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The cell cycle of archaea

Ann-Christin Linds and Rolf Bernander

Abstract | Growth and proliferation of all cell types require intricate regulation and coordination of chromosome replication, genome segregation, cell division and the systems that determine cell shape. Recent findings have provided insight into the cell cycle of archaea, including the multiple-origin mode of DNA replication, the initial characterization of a genome segregation machinery and the discovery of a novel cell division system. The first archaeal cytoskeletal protein, crenactin, was also recently described and shown to function in cell shape determination. Here, we outline the current understanding of the archaeal cell cycle and cytoskeleton, with an emphasis on species in the genus Sulfolobus, and consider the major outstanding questions in the field.
Since the recognition of Archaea as a separate domain of life1, interest in the fascinating organisms in this unique evolutionary lineage has continued to grow. Archaea are found in a wide range of extreme environments, including those with high temperature (hyperthermophiles), high osmotic pressure (that is, saturated salt conditions; halophiles) and extreme pH (acidophiles and alkalophiles), often in combination with anaerobic conditions2. In addition, non-extremophilic archaea are globally distributed and abundant in both marine and terrestrial environments3. Currently, three main branches are recog nized at the phylum level within the domain Archaea: Euryarchaeota4, Crenarchaeota4 and Thaumarchaeota5. Several other candidate phyla have also been proposed, including Aigarchaeota (REF.6), Geoarchaeota (REF.7), Korarchaeota (REF.8) and Nanoarchaeota (REF.9), but their phylum status awaits confirmation. Archaeal organisms have several distinguishing features, including unique membrane constituents10, whereas other characteristics are shared either with bacteria or with eukaryotes11,12. For example, despite the fact that archaeal cells lack a nucleus, the majority of archaeal proteins involved in replication, transcription and translation have homology to eukaryotic counterparts, whereas the corresponding bacterial proteins are significantly more distantly related, if at all. The crenarchaeotal cell division13,14 and cytoskeletal15 complexes also comprise proteins with eukaryotic homologues. Bacterial-like features include the presence of single circular chromosomes (although exceptions exist), the organization of a large fraction of genes into operons, the euryarchaeotal division machinery 1619, and the presence of a wide range of bacterial-type transcription factors20. In all organisms, the cell cycle is divided into a series of main stages during which the central processes occur, and in archaea this cycle is best characterized in species of the crenarchaeotal genus Sulfolobus 21 (FIG.1), which are hyperthermophiles that grow optimally at 80C and pH3. An exponentially growing cell goes through a prereplicative period called the G1 phase, during which the cell increases in size and prepares for chromosome replication. G1 is short in Sulfolobus spp. (it occupies <5% of the cell cycle22) and is followed by the chromosome replication stage, S phase, which lasts for 3035% of the cell cycle22. A second period of cellular growth, known as G2, occupies >50% of the cell cycle22,23 and prepares the cell for genome segregation, or M phase. At this stage, the cell contains two complete copies of the chromosome. During Mphase, the chromosomes align and the genomes segregate, which subsequently leads to cell division, called D phase. Both the M and D phases are short in Sulfolobusspp., each occupying approximately 5% of the cell cycle23,24. Organisms from all three domains of life display great variation in the organization of the cell cycle, and caution therefore needs to be exercised in attempts to generalize. Nevertheless, the Sulfolobusspp. cell cycle, containing a short pre-replicative period and a long postreplicative stage, appears to be widespread among cren archaeotes, including Acidianus hospitalis 25, Aeropyrum pernix 25, Pyrobaculum aerophilum 25 and Pyrobaculum calidifontis 25, and is also evident in the eury archaeote Archaeoglobus fulgidus 26. By contrast, the pre- and post-replicative periods of the non-extremophilic thaumarchaeote Nitrosopumilus maritimus are of similar length, with each stage occupying approximately 25% of the generation time 27. The euryarchaeote
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Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Svante Arrhenius vg 20C, SE106 91, Stockholm, Sweden. Correspondence to R.B. e-mail: Rolf.Bernander@su.se doi:10.1038/nrmicro3077 Published online 29 July 2013

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integrate cytoskeletal functions into the cell cycle and consider the current gaps in our knowledge, emphasizing the main outstanding questions that require further attention from the research community.

G2

G1

G1

G2

Nature Reviews | Microbiology Figure 1 | The Sulfolobus spp. cell cycle. The best characterized archaeal cell cycle is that of species belonging to the crenarchaeotal genus Sulfolobus21. An exponentially growing Sulfolobusspp. cell goes through a short pre-replicative period called G1 before entering into the chromosome replication stage, called Sphase. After an extensive period of cell growth, called G2, which lasts for more than half the cell cycle, the genome segregation and cell division phases, known respectively as M phase and Dphase (both of which are short), occur in rapid succession2224, indicating that there is close positive coupling between these processes. Checkpoint- like inhibition of genome segregation and cell division as a consequence of a blockage in upstream cell cycle processes has been reported109,110 and is indicated.

Polyploid
Containing multiple copies of the chromosome (or chromosomes). In archaea and bacteria, the chromosome copies are identical.

Okazaki fragments
Short single-stranded DNA fragments that are synthesized during DNA replication and are rapidly ligated together to form the lagging strand.

Methanothermobacter thermautotrophicus 28 and the crenarchaeote P. aerophilum 25 seem to lack the G2 period, and in these organisms genome segregation occurs immediately after the termination of, or concomitant with, replication. In the euryarchaeotal genera Halobacterium29, Haloferax 29, Methanococcus 30 and Pyrococcus 31, the cells are polyploid, which has impeded investigations into the organization of the cell cycle owing to difficulties in resolving the replication state of the chromosomes and, thereby, the relative lengths of the cell cycle periods. In this Review, we describe the current knowledge of the central cell cycle processes in archaea, with particular emphasis on Sulfolobusspp. We provide an overview of the composition and activity of the replisome, the multiple-origin mode of chromosome replication, the newly identified Seg system for genome segregation and the mechanisms used for cell division. In addition, we

