Cell Cycle

ISSN: 1538-4101 (Print) 1551-4005 (Online) Journal homepage: https://www.tandfonline.com/loi/kccy20

Comparative and functional analysis of the

archaeal cell cycle

Rolf Bernander, Magnus Lundgren & Thijs J. G. Ettema

To cite this article: Rolf Bernander, Magnus Lundgren & Thijs J. G. Ettema (2010) Comparative
and functional analysis of the archaeal cell cycle, Cell Cycle, 9:4, 795-806, DOI: 10.4161/
cc.9.4.10674

To link to this article: https://doi.org/10.4161/cc.9.4.10674

Published online: 15 Feb 2010.

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 Report Report
Cell Cycle 9:4, 795-806; February 15, 2010; © 2010 Landes Bioscience

Comparative and functional analysis

of the archaeal cell cycle
Rolf Bernander,1 Magnus Lundgren1,2 and Thijs J.G. Ettema1,*
1
Department of Molecular Evolution; Evolutionary Biology Center; 2Department of Cell and Molecular Biology;
Biomedical Center; Uppsala University; Uppsala, Sweden

Key words: archaea, caulobacter, cell cycle, cell division, chromosome replication, crenarchaeota, cytokinesis, genome segregation,
mitosis, saccharomyces, sulfolobus

The temporal and spatial organization of the chromosome replication, genome segregation and cell division processes
is less well understood in species belonging to the Archaea, than in those from the Bacteria and Eukarya domains. Novel
insights into the regulation and key components of the Sulfolobus acidocaldarius cell cycle have been obtained through
genome-wide analysis of cell cycle-specific gene expression, followed by cloning and characterization of gene products
expressed at different cell cycle stages. Here, the results of the transcript profiling are further explored, and potential
key players in archaeal cell cycle progression are highlighted in an evolutionary context, by comparing gene expression
patterns and gene conservation between three selected microbial species from different domains of life. We draw
attention to novel putative nucleases and helicases implicated in DNA replication, recombination and repair, as well as to
potential genome segregation factors. Focus is also placed upon regulatory features, including transcription factors and
protein kinases inferred to be involved in the execution of specific cell cycle stages, and regulation through metabolic
coupling is discussed.

Introduction detail.4 Information about general cell cycle characteristics of

The Archaea domain comprises an evolutionary lineage that has
evolved in parallel with Bacteria and Eukarya.1 The domain is
currently divided into two main phyla, the Crenarchaeota and
the Euryarchaeota and a third, composed mainly of low-tem-
perature species, has been suggested (Thaumarchaeota 2). Each
lineage displays distinct physiological and cellular characteris-
tics, while joined together by features common to all archaea.
The organisms thrive under a wide variety of conditions, from
vast oceanic and soil environments to geographically restricted
ecosystems characterized by extremes in terms of temperature,
pH or salt concentration. While several aspects of the biology of
archaeal species appear to be unique, other traits resemble those
in eukaryotes, including the machineries that govern information
storage, maintenance and processing. Conversely, other archaeal
characters are typically shared with the bacterial domain, such
as chromosome organisation, transcription regulation and co-
transcriptional translation.
The temporal and spatial organization of the chromosome
replication, genome segregation and cell division processes is less
well characterized in archaea than in bacteria and eukaryotes.
The organization and regulatory features of the cell cycle is cur-
rently best understood for the crenarchaeal genus Sulfolobus,3
and the biochemical and molecular properties of the chromo-
some replication process have been studied in considerable
several other crenarchaeal and euryarchaeal species is also avail-
able.5,6 Recently, a novel cell division machinery, the Cdv system,
was identified in crenarchaea.7,8 In contrast, euryarchaea divide
using an FtsZ-dependent mechanism, similar to bacteria, while
thaumarchaea contain a combination of ftsZ and cdv genes.7 In
contrast to replication and cell division, the genome segregation
(mitosis) process remains entirely uncharacterized in archaea.
Genes and proteins involved in the main cell cycle processes
can be recognized through the use of cell cultures that progress in
synchrony through the different cell cycle periods. Genes induced
at specific stages are identified by measuring relative RNA abun-
dance at different time points, through whole-genome microarray
hybridization. This results in a battery of candidate gene prod-
ucts for involvement in regulatory and mechanistic aspects of
chromosome replication, mitosis and cytokinesis. In such a study
of Sulfolobus acidocaldarius, >160 genes that exhibit distinct cell
cycle-dependent expression patterns were identified.9 Many pre-
viously known replication factors were among the cell cycle-in-
duced genes, as was the Cdv division system (above). Conversely,
a variety of genes encoded proteins of unknown function, pro-
viding targets for identification of additional key features of the
archaeal cell cycle, such as the elusive mitosis machinery. Thus,
a detailed analysis of these genes should generate a more compre-
hensive picture of the replication and division machineries, and
of the regulatory features of the cell cycle.

*Correspondence to: Thijs J.G. Ettema; Email: Thijs.Ettema@ebc.uu.se

Submitted: 10/14/09; Revised: 11/17/09; Accepted: 11/17/09
Previously published online: www.landesbioscience.com/journals/cc/article/10674

