In stark contrast with eukaryotes and bacteria, our knowledge on chromosome segregation in archaea is very rudimentary, partly due to the fact that most archaeal genomes have been sequenced only in the last decade, but also attributable to the development of genetic tools to manipulate some archaea only in recent years. I report the findings of the few investigations conducted so far, discussing implications and questions that still need to be addressed.

Despite the paucity of information thus far, SMC proteins are anticipated to play a significant role in chromosome segregation, based on the high level of conservation and by analogy with the mechanisms uncovered in bacteria.

Soppa and colleagues characterized the cell-cycle profile of an SMC-like protein, named Sph1, in H. salinarum []. Using synchronized cultures, sph1 gene expression was shown to be cell-cycle-regulated with a peak at the stage of cell division septum formation. Given that maximal expression is reached at a late stage of the cell cycle when chromosome segregation is nearly completed, Sph1 might be involved in DNA repair in a final step of chromosome replication []. Whether this SMC factor has a role in genome segregation remains to be elucidated.

SMC condensins are widespread across archaeal phyla. Early studies analysed the possible involvement of the SMC protein of Methanococcus voltae in chromosome segregation. Inactivation of the smc gene in this euryarchaeon resulted in aberrant genome partition and cell morphology []. Approximately 20% of the cells harboured no chromosome, and around 2% displayed a size that was three to four times larger than that of wild-type cells. Quantitation of the DNA of these so-called titan cells showed a content 10–20-fold higher than that present in normal cells. This phenotype indicates that the SMC protein presides over an important cell-cycle checkpoint in M. voltae and plays a crucial role in chromosome segregation [].

Anucleate and titan cell phenotypes caused by insertional inactivation of the structural maintenance of chromosome (smc) gene in the archaeon Methanococcus voltae.

Anucleate and titan cell phenotypes caused by insertional inactivation of the structural maintenance of chromosome (smc) gene in the archaeon Methanococcus voltae.

Microscopy investigations have revealed that increased expression of segAB in S. solfataricus cells disrupts chromosome segregation, as evidenced by the presence of anucleate cells, highly condensed nucleoids squeezed into one-half of the cell volume and split, guillotined chromosomes []. These findings indicate that SegA and SegB play a key role in chromosome segregation. Further support comes from the observations that segAB are highly repressed upon UV irradiation [] and that their expression starts concurrently with the initiation of DNA replication [], both of which underscore a function in chromosome segregation. The mechanism underpinning how the SegAB complex drives sister chromatids apart remains to be elucidated.

SegA is an ATPase assembling into higher-order structures in vitro upon ATP binding, while SegB is a site-specific DNA-binding protein contacting palindromic sequences located upstream of the segAB cassette []. The two proteins interact with one another, and SegB synergistically affects SegA self-assembly dynamics, perhaps acting as a nucleator protein. SegB is a dimeric protein that binds specifically to an imperfect palindromic motif located upstream of the segA start codon (site 1) and then at position –59 with respect to the same start codon (site 2) ( Figure 2 A) []. These sites might be archaeal centromere analogs. However, at this stage it cannot be ruled out that the sites might also act as regulatory regions that control the expression of the segAB cassette. Whether additional sites are scattered across the chromosome is currently unknown.

A recent study has reported the identification and initial characterization of a dedicated chromosome-segregation system in the thermophilic crenarchaeon S. solfataricus []. This genome-partitioning apparatus consists of two proteins, SegA and SegB, and a cis-acting centromere-like region ( Figure 2 A) . Intriguingly, the complex is a hybrid partition machine: SegA is an ortholog of bacterial, Walker-type chromosome-encoded ParA proteins, whereas SegB is an archaea-specific factor lacking any sequence identity to either eukaryotic or bacterial proteins. However, SegB displays sequence identity to a group of conserved, uncharacterized proteins present in both Crenarchaea (∼80% identity) and some Euryarchaea (30–46% identity) ( Figure 2 B). Interestingly, the genes encoding SegB proteins are located invariably downstream of segA orthologs. BLAST searches against archaeal genomes available so far indicated that the segAB cassette is present in an array of archaeal genera belonging to both Crenarchaea and Euryarchaea phyla ( Figure 2 B). Although the ploidy and genome content has not been determined for all these genera, what is tantalizing is that the majority of these Archaea are monoploid, or at most diploid such as M. thermautotrophicus. If a cell harbours only one or two copies of the chromosome, then a rigorous toolkit to segregate DNA at cell division is a stringent sine qua non. The 3′ end of segA overlaps with the 5′ end of segB: this arrangement suggests that the genes may be part of a single transcriptional unit implying that SegA and SegB work together to effect the same biological process. Supporting evidence derives from a transcription profiling study showing that the Sulfolobus acidocaldarius homologues of segA and segB are coexpressed in a cell-cycle-regulated fashion [].