Chromosome replication Replication initiation and the replisome. Chromosome replication in archaea is initiated at specific sites consisting of two or more short repetitive DNA sequences known as origin recognition boxes (ORBs)32. Each ORB is bound by an Orc1Cdc6 (origin recognition complex1cell division cycle6) family replication initiator protein, which shows homology to both the ORC family of eukaryotic initiator proteins and the eukaryotic protein CDC6, which is involved in loading of the minichromosome maintenance (MCM) DNA helicase complex (see below)3335. In addition to providing specificity, binding of the Orc1Cdc6 protein is the main regulatory step in the replication process, as initiation occurs only when multiple ATP-bound Orc1Cdc6 monomers form a nucleoprotein complex with the ORBs36,37. Orc1Cdc6 protein binding provides a platform for assembly of the complete replication machinery, the replisome (FIG.2), and promotes association of the hexameric Mcm DNA helicase, which unwinds the double-stranded DNA to form a single-stranded template for DNA polymerization33,36,38. Sulfolobussolfataricus Mcm was recently shown to be able to form a left-handed filamentous structure that dramatically changes the topological structure of boundDNA39. In terms of proteins participating in the elongation stage of replication (FIG.2), the archaeal machinery is entirely homologous to that of eukaryotes38,40, with the exception of the DNA polymerase PolD, which is not found in eukaryotes and seems to be the main replicative polymerase in euryarchaeotes41,42. Crenarchaeotes lack PolD and instead use eukaryotic family Btype DNA polymerases for leading-strand replication. Most archaeal genomes encode multiple DNA polymerase variants, and it is not clear whether leading- and lagging-strand DNA synthesis are carried out by two identical proteins, or whether a specific polymerase is dedicated to the synthesis of Okazaki fragments on the lagging-strand template. Archaeal Okazaki fragments are similar in length to those of eukaryotes43 (around 100nucleotides), whereas bacterial Okazaki fragments are in the order of 1,000nucleotides. The trimeric processivity factor, proliferating cell nuclear antigen (Pcna), is loaded onto the DNA by the pentameric replication factorC (Rfc) complex in an ATP-dependent manner and encircles the DNA to firmly attach the polymerase to the template. The heterodimeric DNA primase, PriSL (also known as PriAB), generates RNA primers for the synthesis of lagging-strand Okazaki fragments, and RNA primer removal and Okazaki fragment ligation is carried out by the nuclease Fen1 and ATP-dependent DNA ligase1 (Lig1), respectively. Fen1, Lig1 and the DNA polymerase all interact with Pcna through a short protein epitope called a Pcna-interacting protein (PIP) box 38,40. The Gins23 and Gins51 proteins interact with PriSL and Mcm through Gins-associated nuclease (Gan; known as RecJ in several
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3 5 Leading strand Pcna 3

Rpa Mcm

3 Rfc DNA pol

5 Gan PriSL Fen1 Lig1 Gins23 Gins51 3

3 5 Lagging strand

RNA primer

Figure 2 | The archaeal replisome. The archaeal replication elongation complex, which is known as the replisome, is eukaryotic in nature36,38. The overall architecture of the leading- and lagging-strand replication complexes, as well as the relative positions of the participating components, is schematically outlined. The trimeric processivity factor proliferating-cell nuclear antigen (Pcna) is loaded onto the DNA by the pentameric replication factor C (Rfc) complex in an ATP-dependent manner. Pcna encircles the DNA and firmly attaches the DNA polymerase (DNA pol) to the template. The heterodimeric DNA primase, PriSL, synthesizes RNA primers for lagging-strand Okazaki fragment synthesis, and RNA primer removal and Okazaki fragment ligation are carried out by the nuclease Fen1 and ATP-dependent DNA ligase (Lig1), respectively. Gins23 and Gins51 interact with PriSL and with the minichromosome maintenance (Mcm) complex through Gins-associated nuclease (Gan), thereby contributing to the structural integrity of the replisome. Single-stranded DNA generated during unwinding is stabilized and protected from endonucleolytic degradation by replication protein A (Rpa).

Nature Reviews | Microbiology

archaeal species38), thereby contributing to the structural integrity of the replisome. The Gins complex might also contribute to DNA unwinding 38, together with Mcm. Single-stranded DNA generated during unwinding is stabilized and protected from endonucleolytic degradation by replication proteinA (Rpa). Most crenarchaeotes and thaumarchaeotes also contain a homologue of bacterial single-stranded-DNA-binding protein (Ssb)44,45. Chromosome replication has been shown to be bidirectional in all archaeal species examined, and invivo DNA polymerization rates vary from 15 to 340bp per second, depending on the organism27,4648. The widely differing rates are likely to reflect adaptation to different environmental conditions (such as growth temperature and nutrient availability), with consequent effects on enzyme kinetics, intracellular energy supply and nucleotidepools. Archaea contain multiple replication origins. All bacteria characterized to date use a single replication origin, from which bidirectional replication is initiated49 (FIG.3a). By contrast, eukaryotic chromosome replication is initiated at multiple sites, which reduces the time required for the duplication of large genomes (Sphase)50. The discovery that Sulfolobusacidocaldarius and S.solfataricus both contain three origins in their single chromosome46 (FIG.3b) provided the first example of prokaryotic replicons with multiple origins. Each origin is recognized by a specific initiator protein, two of which (Cdc61 and Cdc63) belong to the Orc1Cdc6 family 32, and the third
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of which, WhiP, together with the adjacent origin, has been suggested to originate from an integrated extrachromosomal element51. All origins specify bidirectional replication in all Sulfolobus spp. studied to date, and all six replication forks progress at a rate of approximately 90bp per second46. The gene encoding each initiator is located immediately adjacent to the origin at all three Sulfolobus origins32,51. Coupled transcriptiontranslation occurs in archaea52, and concomitant transcription and translation of an orc1cdc6 gene located next to an origin increases the likelihood of binding to the ORBs of the adjacent origin. Consistent with this, individual deletion of each of the three orc1cdc6 genes in Sulfolobusislandicus abolishes initiation only at the origin immediately adjacent to the deleted gene53. Furthermore, the structural rearrangements that occur during formation of the nucleoprotein complex between the Orc1Cdc6 protein and ORBs, together with the assembly of the replication machinery across the origin, might block transcription of the adjacent orc1cdc6 gene. This would provide a feedback mechanism to transiently turn off initiator production when replication has been initiated, thereby preventing unscheduled initiation events. We postulate that the two mechanisms together provide the selective pressure to account for the initiator gene being located immediately adjacent to the replicationorigin. The multiple-origin trait is found in a range of species from the phyla Crenarchaeota and Euryarchaeota26,48,51,54,55; archaeal species with a single replication origin also
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a
Replication origin Terminus region dif site