www.landesbioscience.com Cell Cycle 795

 Here, we report an in-depth comparative analysis of cell cycle-
induced genes of S. acidocaldarius. First, we compare induction
responses of S. acidocaldarius with similar datasets from two other
unicellular organisms, the alpha-proteobacterium Caulobacter
crescentus and the fungus Saccharomyces cerevisiae, to identify gen-
eral principles of cell cycle regulation. Second, we investigate the
phylogenetic distribution patterns of cell cycle-induced genes from
S. acidocaldarius across archaeal, bacterial and eukaryotic genomes,
to identify potential key factors involved in cell cycle progression.
Cell Cycle-Dependent Expression Patterns in
Different Gene Classes
The study of S. acidocaldarius cell cycle-dependent gene expres-
sion9 allows for genome-wide comparative analyses with similar
data sets. High-quality data sets representing unicellular species
from the other two domains of life, the alpha-proteobacterium
C. crescentus10 and the eukaryote S. cerevisiae,11,12 were chosen for
this purpose. In this way, features specific to multi-cellularity are
avoided, while gene products and expression patterns that are
conserved between widely diverged organisms, and thus likely to
represent core cell cycle features, can be identified.
When comparing at the level of protein orthology (Fig. 1A),
it becomes clear that expression patterns are poorly conserved,
as previously noted in comparisons of cell cycle-dependent
gene expression among distant eukaryotic species.13 Thus, only
seven COGs (clusters of orthologous groups) contain genes that
are cell cycle-dependently expressed in all three organisms (see
Suppl. data for full overview of cyclic genes in S. acidocaldarius,
C. crescentus and S. cerevisiae). These include the alpha subunit
of ribonucleotide reductase (COG0209), the DNA polymerase
sliding clamp (beta subunit of DNA polymerase III in bacteria;
COG0592) and members of the PAPS reductase/FAD synthetase
family (COG0175; see below). In addition, genes encoding sub-
units of ABC-type transporters (COG0842; COG1131), per-
meases of the major facilitator superfamily (COG0477) and
members of the alpha/beta hydrolase superfamily (COG0596)
are subject to cell cycle-dependent transcriptional regulation in
all three organisms (see Suppl. data). It should be noted, however,
that these latter COGs consist of large multi-gene families, usu-
ally represented by multiple copies in the genome. For example,
the S. acidocaldarius genome contains 40 major facilitator per-
mease genes, only one of which (Saci_1789) displayed cell cycle-
dependent regulation among those included in the microarray
analysis.9 The C. crescentus and S. cerevisiae genomes encode 26
(2 cyclic) and 73 (12 cyclic) such genes, respectively, and the pre-
cise orthology relations between these are difficult to determine,
rendering detailed comparisons with respect to cell cycle func-
tion complicated.
A functional breakdown of genes that display cell cycle-de-
pendent expression in the three organisms revealed both similari-
ties and disparities in terms of expression features (Fig. 1B). Not
unexpectedly, genes belonging to the class ‘Cell cycle control,
cell division, chromosome partitioning’ were overrepresented in
the data sets from all three organisms, as were genes belonging
to ‘Replication, recombination and repair’. In addition, genes
796 Cell Cycle Volume 9 Issue 4
involved in ‘Cell motility’ were overrepresented in all data sets,
although the gene number in this class is small, particularly in
S. acidocaldarius. In this organism, expression of an operon that
encodes all known crenarchaeal flagella components, including
the FlaFIJH proteins and archaeal flagellin, is induced during S
phase, indicating that the assembly of the motility machinery is a
cell cycle-dependent process. It is currently not clear how flagel-
lar positioning on S. acidocaldarius cells is controlled, nor how
flagella are distributed between daughter cells upon cell division.
Regardless of distribution, new flagella need to be synthesized
in each cell generation to prevent a gradual reduction in number
and, since flagella are complex structures that need to be fully
assembled to be functional, a cell cycle-specific coupling may be
the most efficient way to coordinate the process. In Caulobacter,
the expression of flagella- and pili-encoding genes is strongly cor-
related with the cellular life cycle,14 during which the organism
alternates between a flagellated, motile, state and a stalked, non-
motile, stage during which the cell is attached to a solid support.
Thus, transitions between motile and stalked cell morphologies
add to the requirement for a coupling between flagellar synthesis
and cell cycle progression in this organism.
In contrast, other functional classes are differentially repre-
sented as cell cycle-dependent in the respective data sets. Genes
belonging to ‘Carbohydrate transport and metabolism’ are, for
example, underrepresented in S. acidocaldarius and C. crescentus,
but slightly overrepresented in S. cerevisiae (Fig. 1B). S. cerevi-
siae cultures grown under nutrient-limiting conditions have been
shown to display strong periodicity, denoted ‘metabolic cycles’.15,16
During these 4–5 hr periods, which occur in synchrony with the
cell cycle, essential cellular and metabolic events are compart-
mentalized in time, to preserve genome integrity by prevent-
ing oxidative DNA damage.17 Furthermore, in Bacillus subtilis,
coupling between glycolysis and DNA replication,18 as well as
between nutritional status and both chromosome replication19
and cell division,20 has been demonstrated, providing additional
examples of active interplay between metabolic processes and cell
cycle progression. The fact that genes encoding metabolic func-
tions are under-represented in the S. acidocaldarius data set could
indicate that metabolic coupling is of limited importance to this
organism, at least under the conditions tested.
Genes belonging to ‘Translation, ribosomal structure and bio-
genesis’ are under-represented in the S. acidocaldarius cell cycle-
dependent data set. For example, no ribosomal protein-encoding
gene is cyclically expressed, whereas in C. crescentus 16 such genes
display cell cycle-dependent expression (rps2, 3, 5, 8, 11, 13, 17,
20; rpl2, 4, 5, 6, 10, 14, 15, 18). Apparently, protein synthesis
is a strongly cell cycle-dependent process in C. crescentus and,
to a lesser extent, in S. cerevisiae, in contrast to the situation in
S. acidocaldarius. It is possible that in C. crescentus this may, again,
be explained by the bimodal life cycle of the organism, in which
cell proliferation only occurs in the attached state. There may,
thus, be a higher requirement for ribosome biogenesis during cell
cycle progression, which occurs in the stalked cell under condi-
tions favourable for cellular mass increase, whereas the flagellated
swarmer cell primarily may be occupied with maintenance of cel-
lular homeostasis under nutrient-poor conditions.
 Figure 1. (A) Cell cycle-dependent expression is poorly conserved among microorganisms from different domains of life. The Venn diagram shows the
distribution of 131 COGs represented in S. acidocaldarius (yellow), C. crescentus (blue) or S. cerevisiae (orange) that display cell cycle-dependent expres-
sion in at least one of the organisms. (B) Relative over- and under-representation of cell cycle-dependently expressed genes in different functional
classes for S. acidocaldarius (yellow bars), C. crescentus (blue bars) and S. cerevisiae (orangev bars).

