foundations that sponsored my work in Sweden.
My family, for their love and support during my studies.
48
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