Replisome

Recombination event

XerCD

XerA

Dimer resolution

Dimer resolution

Figure3 | Replication, recombination and resolution of circular chromosomes. a|Replication from a single Nature Reviews | Microbiology origin. In all bacteria49 and some archaea26,47, bidirectional replication is initiated from a single site in the chromosome. If recombination between the duplicated DNA does not occur (left pathway), the replication forks meet in the termination region and collide, generating two segregated daughter chromosomes. If homologous recombination occurs between the two daughter chromosomes (right pathway), a chromosome dimer is formed and needs to be resolved to allow the two duplicated chromosomes to segregate. In bacteria, resolution is mediated by the tyrosine recombinase XerCD, which binds to a specific site called the dif site, at which XerCD-mediated site-specific recombination occurs49. Genome segregation occurs concomitant with, or shortly after, nucleoid partition in bacteria and is therefore completed shortly after replication termination49,70. b|Replication from multiple origins. The replication process is more complex in many archaea, as exemplified by the three bidirectional replication initiation events that occur in synchrony in Sulfolobusacidocaldarius46. Thus, the chromosome is replicated by six replisomes, followed by three asynchronous replication termination events (left pathway). If homologous recombination has taken place (right pathway), a chromosome dimer is produced. A single dif site is sufficient for XerA-mediated dimer resolution, despite the increased complexity of the replication process compared with the single-origin mode. Rather than being located at the last chromosomal position to be replicated, as it is in bacteria, in S.acidocaldarius the dif site apparently does not need to be symmetrically positioned between two origins, nor does it need to be located within the longest inter-origin region60,62. After replication termination, daughter chromosomes remain associated during an extensive G2 period (indicated by the two superposed chromosomes). It is possible that the long G2 period provides a selective advantage by allowing sufficient time for XerA-mediated recombination after replication termination in the multiple-origin replication mode.

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exist, although so far the examples are restricted to euryarchaeotes26,47. The existence of multiple origins in archaea seems to be advantageous for reasons other than reducing the duration of S phase; the origins are often unevenly spaced in the genome, and although reducing the number of origins results in a longer replication period, this can still be accommodated within the minimal length of a cell generation. This has been demonstrated experimentally in S.islandicus, in which deletion of each of two orc1cdc6 genes (thereby inhibiting use of the respective adjacent origin) was shown to have no effect on growthrate53. A correlation between core gene distribution and replication origin location has been demonstrated48,56, such that core genes are preferentially located in originproximal regions of the chromosome. Such genes are replicated earliest, which contributes to their increased expression by increasing the gene dosage (as opposed to genes at origin-distal chromosome locations). However, theoretical calculations show that the maximal gene dosage effect is only 25% in S.acidocaldarius populations, whereas observed differences in expression levels between origin-proximal and origin-distal genes in this species are in the 24fold range, or even higher 56. An alternative reason for this arrangement might be that having core genes located close to the origin provides a rapid backup of essential genetic information during replication, which might be particularly beneficial for extremophilic archaea because of the elevated levels of DNA damage that occur in the environments they inhabit. This damage is likely to result in an increased level of non-functional alleles, and the duplicated gene could thus be used as a backup copy for an inactivated allele or as a template for homologous repair. Considering the seemingly negligible effect that multiple origins have on growth rate53, the availability of a backup supply of essential genetic material might thus be the primary selective advantage56. Using high-throughput sequencing-based marker frequency analysis, the first archaeon with four replication origins was recently reported48. In P.calidifontis, a crenarchaeote that thrives at 90C, bidirectional replication is initiated at all four origins, and all replication forks travel at a similar velocity (35bp per second). In contrast to the three identified initiator proteins of Sulfolobusspp., only one has been identified in P.calidifontis. This protein, Cdc6, is the only Orc1Cdc6 family member encoded in the genome, and it binds to ORBs located immediately upstream of the cdc6 gene. The ORBs consist of a short conserved motif (8bp)48 contained within the longer motif (2025bp) typical of other archaea32,35. The fact that no common ORBs are present in the four P.calidi fontis origins48 suggests that each origin is recognized by its own specific initiator protein, which means that three putative initiators have yet to be discovered. These findings underscore the general nature of the multiple-origin mode of replication in archaea, and it will be of considerable interest to determine the replication mode of a broader selection of species, from all the main archaeal taxa, to further investigate the generality and evolutionary history of thistrait. Initiation synchrony and replisome localization. In terms of origin usage, theoretical simulations of marker frequency distributions, from both exponentially growing and synchronized cell cultures, demonstrate that all replication origins are used by all cells in S. acido caldarius 46, S.solfataricus 46 and P.calidifontis 48 populations. Accordingly, expression of all three initiator genes is induced in a cell cycle-specific manner shortly before Sphase in S.acidocaldarius57 and in S.islandicus53. This has also been shown by replication initiation point mapping (RIP mapping) and two-dimensional gel electrophoresis of replication intermediates, both of which demonstrate comparable initiation activity at all three origin regions32,51,53. Synchronized initiation brings up interesting questions regarding how replication is coordinated between multiple widely spaced origins in the genome, each recognized by a different initiator protein. For example, how are the signals to initiate DNA synthesis transmitted to all initiators and origins, and what are those signals? Transient spatial colocalization of origin regions would provide a possible means for coordination, although transient events are difficult to demonstrate experimentally. Furthermore, it is unclear how initiation is restricted to a single event at each origin in each cell generation. One plausible mechanism is inactivation of the Orc1Cdc6 initiator after replication initiation through hydrolysis of bound ATP, as occurs for the bacterial initiator protein, DnaA49, the ATPase domain of which has extensive structural similarity to the corresponding domain in archaeal and eukaryotic ORCCDC6 family proteins58. Structural rearrangements of the origin region during initiation (see above) might also inhibit expression of the immediately adjacent orc1cdc6 gene. Finally, chromosome replication and genome segregation in polyploid archaea are suggested to be cell cycle independent29, in accordance with the idea that stochastic replication and segregation might be sufficient to ensure that each daughter cell receives at least one chromosome copy, provided the overall copy number is sufficiently high. Relaxed copy number control and a lack of coupling of replication initiation to a particular cell cycle stage would indicate that novel principles for cell cycle control and coordination await characterization in these species. After initiation, the replication elongation stage ensues. The subcellular localization of replisomes and nascent DNA has been studied in S.acidocaldarius by immunolocalization59. Despite the inherent limitations in determining the spatial distribution of intracellular structures in small coccoid cells, the data support a model in which the two replisomes that assemble at each origin stay together after initiation, indicating that there is spatial coordination during the elongation stage. However, evidence is lacking to support a model in which there is a higher level of coordination between the replication forks initiated at the three origins, as the three replisome pairs occupy different intracellular locations59. Replication termination and chromosome dimer resolution. In bacteria and archaea with a single replication origin, the two replication forks meet in the termination region47,49,60, which is located on the opposite side
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Core gene
A gene that is conserved in all, or most, sequenced members of an evolutionary lineage. Core genes are often essential and highly expressed.