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 Figure 2. Replisome comparison between the three domains of life indicating the similarity of eukaryal and archaeal replisomes, both in terms of the
participating factors and their organization. The cartoon indicates central components and their interaction with each other and DNA. Orthologous
proteins are indicated in the same colour. Proteins expressed from genes with cell cycle-dependent transcription are indicated with a black-and-white
border. The different components and their interactions were inferred from refs. 22,64,65

Dynamic Formation of Chromosome Replication Integrative Analysis of Proteins Involved in Archaeal

Machineries Cell Cycle Progression

The archaeal and eukaryal replication machineries display
remarkable conservation, to the exclusion of bacteria4 (Fig. 2).
Only the respective DNA polymerase sliding clamp subunits of
the archaeal/eukaryal (PCNA) and bacterial (beta subunit of
bacterial DNA Pol III) systems fall into the same orthologous
group (COG0592). Deep common ancestry between other
components has also been suggested, as indicated by the similar
tertiary structure of the DnaA and Cdc6 proteins.21 Conversely,
the Mcm and DnaB helicases work in mechanistically different
fashions, e.g., by translocating on different DNA strands, sug-
gesting independent origins.22 In eukaryotes, the origin recogni-
tion complex (ORC) remains bound to the origin throughout
the cell cycle,23 and is activated in early G1 to recruit additional
factors for the pre-replicative complex (pre-RC) in a highly reg-
ulated manner. Cdc6 and Cdt1 are central components of the
pre-RC and bind ORC independently. In contrast to the eukary-
otic system, the genes for the origin-recognizing Cdc6-1, Cdc6-3
and WhiP24 proteins in Sulfolobus are transcribed just in time
for replication.9 The order and manner of replisome assem-
bly in archaea is not known, nor how the process is regulated,
though protein phosphorylation is likely to play a role, analogous
to cyclin-dependent kinases in eukaryotes, as indicated by cell
cycle-specific transcription of several protein serine-threonine
kinases in Sulfolobus.9 The expression of different central repli-
cative factors, such as helicases, replicative polymerase and pro-
cessivity components is, to varying degree, cell cycle-dependent
in all three organisms, as is that of several additional factors
(Fig. 2). In certain cases only one or a few subunits of a multi-
subunit protein are cyclically expressed, reminiscent of the “just-
in-time” complex assembly employed by different eukaryotes,25
possibly to simplify regulation. Notably, most factors associated
with lagging strand replication in all three domains do not dis-
play cyclic transcription (Fig. 2), in agreement with a role in
other, cell cycle-independent, processes, such as DNA repair,
during cell cycle periods other than S phase.3,9
In the following, we discuss proteins and genes that, based on
integrative analysis of cell cycle-specific expression data, homol-
ogy searches, phylogenetic distribution patterns, and experimen-
tal and comparative genomics data,26,27 are likely to constitute
novel players in the archaeal cell cycle. The examples are orga-
nized according to phylogenetic conservation, starting with pro-
teins specific to S. acidocaldarius or (cren)archaea and ending
with cell cycle factors found in all three domains of life. It should
be noted that all genes discussed display cell cycle-specific expres-
sion in S. acidocaldarius, whereas conservation in other organisms
only refers to gene presence or absence (conservation of expres-
sion patterns is discussed above).
(Cren)Archaeal-Specific Patterns
This class comprises synapomorphic features of the (cren)archaeal
cell cycle. Interestingly, among genes that fit this phylogenetic
distribution are those with the highest cell cycle-dependent
induction levels observed in the entire S. acidocaldarius data set,9
e.g., Saci_0276 (NOG75842)28 (NOG; non-supervised ortholo-
gous group) and Saci_0277 (COG2248), as well as Saci_0743
and Saci_0744 (NOG12498), both organized into putative
operon structures. Saci_0277 has orthologs in all crenarchaea,
several euryarchaea, as well as in the highly reduced genome of
the parasitic archaeon Nanoarchaeum equitans, and encodes an
uncharacterized protein of the metallo-beta-lactamase superfam-
ily. Saci_0276, Saci_0743 and Saci_0744, of which the former
two display the highest differential expression and thus would
be anticipated to play pivotal roles in mitosis, cytokinesis or early
replication events, encode proteins that are restricted to S. acido-
caldarius (Saci_0743), or to the Sulfolobales order (S. acidocal-
darius, S. islandicus, S. tokodaii, S. solfataricus and Metallosphaera
sedula). Ironically, all three gene products lack similarity to any
protein of known function.

798 Cell Cycle Volume 9 Issue 4

 Figure 3. Structural similarity between A. fulgidus COG1628 protein AF1433 and the Thermotoga maritima endonuclease V (Tm-EndoV). (A) Structural
alignment between AF1433 Tm-EndoV based on superimposition of the respective structures. Grey boxes delineate residues that could be superim-
posed with a root mean square value of less than 1.35 Å. Residues that constitute the active site pocket of Tm-EndoV are shaded in red, and dark blue
shading indicates cases where active site residues are conserved in AF1628. Secondary structure elements are indicated as follows: H, alpha helix; S,
beta strand; t, turns. Note that the strand-separating PYIP wedge of Tm-EndoV (Pro79, Tyr80, Ile81 and Pro82, shaded in yellow) is absent in the AF1628
structure. (B) Superimposition of Tm-EndoV (yellow) and AF1628 (cyan) structures. The superimposition is based on the α-carbon atoms of 94 residues
that could be imposed with a root mean square value of less than 1.35 Å (shaded grey in A). Active site residues conserved between Tm-EndoV and
AF1628 are shaded in red and blue for Tm-EndoV and AF1628, respectively. The ball-and-stick model depicted next to the imposition represents the
hypoxanthine lesion DNA substrate of Tm-EndoV (only the nicked strand is visible).

Another gene of significant interest is Saci_0721 (COG1628).
The gene is located in a potential operon with the replication
initiator cdc6-1 (Saci_0722; COG1474), and both genes display
virtually identical temporal patterns of cyclic induction at the
onset of S phase, although the Saci_0721 response is lower in
magnitude. Detailed homology searches reveal that Saci_0721
orthologs are present in all archaeal genomes and that the protein
is distantly homologous to endonuclease V (COG1515), which is
involved in removal of deaminated bases (e.g., uracil, hypoxan-
thine and xanthine) from damaged DNA, and displays activity
towards base pair mismatches, flap- and pseudo-Y structures as
well as to small insertions/deletions in DNA molecules. A PSI-
BLAST search seeded with Saci_0721 (BLOSUM62 matrix,
e-value inclusion threshold 0.005, low complexity filter enabled)
starts retrieving significant (e-value < 0.005) hits with proteins
belonging to COG1515 in the third iteration (e.g., Thermofilum
pendens endonuclease V, Tpen_0682, at an e-value of 4E-04).
Additional iterations retrieve other significant hits with endonu-
clease V proteins, revealing a robustness of homology between
these distant protein families. The proposed relatedness is further
supported by the significant similarity of 3D structures of AF1433,
the Archaeoglobus fulgidus representative of COG1628 (2QH9)29
and the Thermotoga maritima endonuclease V (Tm-EndoV;
COG1515; 2W36;30 Fig. 3A and B). Apart from the structural
conservation, some of the residues that form the active site pocket
in Tm-EndoV are conserved in AF1433, including those involved
in the interaction with the 5'-end of the phosphate groups of the
nicked product DNA (Asp43, Lys139 and His214 in Tm-EndoV;
Fig. 3A), supporting a role in DNA repair for AF1433 and its
orthologs. Yet, the strand-separating PYIP wedge (Pro79, Tyr80,
Ile81 and Pro82 in Tm-EndoV) which is a strongly conserved
element within proteins belonging to the EndoV family, seems to

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 Figure 4. Conserved gene order and sequence analysis alludes to a role in genome partition for the Saci_0203 gene product. (A) Conserved gene
order of Saci_0203 orthologs (blue) with ParA-encoding genes (orange) in multiple archaeal genomes. Genes are annotated with their respective
locus tags. (B) Sequence homology between the N-terminus of the Saci_0203 ortholog of Methanobrevibacter smithii (Msm_1240) and ParB proteins.
Consensus sequences are depicted below the alignment using the physico-chemical classification for amino acids as indicated below, except that fully
conserved residues are depicted in boldface. Amino acid residue abbreviation and shading: l, aliphatic (ILV, red font on grey shading); t, tiny (AGTS,
blue font on yellow shading); a, amphoteric (HREQDN, red font on green shading); h, hydrophobic (MILVCFYWHKAG, white font on black shading); p,
polar (YWHKRDETSNQ, black font on green shading); +, positive (KRH, red font on blue shading); s, small (TSCAGPDN, green font on yellow shading).
Proteins are identified by a species abbreviation and the gene identifier. Species abbreviations: Metsm, Methanobrevibacter smithii; Helmo, Heliobac-
terium modesticaldum; Theth, Thermoanaerobacterium thermosaccharolyticum; Clobo, Clostridium botulinum; Rumgn, Ruminococcus gnavus; Mycsp,
Mycobacterium sp. MCS; Neisu, Neisseria subflava; Psefl, Pseudomonas fluorescens; Theaq, Thermus aquaticus; Bacce, Bacteroides cellulosilyticus.

be absent from COG1628 members, indicating mechanistic dif-
ferences between COG1628 and EndoV proteins. In conclusion,
given the structural and distant sequence homology to EndoV in
conjunction with the cell cycle-dependent induction at early S
phase, as well as the observed clustering with cdc6-1, it is tempt-
ing to speculate that the Saci_0721 gene product is involved in
DNA repair during genome replication and, as such, represents a
novel archaeal replisome component.
Saci_1228 and Saci_1229 exhibit a phylogenetic distribution
restricted to the Sulfolobales and Thermoproteales orders, and are
organized into a putative operon structure that is highly induced
at the mitotis/cytokinesis stage. The Saci_1228 gene product
and its archaeal orthologs contain an AAA+ ATPase domain and
display distant homology to ATP-dependent HrpA-like helicases
(COG1643), which are involved in RNA unwinding in a num-
ber of processes, including mRNA processing and rRNA matura-
tion. Interestingly, HrpA-like RNA helicases are not encoded by
archaeal genomes, and hence the Saci_1228 gene product might
constitute an archaeal HrpA counterpart that affects cell cycle
progression.
Saci_0203 (NOG09521) is a candidate gene for involvement
in genome partition. The gene, located in a putative operon with
the ParA-encoding gene Saci_0204 (see below), displays dis-
tinct cyclic induction at the mitotic cell cycle stage similar to
Saci_0204, although at lower magnitude. Whereas parA appears
to be ubiquitous in archaea, Saci_0203 is restricted to members
of the Sulfolobales and certain methanogens. Interestingly, those
species that do contain an Saci_0203 ortholog, this gene is always
located directly adjacent to the ParA-encoding gene (Fig. 4A).
Saci_0203 and its archaeal orthologs lack significant similarity to
other proteins, and among the Saci_0203 orthologs the homology
is restricted to the C-terminal domain (not shown). Intriguingly,
the N-terminus of the Saci_0203 ortholog in Methanobrevibacter
smithii (Msm_1240) displays weak similarity to the partition pro-
tein ParB (Fig. 4B). Although true ParB orthologs are present in
most archaeal genomes, the S. acidocaldarius ParB-encoding gene
(Saci_1498, COG1475) does not display a cell cycle-dependent
expression pattern. Irrespective of putative ParB similarities, the
conserved clustering of Saci_0203 orthologs with ParA-encoding
genes, as well as the observed induction during mitosis, strongly
implicate Saci_0203 and its orthologs in genome partition.