Marker frequency analysis

An assay in which the relative copy number of markers (short DNA sequences) distributed along the chromosome is measured. In DNA isolated from an exponentially growing cell population, the markers form a copy number gradient from the replication origin (or origins) to the termination region, provided that replication always initiates from a fixed position.

Replication initiation point mapping

A method for determining the position at which DNA replication is initiated. A short DNA oligonucleotide complementary to a sequence close to a replication origin is hybridized to chromosomal DNA isolated from actively replicating cells. DNA polymerization initiated from the primer continues until the end of the newly synthesized DNA template strand is reached, which marks the replication initiation point.

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of the circular chromosome to the origin (FIG.3a). The final stages of replication seem to take place after simple fork collision49. However, if homologous recombination has taken place between the two identical daughter chromosomes, the resulting dimer needs to be resolved before genome segregation can occur, otherwise the chromosome will be severed when the genome segregation machinery attempts to pull the chromosomes apart into the daughter cells61. In bacteria, the tyrosine recombinase XerCD is devoted to the resolution of chromosome dimers49. XerCD mediates site-specific recombination between 28bp repeat sequences (called dif sites) in the termination region of the chromosome dimer, which then resolves into two monomers (FIG.3a). Because archaea contain a homologue of XerCD, known as XerA, this protein was suggested to fulfil an analogous function60,62 (FIG. 3b). This hypothesis was recently corroborated by protein structure-based studies demonstrating that the catalytic mechanism of Pyrococcus abyssi XerA is similar to that of XerCD63. Furthermore, the catalytic residues in the carboxy terminal domain of XerA are also conserved in XerCD, and the dif-binding motifs of XerA from P.abyssi and other species in the order Thermococcales have 50% identity to the corresponding region in bacterial XerCD60. In addition, a comparison between dif sites from Thermococcales members and a bacterial dif consensus sequence revealed 50% identity at the nucleo tidelevel60. Moreover, species in the Thermococcales taxon contain a single replication origin, and the predicted dif sites are preferentially located in the termination region and have been shown to act as recombination substrates for XerA invitro60,63. In bacteria, the DNA translocase FtsK binds to polar DNA sequence elements called FtsK-orienting polar sequences (KOPS) that flank the dif site and direct FtsK translocation towards the dif locus. The regulatory subdomain of FtsK, called the -region, then activates XerCD to carry out recombination at dif, thereby coordinating chromosome translocation with dimer resolution64. Analogous DNA elements, denoted archaeal short polarized sequences (ASPS), have been found in euryarchaeotes60, but an FtsK homo logue has not been identified, although XerA is not dependent on any protein partner for invivo or in vitro recombination60,62. Despite there being three replication termination regions in the Sulfolobus spp. chromosome, only a single dif site seems to be present, although there is some disparity regarding the exact consensus sequence and location of this site60,62. However, a single site would be sufficient to resolve chromosome dimers after replication termination, despite the increased complexity of the replication process as compared to the single-origin mode (FIG.3b). Resolution might be further facilitated by the extensive G2 period (FIG.1), which could provide sufficient time for XerA-mediated recombination when multiple asynchronous replication termination events occur in widely separated chromosomal locations. Finally, in agreement with a role for XerA at the end of replication, cell cycle-specific induction of xerA occurs during S phase in S.acidocaldarius 57. One question relating to the resolution of replication intermediates is whether daughter chromosomes rapidly segregate away from each other after replication termination or remain associated until the onset of genome segregation. Early studies showed that during the extensive G2 stage in S.acidocaldarius and S.solfataricus, replicated chromosomes are not visible as separate fluorescence foci after DNA staining, suggesting that the chromosomes remain in close proximity 23. Consistent with this interpretation, sister chromatid junctions have been observed between replicated chromosomes, evident as X-shaped molecules in two-dimensional gel electrophoresis65. The persistence of such structures in post-replicative cells might facilitate homologous recombination-mediated repair when the frequency of DNA damage is high under extreme growth conditions. The Sulfolobusspp. model cannot, however, be generalized to all archaea; in both M.thermautotrophicus 28 and P.aerophilum25, nucleoid segregation is concluded shortly after, or concomitant with, replication termination, precluding the existence of an extended period for post-replicative homologous recombination.

Centromere
A chromosomal region that functions as the attachment site for spindle fibres during eukaryotic chromosomal segregation (mitosis) and which also holds replicated sister chromatids together.