800 Cell Cycle Volume 9 Issue 4

 and Saci_1787 genes show little variation in expres-
sion over the cell cycle (Fig. 5). Further, Saci_0006
displays strong induction in response to SSV4 virus
infection (unpublished data), indicating that multiple
signal pathways converge on this transcription regula-
tor. Finally, and in contrast to the other members of the
group, the Saci_1992 homolog is induced around the S/
G2 transition (Fig. 5). Comparative investigation of the
molecular mechanisms behind the differential regula-
tion of this closely related gene set thus opens up pos-
sibilities for detailed elucidation of cell cycle-dependent
control mechanisms.
Within the subset of cell cycle-regulated S. acidocal-
darius genes with orthologs only in other archaea and
in bacteria several ATPases were identified, including a
member of the ParA family (Saci_0204, COG1192; also
see above), and several predicted ATPases of unknown
function, including Saci_0053, Saci_1128 (COG0433)
and Saci_1773 (COG1373). The parA gene is of spe-
Figure 5. S. acidocaldarius ArsR-type transcriptional regulators display distinctly cial interest, as bacterial ParA proteins have been impli-
differential cell cycle-specific induction patterns. Saci_0800 (filled square) is cycli- cated in segregation of chromosomes and plasmids.36
cally induced in early S phase, Saci_0006 (filled diamond) displays an inverted
response with strong repression in early S, and Saci_1992 (filled triangle) is in-
For instance, the ParA partitioning protein in C. cres-
duced around the S/G2 transition. Saci_1710 and Saci_1787 (filled and open circle, centus, encoded by cell cycle-dependently expressed
respectively) genes show little variation in expression over the cell cycle. (AU; gene CC3753, acts as a molecular switch that couples
arbitrary units.) chromosome partitioning to cytokinesis together with
a second partitioning protein, ParB.37 Thus, it is con-
Factors Shared between Archaea and Bacteria ceivable that the ParA ortholog in S. acidocaldarius,
Saci_0204, also participates in genome segregation and/or cell
Whereas archaea and eukaryotes share highly similar basal tran- division, particularly since the gene shows strong cyclic induction
scription machineries, the archaeal complement of transcription at the mitotic cell cycle stage.9 This would indicate that mitotic
regulators is generally bacterial-like, including a broad set of fac- processes in archaea and bacteria share common features, in line
tors containing a HTH (helix-turn-helix) DNA-binding domain with the similarities in chromatin organization and cell structure
combined with a regulatory domain.31 Several such regulators are in organisms from both domains. These include circular chro-
found among the cell cycle-dependently expressed genes in S. aci- mosomes (exceptions exist), absence of eukaryotic-type telomers
docaldarius, including members of the Lrp (Saci_2136, Saci_0944, and centromers, as well as absence of a nuclear membrane with
COG1522), DtxR (Saci_1012, COG1321), TetR (Saci_1107, consequent opportunities for coupled transcription-translation
COG1309) and ArsR families. Several ‘master regulators’ have that affects chromatin structure and nucleoid morphology.
been identified in bacteria, including an activator (Spo0A) of the Among the COG0433 ATPases, orthologs of the Saci_0053
sporulation pathway in Clostridia and Bacillus species,32 a global gene product have been suggested as archaeal functional analogs
regulator (Lrp) of cellular metabolism in Escherichia coli33 and of the bacterial FtsK family of genome-partitioning proteins.38
two regulators (CtrA14 and GcrA 34) that drive cell cycle progres- In bacteria, FtsK is required for septum formation and acts as
sion in Caulobacter. A global metabolic regulator was recently a DNA translocase for chromosome processing during septum
described in the halophilic archaeon Halobacterium salinarum closure. Moreover, E. coli FtsK is involved in the control of
NRC-1,35 and it is possible that regulators with a broad range metastable recombination events provoked by rearrangements at
of targets could be present also among the cell cycle-specifically the chromosome replication termination site.39 Analysis of con-
expressed transcription factors in S. acidocaldarius. The concept is served gene context in archaea strongly links the Saci_0053 gene
supported by the presence of common regulatory elements, CCR product to the adjacent mre11 gene (Saci_0052, COG0420),
(Cell Cycle Regulon) boxes,9 upstream of gene sets with near- encoding a repair-associated nuclease implicated in checkpoint
identical induction profiles, and the Lrp-like regulators would be signalling and DNA replication.40 In several archaeal genomes,
particularly interesting to investigate in this respect. orthologs of Saci_0052 and Saci_0053 are positioned adjacently
The ArsR-like transcription regulators belonging to COG0640 and, in Methanothermobacter thermautotrophicus, the genes are
provide another example of interesting regulatory issues. The even fused (MTH541). Moreover, it has been suggested that the
group consists of five homologs that display distinctly differential ATPase forms a protein complex with Mre11 that is required for
induction patterns: whereas Saci_0800 is cyclically induced in the processing of DNA double-stranded breaks and recombina-
early S phase, the Saci_0006 gene displays an inverted response tion intermediates.41 Given the induction of Saci_0053 prior
with strong downregulation in early S, while the Saci_1710 to the S/G2 transition,9 together with the information outlined