Genome segregation Chromosome replication is followed by genome segregation (FIG.1). In bacteria, the ParAB system is involved in plasmid and nucleoid segregation61. parA and parB are usually located in an operon (FIG.4a) that is autoregulated by ParB, and encode a filament-forming ParAfamily ATPase and a DNA-binding protein, ParB. These proteins act in conjunction with a centromere-like DNA-binding site called parS, which is located in the origin-proximal part of the genome61. All three components are needed for proper genome segregation. ParB binds parS, after which ParA binds to ParB and polymerizes into dynamic filaments66. Currently, the best understood bacterial genome segregation system is that of Caulobacter crescentus, in which the parS-containing region of one chromosome copy is translocated by a pulling mechanism powered by depolymerization of the ParA filaments that extend between the ParB-bound parS site and the distal cell pole67 (FIG.4a). A curious feature of this partitioning system is the intracellular poletopole oscillation that has been observed for certain ParA-family proteins, such as MinD68 and Soj69 of Escherichia coli and Bacillus subtilis, respectively; however, it is unclear whether this activity affects genome segregation. Finally, ParAB-mediated segregation is restricted to the origin-proximal (parScontaining) part of the chromosome, whereas the bulk of the bacterial genome seems to gradually segregate concomitantly with replication, through condensation onto the initially segregated DNA70 by a mechanism that is not well understood.
The Seg system. An initial characterization of a machinery that affects genome segregation in S.solfataricus was recently reported71, providing the first insights into archaeal genome segregation components. Similarly to the ParAB system, two genes are involved in S.solfa taricus segregation, segA and segB, which are located
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MinD
A protein belonging to the bacterial MinCDE complex, which prevents cell division from occurring at the cell poles by blocking FtsZ polymerization.

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a
ParB

parS site
ParB

b
Seg B Seg B

parA

parB

CGTTTCACGTGAAACA TGTTCCACGTGAAACA Replisome ParA Replicated DNA parS sites oriC ParB

segA TATA box GTGGACTCGTC GAAGAGTCTAG GTGGACTAGTA GAAGAGTCTAG GAATAGTCTAG TAAGACTCTAG TAAGAGTCTAT Pulling model SegA Chromosome

segB

S. solfataricus S. islandicus M. sedula S. tokodaii S. acidocaldarius

Pushing model

SegB

Figure 4 | Genome segregation in bacteria and archaea. a|ParAB-mediated chromosomeNature segregation in Caulobacter Reviews | Microbiology crescentus. ParB binds to repeat sequences called parS sites, which are located several kilobases upstream of the parAB operon111 (not drawn to scale). The parS sites function as centromeric elements early in the genome segregation process. Before replication initiation, the chromosome replication origin (oriC) is positioned near the oldest cell pole (that is, the most recent cell division event occurred at the opposite pole)67,70. As the parS sites are located in the origin-proximal part of the chromosome, they are duplicated shortly after replication initiation (in the figure, replication has proceeded about one-third of the way along the chromosome; black DNA represents replicated DNA, and blue DNA is not yet replicated). ParB binds to the two parS sites, and one copy of parS is sequestered at the old pole; the other copy is not sequestered, so that ParA can bind ParB and then form filament bundles that extend from parS to the distal cell pole. Depolymerization of the ParA filaments results in rapid movement of the parS-containing region of the replicated chromosome to the distal cell pole111. b|SegAB-mediated chromosome segregation in Sulfolobus solfataricus. The S.solfataricus segAB operon (not drawn to scale) has a similar genetic organization to the parAB operon, and SegA and ParA belong to the same protein family. However, SegB has no sequence similarity to ParB. Transcription of segA and segB, which overlap by 4bp, is autoregulated through binding of SegB to repeat sequences located on either side of the TATA box71. The repeat located between the TATA box and the transcription start site is highly conserved within the order Sulfolobales (deviations from the consensus sequence are indicated in pink in the alignment), whereas the repeat upstream of the TATA box is present in only S.solfataricus and Sulfolobusislandicus. It is currently unknown whether the SegB-binding sites function as centromeric elements for the chromosome segregation machinery. In a pulling model for SegAB-mediated chromosome segregation, analogous to the ParA-mediated chromosome segregation of C.crescentus and the eukaryotic mitotic process72, SegB binding to the chromosome (multiple binding sites are assumed) is followed by SegA binding to SegB. SegA polymerization results in filaments stretching from the chromosomes to anchoring sites at the cell membrane. Depolymerization of SegA filaments then results in a pulling force that segregates the chromosomes. In a pushing model, similar to the ParB partitioning system of plasmid R1 in Escherichiacoli61, SegB binding to multiple sites in the chromosome is followed by SegA binding to SegB, and then SegA polymerization between the two replicated and aligned chromosomes results in a pushing force that moves the chromosomes apart. Alternatively, SegAB might segregate only the parS-containing region of the chromosome, similarly to the C.crescentus ParAB system. M.sedula, Metallosphaera sedula; S.acidocaldarius, Sulfolobus acidocaldarius; S.tokodaii, Sulfolobus tokodaii.

near replication origin ori1. These two genes form an operon in which the 3end of segA overlaps the 5end of segB by 4bp (FIG.4b). SegA has about 30% identity to members of the ParA family, whereas SegB is specific to archaea and has no sequence similarity to bacterial ParB proteins71, although structural similarity cannot be ruled out. Similarly to ParA, SegA polymerizes in the presence of ATP, and this is enhanced by SegB71, which seems to be a functional analogue of ParB. Similarly to bacterial ParB, SegB forms dimers and is likely to provide autoregulation of the segAB operon by binding to sequence elements immediately upstream of segA71 (FIG.4b). Whether these sequences also act as centromeric elements during chromosome segregation is not known. Expression of segAB is strongly induced around the replication initiation stage in S.acidocaldarius57, consistent with a role in genome segregation. In an S.solfataricus strain overexpressing SegAB, and in a SegA(K14Q) mutant strain, aberrant nucleoid
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morphology and anucleate cells are observed71, demonstrating that SegA and SegB affect nucleoid conformation and proper genome segregation. It is currently unknown whether the SegAB system exerts segregational force through a pulling mechanism (FIG.4b) similarly to the C.crescentus ParAB system (FIG.4a), or possibly through a pushing mechanism, as has been observed for certain bacterial plasmids (FIG.4b), nor whether intracellular oscillation of SegA occurs. Irrespective of the mechanistic details, the existence of bacterial-like chromosome resolution and genome segregation components in Sulfolobusspp. contrasts sharply with the eukaryotic-like replication machinery, raising interesting questions regarding the origin and evolution of the replication and chromosome processing machineries in all three domains oflife. Although several features of the SegAB system are similar to aspects of the ParAB system, other observations indicate that they operate differently. First,
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in Sulfolobus spp., genome segregation is separated from chromosome replication by an extensive G2 period23 (FIG. 1), whereas in C.crescentus and other bacteria, the partitioning process occurs concomitantly with replication67, suggesting that there are fundamental regulatory differences between the two systems. Second, chromosome alignment and the
Box 1 | The ESCRT-III system
Viral budding