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above, it is conceivable that the Saci_0053 and Saci_0052 gene
products may be linked to cell cycle-dependent recombination
functions, potentially in conjunction with chromosome replica-
tion termination.
NucS, a structure-specific single-stranded-DNA endonuclease
of the RecB family, is encoded by the Saci_0200 gene (COG1637).
The protein is also present in other archaea and several bacteria,
and the Pyrococcus abyssi homolog was recently shown to lack
structural similarities to other known nucleases, including flap
endonucleases.42 The P. abyssi enzyme interacts with the Hef
endonuclease, a homolog of the eukaryotic Xpf endonuclease, and
displays increased DNA cleavage efficiency in association with the
Pcna sliding clamp. S. acidocaldarius nucS displays a transcription
pattern opposite to those of other structure-specific endonucleases
such as xpf (Saci_0604, COG1948) and hel308 (Saci_0263,
COG1204) (also see below) suggesting that, instead of replica-
tion-associated repair, this gene product could be involved in
other processes, e.g., recombination, in this species.
Archaeo-Eukaryotic Factors
The molecular machineries that govern information storage and
processing in archaea and eukaryotes share common ancestry.
Recently, we have shown that also the crenarchaeal Cdv divi-
sion system and the eukaryotic type III vesicle-mediated proteins
sorting machinery (ESCRT-III) are evolutionarily related.7,43 In
general, it is expected that patterns that specifically unite cell
cycle-specifically expressed genes from S. acidocaldarius with
eukaryotes, to the exclusion of bacteria, will include factors
involved in DNA replication, chromatin organization and, pos-
sibly, genome segregation (mitosis).
A cyclically expressed gene that fits this pattern is pelota
(Saci_0072; COG1537) orthologs of which are present in all
archaeal and eukaryotic genomes sequenced thus far. This
includes the highly reduced genome of N. equitans (NEQ109),
suggesting an essential function for pelota. The function of Pelota
is currently best understood for the yeast ortholog, Dom34,
which physically interacts with Hbs1, a protein homologous to
elongation factor 1-alpha (EF-Tu).44 Together, Dom34 and Hbs1
are responsible for endonucleolytic cleavage of mRNAs that
stall in the translation elongation stage, in a process known as
No-Go decay.45,46 As for S. acidocaldarius Pelota, there are indica-
tions that DOM34 is subjected to cell cycle-dependent regula-
tion, although not at the transcription level. Rather, DOM34 is
a target for the YCK2 protein kinase, which displays dynamic
membrane localization during the cell cycle,47 indicating that cell
cycle-specific regulation of DOM34 occurs at the protein level.
Given the indications of participation in the progression of the
eukaryotic cell cycle, together with the observed cyclic expres-
sion of the S. acidocaldarius ortholog, evidence is building for a
central cell cycle function also of archaeal Pelota.48 As discussed
above, the data available for yeast DOM34 suggests a function
in mRNA decay, although additional experimental work will be
required to reveal the precise molecular role in archaea.
As pointed out above, an interesting class of genes implicated
in cell cycle-regulatory functions in all three domains of life is
802 Cell Cycle Volume 9 Issue 4
protein serine/threonine kinases, several of which display cell
cycle-specific expression in S. acidocaldarius,9 e.g., Saci_1194 and
Saci_1694, both belonging to COG0515. Here, we report the
identification of yet another protein kinase, Saci_1477, which is
present in several members of the crenarchaeota and which rep-
resents a distant member of the Rio1-family of serine/threonine
protein kinases (COG3642; Fig. 6). The Saci_1477 gene dis-
plays cyclic expression that peaks during S phase. Genes encod-
ing Rio1-type kinases are restricted to Archaea and Eukarya,
and the best-studied member of this orthologous group, yeast
protein BUD32, forms part of EKC/KEOPS, a protein complex
that exerts essential regulatory functions in bud-site selection,
telomere uncapping and elongation, as well as in transcrip-
tion.49 Apart from Bud32, the kinase-associated endopeptidase
1 (Kae1; COG0533) component of the EKC/KEOPS complex
is also conserved between Eukarya and Archaea. The genes are
often clustered in archaeal genomes, and are even fused in sev-
eral euryarchaeal genomes, suggesting physical interaction. A
recent structural study of the Methanococcus jannaschii Bud32/
Kae1 fusion protein revealed that this is indeed the case, identi-
fying several residues that constitute the Bud32/Kae1 interface,
most of which are conserved in both the archaeal and eukaryotic
Bud32 and Kae1 proteins.50 Furthermore, the association with
Kae1 was proposed to maintain the Bud32 kinase in an inactive
state.50 Interestingly, several of the Mj-Bud32 residues that form
part of the Bud32/Kae1 interface are conserved in Saci_1477 and
its crenarchaeal orthologs, as well as residues that constitute the
active site loop (Fig. 6) and, hence, it is tempting to speculate
that the Saci_1477 gene product is able to interact with the S.
acidocaldarius Kae1 protein (Saci_0851), thus constituting a cell
cycle-dependent subunit of the EKC/KEOPS complex in several
crenarchaea. Given the absence of telomeres, and the different
chromatin structure in archaea relative to eukaryotes, it appears
likely that the molecular function of the complex differs between
the two domains. This is supported by the fact that the Kae1 pro-
tein from Pyrococcus abyssi recently was shown to display DNA-
binding activity, as well as an apurinic lyase activity, as opposed
to the O-sialoglycoprotein endopeptidase activity reported for
yeast Kae1.51 This finding, in combination with the universal
distribution of Kae1 and Bud32 across archaeal and eukaryotic
genomes, leads to the suggestion that EKC/KEOPS is likely to
be essential for genome maintenance in these organisms. The fact
that the BUD32 paralog Saci_1477 displays a cell cycle-depen-
dent expression pattern strengthens this idea, and it is likely that
exciting findings will result from additional investigations into
the molecular function and mechanism of action of the archaeal
EKC/KEOPS complex.
The Xpf endonuclease, encoded by the Saci_0604 gene
(COG1948), is transcriptionally induced during S phase in S.
acidocaldarius. The eukaryotic XPF/MUS81 protein family
includes both catalytic and non-catalytic members and euryar-
chaeal orthologs display similarity to eukaryal Xpf architec-
ture with both a N-terminal helicase and C-terminal nuclease
domains, while crenarchaeal Xpf generally lacks the helicase
domain. In S. solfataricus, endonuclease activity has been demon-
strated on a range of DNA flap, bubble and junction substrates,
 Figure 6. Multiple sequence alignment of BUD32 with distant crenarchaea-specific BUD32 paralogs. Residues that comprise the active site are
indicated with filled circles (●). Consensus sequences are depicted below the alignment using the physico-chemical classification for amino acids as
specified in the legend to Figure 4. Proteins are identified by a species abbreviation and the gene identifier. Species abbreviations: Hsa, Homo sapiens;
Sce, Saccharomyces cerevisiae; Ath, Arabidopsis thaliana; Pae, Pyrobaculum aerophilum; Sso, Sulfolobus solfataricus; Sac, Sulfolobus acidocaldarius; Sto,
Sulfolobus tokodaii; Ape, Aeropyrum pernix; Pfu, Pyrococcus furiosus; Mja, Methanococcus janaschii; Tac, Thermoplasma acidophilum; Sma, Staphylother-
mus marinus; Mse, Metallosphaera sedula.