ESCRT-III Endosomal sorting

VPS4 Cytokinesis Nucleus

rapid segregation of entire chromosomes is observed in Sulfolobusspp.23 (FIG.1), whereas in C.crescentus, gradual segregation of the nucleoid occurs70 after the initial ParAB-mediated segregation of a limited chromosomal region proximal to the origin. The Sulfolobusspp. characteristics are indicative of an active, cell cycle-coupled segregation mechanism21, reminiscent of the eukaryotic mitotic process72. The SegAB system seems to be phylo genetically restricted to the order Sulfolobales within the phylum Crenarchaeota71 (except for a few euryarchaeotes that contain distant homologues of either SegA or SegB), suggesting that additional genome segregation mechanisms exist in archaea. Other candidate genome segregation proteins include homologues of the bacterial protein MinD73 (see below) and the structural maintenance of chromosomes superfamily (Smc super family) of ATPases7476. Exciting discoveries are to be expected from further investigations into the molecular, functional and regulatory features of archaeal genome segregation.

ESCRT-0

ESCRT-I ESCRT-II

Cell division and the cytoskeleton Genome segregation is followed by cell division, during which cell constriction occurs between the segregated chromosomes, and two new daughter cells are formed. The two main cell division systems in archaea are FtsZ and Cdv (cell division), and their occurrence is correlated to phylogeny 77. This demonstrates that there are important differences in fundamental cellular processes between organisms from the main archaeal phyla, providing strong support for this classification.
FtsZ-mediated division. In bacteria, the most common division mechanism relies on FtsZ, a member of the tubulin family78. An inner membrane-associated circumferential ring-shaped structure called the Zring is formed at mid-cell through the polymerization of FtsZ monomers. Ring formation is initiated during the replication elongation stage79, thereby tightly linking division to replication and genome segregation. Division occurs through gradual contraction of the Zring, powered by GTP hydrolysis80. The division process involves a large protein complex called the divisome, through which FtsZ-mediated membrane invagination is coordinated with inward growth of the septal cell wall and FtsK-mediated translocation (see above) of the remaining chromosomal DNA out of the way of the closing septum64,80. Archaea belonging to the phylum Euryarchaeota (with the exception of the Picrophilus genus81) also contain FtsZ homologues that are used for division1619, as do species belonging to the candidate phylum Nanoarchaeota9. Although detailed molecular studies of archaeal FtsZ-mediated division have not been reported, the presence of a centrally located Zring in immunostained haloarchaeal cells18,19 suggests that they use a constriction mechanism similar to that of bacteria. Homologues of bacterial MinD have been identified in several euryarchaeotal and thaumarchaeotal species, and a role for these proteins in genome segregation or cell division has been proposed73. In this context, it should be noted that one of the four minD genes in the eury archaeote P.abyssi is organized into an operon together
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Ubiquitylated cargo Nature Reviews |pathway Microbiology The eukaryotic ESCRT (endosomal sorting complex required for transport) was initially identified as a system involved in protein sorting. ESCRT complexes recognize and sort ubiquitylated proteins (cargo) into late endosomes (also known as multivesicular bodies) for trafficking to the lysosome for degradation93,94 (see the figure). The system consists of the four protein complexes ESCRT0, ESCRT-I, ESCRT-II and ESCRT-III along with the protein vacuolar protein sorting4 (VPS4), a member of the AAA+ ATPase family34. ESCRT0, ESCRT-I and ESCRT-II are involved in cargo recognition at the endosomal membrane, which is followed by association of the ESCRT-III complex. ESCRT-III polymerizes on the outer endosomal membrane surface, and this gives rise to membrane deformation and budding away from the cytoplasm into the lumen of the endosome, along with ESCRT-bound cargo, resulting in the generation of intraluminal cargo-containing vesicles93,94. VPS4 is recruited to the endosomal membrane by interactions between the microtubule interaction and transport (MIT) domain of VPS4 and the MIT-interacting motif (MIM) domain of ESCRT-III proteins93,94. The ATPase activity of VPS4 provides the energy required to disassemble the ESCRT-III complex during membrane invagination. ESCRT-III is a multipurpose protein complex involved in viral release, cytokinesis (eukaryotic cell division), exosome biogenesis and autophagy84,93. A variety of enveloped viruses recruit ESCRT-III components to their budding sites at the host cell membrane (see the figure); ESCRT-III and VPS4 are then used in the release of new virions from the cell84. Recently, it was also shown that ESCRT-III and VPS4 are involved in the final abscission stage of cytokinesis (see the figure)84. The initial invagination and constriction of the cell membrane is induced by the formation of an actinmyosin ring, after which ESCRT-III and VPS4 are recruited for the final scission and separation of the daughter cells95,96. Because of the homology between ESCRT proteins and the Cdv (cell division) proteins of archaea, this finding suggests that the systems served a primordial cell division function in an ancient progenitor of the Archaea and Eukarya lineages, and have since evolved to carry out other cellular functions in eukaryotes when the actin and myosin families were adopted for cell constriction.

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Late endosomes
Membrane-bound compartments in which material destined for degradation in the lysosome is sorted in eukaryotic cells. Late endosomes have passed through several stages of endosome maturation.

Exosome
A multiprotein complex that is involved in RNA degradation in eukaryotes and archaea.

with an adjacent ftsZ gene82, suggesting that they cooperate during cell division. Because most archaea contain a pseudomurein cell wall or a proteinaceous S layer (surface layer) outside the cell membrane10, it is likely that divisome-like complexes which coordinate septationrelated activities are used, although the components of such complexes have yet to be identified. Haloquadratum walsbyi 83, a haloarchaeon with square-shaped cells that grow in leaf-thin structures, represents a particularly intriguing organism for cell division studies. A key question is how FtsZ activity is coordinated in time and space to produce the remarkable square cells, which are often associated in large multicellularsheets.