similar to eukaryotic counterparts.52 However, the Pcna sliding
clamp is required for catalytic function, an association that could
have the same role as the missing helicase domain, and indicates a
function for the Saci_0604 gene product in repair of replication-
associated DNA damage,53 supported by the S-phase-specific
transcriptional activation.
Universal Factors
The hallmark example of a universally conserved cell cycle reg-
ulated gene is ribonucleotide reductase (above; Fig. 1A). The
S-phase-coupled expression of both this and the likewise con-
served DNA polymerase sliding clamp are easily understood as
an increased demand for DNA synthesis precursors and replica-
tion factors. In contrast, other examples of cell cycle-specificially
expressed genes in S. acidocaldarius that show conservation also
in the other two domains of life are less readily explained.
Among the genes that showed cell cycle dependent expres-
sion in S. acidocaldarius, as well as universal conservation,
were those belonging to COG0175, which includes Saci_2202
and Saci_0474 and counterparts in S. cerevisiae (MET16) and
C. crescentus (CC1121; Fig. 7). COG0175 encompasses enzymes
implicated in sulphate assimilation steps in methionine metab-
olism (CysD and CysH) and in FAD biosynthesis. Linkage
between methionine metabolism and cell cycle progression
has been observed in other organisms, but is currently not well
understood. For example, methionine deprivation is widely used
as an anti-cancer treatment in human, as this has been shown
to arrest the cell cycle,54 and methionine exposure has been
reported to induce G1 arrest in Schizosaccharomyces pombe.55 Yeast
protein MET16, which in S. acidocaldarius is closest related to
Saci_2202, has been shown to interact with the cytoplasmic
thioredoxin iso-enzymes TRX1 and TRX2, which protect
cells against both oxidative and reductive stress.56 The S. acido-
caldarius genome contains 5 such thioredoxin-encoding genes
(Saci_0041, Saci_0340, Saci_0791, Saci_1485, Saci_1823).
Notably, Saci_1823 shows strong cell cycle-dependent reduc-
tion of expression in S phase, indicative of a coupling between
methionine metabolism, stress responses and cell cycle progres-
sion also in this organism. Altogether, the observation that genes
involved in methionine metabolism in S. acidocaldarius display
cell cycle dependent regulation, as in other organisms (Fig. 7),
alludes to a role in prevention of oxidative damage to the cell,
presumably at the DNA level.
Another interesting universal class of cell cycle-regulated pro-
teins is represented by Saci_0046 and Saci_2119, which display
distant homology to various members of the SMC protein fam-
ily. SMC proteins are a large, diverse, family of ATPases that

www.landesbioscience.com Cell Cycle 803

 Figure 7. Phylogenetic tree of sequences of selected species of COG0175, comprising enzymes implicated in sulfate assimilation steps in methion-
ine metabolism (CysD and CysH), as well as eukaryotic FAD synthetase (FAD1). This orthologous group is one of the few that contains members that
display cell cycle-dependent expression in organisms that represent all three domains of life. Genes displaying cell cycle-dependent expression in
the S. acidocaldarius, S. cerevisiae and C. crescentus data sets are boxed. The tree and bootstrap support values were inferred using PHYML.66 Values for
partitions supported above 50% (out of 100 replicates) are displayed.

participate in different aspects of higher-order chromosome orga-
nization and dynamics. The N-terminal domain of Saci_0046,
which displays induction during the S/G2 transition, displays
distant homology to the (AAA+) ATPase domain of SMC pro-
teins, including conservation of residues critical for ATPase activ-
ity (Fig. 8A). Detailed analysis of the C-terminal part suggests a
left-handed coiled-coil structure with a periodicity of about 3.5
residues (not shown). The Saci_2119 gene product also shows
distant homology to SMC proteins and is also induced during
the S/G2 transition, resembling Saci_0046 in terms of expression
kinetics. The N-terminal region of Saci_2119 is dominated by a
highly charged 14 amino acid repeat (NTKAI(A/I)ELRK(M/Q)
TEE) (Fig. 8B), again predicted to adopt a coiled-coil structure
(not shown). Together, the observed (distant) homology to SMC-
like ATPases, as well as the induction during the S/G2 transi-
tion stage, indicate a potential involvement in the chromosome
condensation and segregation processes for the Saci_0046 and
Saci_2119 gene products.
The Saci_0263 (COG1204) protein is similar to Hel308,
a member of the superfamily class 2 (SF2) helicases which are
widely distributed across all three domains of life, as well as in
viruses. Eukaryotic Hel308 has been shown to be involved in
genome maintenance by stabilizing stalled replication forks and
removing recombination intermediates and recent biochemi-
cal data from other organisms support this RecQ-like role. A
euryarchaeal Hel308 homolog (Hjm) has been shown to partially
complement the replication fork-remodelling RecQ helicase in
E. coli.57 The S. solfataricus Hel308 ortholog is able to remove
proteins and complementary DNA from the lagging strand of a
replication fork in the 3'-5' direction, enabling replication restart
or recombination initiation.58 Furthermore, Sulfolobus tokodaii
Hel308 has been shown to associate with the Holliday junction-
specific endonuclease Hjc59 which, together with the observed
cyclic transcription of Saci_0263 during cell cycle progression,
with upregulation in early S phase, supports a function for the
Sulfolobus orthologues in replication-coupled repair.
The Archaeal Cell Cycle goes Full Circle
To obtain a comprehensive picture of the archaeal cell cycle, pro-
cesses and factors that remain unexplored require increased atten-
tion. Comparative investigations of cell cycle components and
expression patterns between organisms from all three domains
of life provide valuable tools for further evaluation of existing
data sets, and the genes and proteins highlighted here are candi-
dates to fulfil pivotal, yet unexplored, roles in archaeal cell cycle
progression. Further investigation of these thus open up exciting
possibilites to fill in major remaining gaps in our understand-
ing, such as the molecular details and regulatory features of the
elusive archaeal mitotic process. Elucidation of the fundamental

804 Cell Cycle Volume 9 Issue 4

 Figure 8. Sequence analysis of S. acidocaldarius SMC-like proteins displaying cell cycle-dependent expression. (A) Alignment of the Saci_0046 protein
product with the ATPase domain from the S. cerevisiae SMC1 protein (SMC1_YEAST). Residues that match the ABC_SMC1_euk profile from the CDD
database,67 which encompasses eukaryotic SMC1 proteins, are boxed. Motifs important for ATPase activity which are conserved in the Saci_0046
protein are indicated as follows: 1, ABC transporter motif; 2, ATP binding motif; 3, Walker B motif; 4, D-loop motif. Secondary structure elements are
indicated as follows: H, alpha helix; S, beta strand; T, turns. (B) Sequence analysis of the N-terminus of Saci_2119 reveals a highly charged 14 amino
acid repeat with a predicted coiled-coil structure. Positively and negatively charged amino acid residues are highlighted by grey and black shading,
respectively.

regulatory signals and networks that govern archaeal cell cycle
progression is also within reach, and would have far-reaching
implications both for fundamental aspects of archaeal biology
and for the cell cycle field in general. In particular, the extensive
similarities between archaea and eukaryotes suggest that insights
into core features of the eukaryotic cell cycle, including cancer-
related issues, may be gained from results obtained with archaeal
model systems. Several surprising findings have been reported
during recent years, including the first example of multiple chro-
mosome replication origins in prokaryotes and a novel cell divi-
sion mechanism, and we are confident that additional unexpected
findings with wide implications are likely to emerge from the rap-
idly progressing archaeal cell cycle field.
Materials and Methods
classification of the cell cycle-dependent genes was assigned using
the COG data base classification scheme downloaded from ftp://
ftp.ncbi.nih.gov/pub/COG/COG/.60
Protein sequence and structure analysis. Protein homology
searches were performed using PSI-BLAST using default settings
except that the low-complexity filter was enabled. Multiple
sequence alignments were performed using Kalign61 followed by
manual adjustment whenever necessary. Comparisons of protein
structures, including structural alignments and structural super-
imposition was based on 3D structural data obtained from PDB
(http://www.rcsb.org/pdb/) 62 and performed using the Swiss
PDB viewer.63
Supplementary data. A file comprising a dataset of cell cycle-
dependently regulated genes in S. acidocaldarius, C. crescentus
and S. cerevisiae is available as a Supplementary file.

Datasets used. Genome-wide cell cycle-specific gene expression Acknowledgements

datasets used in this study were obtained from http://devbio1.
stanford.edu/Caulo/14 (C. crescentus) and http://www.egs.uu.se/
molev/suppl.data/lundgren2007pnas/9 (S. acidocaldarius). The
yeast dataset was extracted from a curated dataset published
by Jensen et al. (http://www.cbs.dtu.dk/cellcycle/compsys/
suppl/).13 Orthologous relations between cell cycle-dependently
expressed genes in S. acidocaldarius, C. crescentus and S. cerevi-
siae were extracted from the STRING data base, version 8.0, at
http://string.embl.de/,28 and information regarding functional
This work was supported by Rubicon (Netherlands organization
for scientific research) and Marie Curie-IEF (European union)
grants to T.J.G.E., a Wenner-Gren fellowship to M.L., and by
grants from the Swedish Graduate Research School in Genomics
and Bioinformatics and the Swedish Research Council to R.B.
Note
Supplementary materials can be found at:
www.landesbioscience.com/supplement/BernanderCC9-4-Sup.xls

www.landesbioscience.com Cell Cycle 805


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