Box 2 | Alternative division modes and cytoskeletal elements

The FtsZ and Cdv (cell division) systems are absent in organisms belonging to the order Thermoproteales (in the phylum Crenarchaeota)13, and constricted cells have not been observed in microscopy studies of actively growing Pyrobaculum spp. cell cultures25,97,98. Furthermore, an unusual snapping mode of cell fission has been observed in Thermoproteus tenax (also a member of the order Thermoproteales), in which cells rapidly split in the centre after a period of intense vibration and form a Vshaped structure before the two cells separate a few minutes later99. Thermoproteales members contain an actin homologue known as crenactin15, encoded by cren1. As a contractile actinmyosin ring forms the core of eukaryotic cytokinesis100, these archaea were expected to possess an actin-based division machinery77. However, crenactin forms spiral-shaped intracellular structures that function in cell shape determination15. Consistent with this finding, the phylogenetic distribution of cren1 is strongly correlated with elongated cell morphologies15. Furthermore, the bacterial protein MreB, which also belongs to the actin family, forms similar spiral-shaped intracellular structures101 (the detailed structure of bacterial MreB filaments is currently under debate102) and has a key role in bacterial cell shape determination103. Rearrangement of crenactin15 and MreB101 spirals in archaea and bacteria, respectively, into parallel ring-shaped structures has been observed, presumably in preparation for cell division, which might suggest that they have similar functional roles. Thus, a functional role for crenactin in cell division cannot be ruled out. Moreover, in Escherichiacoli, MreB ring structures interact with FtsZ rings104, and in Chlamydiaspp. (which lack ftsZ), MreB is recruited to a ring-shaped division structure105, although MreB-dependent constriction activity has not been demonstrated. However, the fact that E.coli FtsZ can mediate vesicle contraction in the absence of MreB106 argues against an essential role for MreB and, by analogy, crenactin, in division. Additional candidates for division factors include the recently discovered arcadin proteins, which are encoded together with crenactin in a conserved Thermoprotealesspecific operon15. Although three of the four arcadins in Pyrobaculumcalidifontis show a spiral-shaped pattern when immunostained, suggesting that they act in conjunction with crenactin in shape determination, arcadin2 displays a punctuate intracellular distribution15. The fluorescence foci of arcadin2 are often localized between segregated nucleoids, and a single focus has been observed at one of the poles in newly divided cells, compatible with a function in the division process. In conclusion, shape determination seems to be the primary function of crenactin, in agreement with the conservation of cren1 in rod-shaped, filamentous and needle-like crenarchaeotes and in Candidatus Korarchaeum cryptophilum, and its absence in lineages with spherical or coccoid morphologies. One exception is the thaumarchaeote Nitrosopumilusmaritimus, which is rod shaped27,107 but lacks cren1 (REF.15). Thus, alternative shape-determining systems seem to exist in thaumarchaeotes, and this area of research requires further study. Finally, species belonging to the crenarchaeotal genus Ignicoccus display a unique intracellular organization with two cytoplasmic membrane systems separated by a large compartment108. During cell proliferation, division initially seems to occur at the innermost membrane, followed by outer-membrane fission108. The regulation and organization of this unusual division mode provides a fascinating topic for future investigation, including the question of whether the same division machinery (presumably Cdv, as no ftsZ gene is present) is used at both membranes.

Cdv-mediated division. The division mechanism of cren archaeotes long remained enigmatic, as genes encoding established cell division proteins were not found in crenarchaeotal genomes. The issue was resolved by the discovery of a new division system, known as Cdv, in S.acidocaldarius 13,14. The machinery consists of CdvA, CdvB and CdvC, which multimerize into a ring-shaped structure situated at the leading edge of the invaginating membrane during constriction13, similarly to the contracting Zring in euryarchaeotes and bacteria. Interest in the Cdv system has been further augmented by the observation that CdvB and CdvC have homology with components of the ESCRT-III (endosomal sorting complex required for transportIII) system84, which is involved in vesicle formation processes in eukaryotic cells (BOX1). CdvB is homologous to members of the vacuolar protein sorting24 (VPS24) family of ESCRTIII proteins, whereas CdvC is homologous to the VPS4 family ESCRT-III associated ATPases13,14. The Cdv machinery has been characterized at the molecular level in S.acidocaldarius 13,14,85,86, S.solfatari cus 14,85 and Metallosphaera sedula 87. CdvB and CdvC interact in vitro, and deletion analysis showed the interaction to be mediated by protein motifs analogous to the microtubule interaction and transport (MIT) and MIT-interacting motif (MIM) domains (BOX1) that are responsible for interactions between the corresponding proteins in the ESCRT-III system85. CdvC has ATPase activity and forms ring-shaped multimers containing 12 or 14 subunits87, the latter being composed of two stacked rings like the yeast counterpart, Vps4. In contrast to CdvB and CdvC, CdvA is not homologous to any ESCRT protein and is unique to archaeal lineages that depend on Cdv for division13. CdvA is a membraneinteracting protein85 with an E3B domain that binds to the winged helix domain of CdvB in vitro, whereas binding to CdvC is not observed87. Binding of CdvA and CdvB to liposomes has been shown to cause membrane deformation85. Interestingly, CdvA forms helical filamentous structures together with chromosomal DNA in M.sedula87, suggesting a possible role for this protein in genome segregation. For example, CdvA might be involved in the reorganization of chromatin structure that is necessary for the chromosome alignment observed before genome segregation, or it might coordinate genome segregation with cell division. The combined data suggest a model in which Cdvmediated constriction involves initial binding of CdvA to the inner surface of the cell membrane85 at the future division site, possibly in conjunction with chromosomal DNA87, followed by recruitment of CdvB and CdvC. The Cdv proteins then multimerize into a spiral-shaped86 structure around the inside of the membrane, and this structure gradually constricts the cell. The constriction force is likely to be generated through ATP binding and hydrolysis by CdvC, similarly to the ATP-dependent activity of VPS4 in ESCRT-III-mediated vesicle formation in eukaryotic cells (BOX1). In terms of regulatory features, strong induction of the cdv operon shortly before entry into the division stage has been demonstrated in S.acidocaldarius 57. This induction has been
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Table 1 | Archaeal cell cycle characteristics
Cell cycle feature Replication
Replication origin Initiator binding sites Specificity factor and initiator Replisome components Chromosome dimer recombinase Recombination site Single* or multiple ORBs

Archaeal phylum Euryarchaeota Crenarchaeota

Multiple ORBs Orc1Cdc6 protein , WhiP

Thaumarchaeota
Unknown Unknown Orc1Cdc6 protein Eukaryotic homologues, PolD XerA* Unknown Unknown Smc

Orc1Cdc6 protein

Eukaryotic homologues , PolD XerA* dif* Unknown MinD*, Smc

Eukaryotic homologues XerA* dif*

Genome segregation
Genome segregation system Other possible genome segregation proteins SegAB (order Sulfolobales)* CdvA , crenactin , arcadin2

Cell division
Division system FtsZ* MreB* Absent CdvABC Crenactin (order Thermoproteales) Arcadin 1arcadin4 (order Thermoproteales) CdvABC Absent Absent

Cytoskeleton
Actin family protein Arcadins

Cdv, cell division; ORBs, origin recognition boxes; Orc1Cdc6, origin recognition complex1cell division cycle 6; PolD, DNA polymerase D; Smc, structural maintenance of chromosomes. *Bacterial trait. Eukaryotic trait. Uniquely archaeal trait.

Autophagy
The lysosomal degradation and recycling of defective cellular components in eukaryotes.

Actinmyosin ring
A contractile ring structure that is made of non-muscle myosin and actin filaments, and is responsible for cell constriction during the division process (cytokinesis) in eukaryotic cells.

Structural maintenance of chromosomes family

A family of ATPases that are involved in different aspects of higher-order chromosomal organization and dynamics in all three domains of life.

Tubulin family
A family of GTP-binding globular proteins, each of which forms the structural subunit of a eukaryotic microtubule. Microtubules are involved in a wide range of cellular functions, including shape maintenance, motility, and formation of the spindle fibres that pull chromosomes apart during mitosis.

corroborated at the protein level by the strict correlation between the presence of Cdv proteins (as detected by immuno staining) and the presence of segregated nucleoids and visible cell constriction13, as well as by the absence of Cdv proteins in cells in which genome segregation and division have not commenced13. Thaumarchaeotes also use the Cdv machinery for division, as demonstrated by the presence of Cdv structures between segregated nucleoids in dividing N.maritimus cells27. Thaumarchaeotes also have a distant FtsZ homologue that seems to have evolved to function in processes unrelated to cell division, as ring-shaped FtsZ structures are not observed in these cells, and FtsZ abundance shows no correlation to cell cycle stage27. This hypothesis is further supported by the observation that the GTP-binding motif, which is a hallmark of the tubulin family, is absent in the thaumarchaeotal FtsZ homologues88. Also, one of the N.maritimus CdvB homologues has been shown to assemble into polymers when heterologously expressed in yeast and mammalian cells, whereas no polymerization is observed for the FtsZ homologue89. Further investigations will be necessary to obtain a full understanding of Cdv-mediated division, which is also likely to provide insights into core features of the eukaryotic ESCRT-III machinery. Efforts are needed to identify additional proteins that interact with Cdv-based divisomes, as well as to further elucidate the structural and functional features of the constriction mechanism, in addition to the regulatory and energetic aspects of the process. Crenarchaeotes encode multiple CdvB para logues90 in which the CdvA-binding epitopes are absent85, and the cellular functions of these proteins should be elucidated, including their putative participation in

intracellular vesicle trafficking, in virion generation and release91, and in vesicle-mediated export of enzymes and other material out of the cell92. Finally, although two evolutionarily unrelated division mechanisms, FtsZ and Cdv, have been identified in archaea, it seems that additional division systems await discovery (BOX2).

Perspectives The main cell cycle processes in archaea display bacterial, eukaryotic and uniquely archaeal traits (TABLE1). The initiation of chromosome replication involves the binding of eukaryotic-type Orc1Cdc6 initiator proteins to ORB sequences in the replication origin. This is a conserved feature of archaea, although ORB consensus sequences vary between phyla. The chromosome replication machinery (FIG.2) is entirely eukaryotic in nature in all phyla, except for PolD, the polymerase in euryarchaeotes. Chromosome dimer resolution seems to occur through a bacterial-like mechanism in which the recombinase, XerA, recognizes a dif site in the chromosome and then catalyses site-specific recombination that resolves the two daughter chromosomes (FIG.3). In terms of genome segregation, the SegAB system is the only genome segregation system identified in archaea (FIG.4), and the data available suggest that it functions in an analogous manner to the bacterial ParAB system. SegAB is restricted to species in the order Sulfolobales, indicating that other genome segregation mechanisms exist. Cell division is mediated by an FtsZ-dependent mechanism in euryarchaeotes, whereas crenarchaeotes and thaumarchaeotes utilize the ESCRT-III-like Cdv system. Both systems are absent in archaea from the order Thermoproteales (within the phylum Crenarchaeota), indicating that additional
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division systems exist. The actin homologue crenactin is specific to archaea from the order Thermoproteales, and although a role in cell division cannot be ruled out for this protein, the spiral-shaped cytoskeletal structures that are formed by crenactin and the strict correlation to elongated cell morphologies indicate that its primary function is in cell shape determination. Despite the considerable advances that have been made in understanding the cell cycle of archaea, elucidation of mechanistic and functional details is still needed in several areas. One of the main issues that remains largely unexplored is the regulatory network and signals that control and coordinate chromosome replication, genome segregation and cell division, with respect to both each other and cellular growth. Insights into this key aspect will be important not only for our understanding of fundamental archaeal biology, but also to help resolve core features of eukaryotic cell cycle regulation. The majority of studies to date have been carried out on members of the genus Sulfolobus, and future work should involve investigations of a wider range of archaeal lineages to determine the generality of our current knowledge of the cell cycle and to uncover additional systems that await discovery. This will require increased efforts to develop growth conditions suitable for obtaining pure cultures from different physio logical groups and candidate phyla. Investigations into the full range of evolutionary branches are particularly important if we are to understand those traits that are general features of archaea, those representing environ mental adaptations and those that are lineage-specific solutions to the problem of how to orchestrate the growth and proliferation of a livingcell.

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Acknowledgements

This work was supported by the Swedish Research Council (grant 62120105551). This Review is dedicated to the memory of Carl Woese, who discovered archaea.

Competing financial interests statement

The authors declare no competing financial interests.

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