The SegAB System Is Widespread across Archaea. (A) Organization of the segAB cassette, including the upstream sso0033 gene and the two DNA sites (in lilac) to which SegB binds. (B) Phylogenetic tree of a nonexhaustive set of SegB orthologs. Genomic context studies show that each segB gene is accompanied by a segA gene. Blue box, crenarchaeal SegB cluster; green box, euryarchaeal SegB orthologs. Within the crenarchaeal cluster the Sulfolobus solfataricus P2 strain, whose SegAB have been characterized, is shown in red.

Figure 2 The SegAB System Is Widespread across Archaea. (A) Organization of the segAB cassette, including the upstream sso0033 gene and the two DNA sites (in lilac) to which SegB binds. (B) Phylogenetic tree of a nonexhaustive set of SegB orthologs. Genomic context studies show that each segB gene is accompanied by a segA gene. Blue box, crenarchaeal SegB cluster; green box, euryarchaeal SegB orthologs. Within the crenarchaeal cluster the Sulfolobus solfataricus P2 strain, whose SegAB have been characterized, is shown in red.

The AspA–ParBA Machinery: Borrowing Building Blocks from Bacteria and Eukaryotes

53 Schleper C.

et al. A multicopy plasmid of the extremely thermophilic archaeon Sulfolobus effects its transfer to recipients by mating. 54 She Q.

et al. Genetic profile of pNOB8 from Sulfolobus: the first conjugative plasmid from an archaeon. 55 Schumacher M.A.

et al. Structures of archaeal DNA segregation machinery reveal bacterial and eukaryotic linkages. 30 Hayes F.

Barillà D. The bacterial segrosome: a dynamic nucleoprotein machine for DNA trafficking and segregation. 31 Baxter J.C.

Funnell B.E. Plasmid partition mechanisms. 53 Schleper C.

et al. A multicopy plasmid of the extremely thermophilic archaeon Sulfolobus effects its transfer to recipients by mating. 54 She Q.

et al. Genetic profile of pNOB8 from Sulfolobus: the first conjugative plasmid from an archaeon. 53 Schleper C.

et al. A multicopy plasmid of the extremely thermophilic archaeon Sulfolobus effects its transfer to recipients by mating. Figure 3 Organization and Structures of the pNOB8 AspA–ParBA System. (A) Schematic diagram of the gene cluster. The 3′ end of aspA overlaps with the 5′ end of parB, and the 3′ end of parB overlaps with the 5′ end of parA. (B) AspA–DNA structure (PDB 5FC0) showing three interacting AspA dimers (in orange, green, and blue) associated with the DNA fragment containing the 23 bp putative centromeric site. (C) Adenylyl-imidodiphosphate (AMP-PNP)-bound ParA dimer structure (PDB 4RU8). The ATP analog AMP-PNP is shown in red. (D) (Left) ParB-N structure (PDB 4RSF); (right) ParB-C dimer structure with one monomer shown in green and the other in magenta (PDB 4RS7). The structural images were generated by using PyMOL version 1.8.0.7 (Schrodinger) using the indicated PDB coordinates. Sulfolobus NOB8H2 is a strain isolated by the archaea pioneer Wolfram Zillig and coworkers from acidic hot springs at Noboribetsu in the island of Hokkaido, Japan []. This strain harbours a 41 kbp conjugative plasmid, pNOB8, whose sequence has been determined []. The plasmid contains ∼50 ORFs including two tandem genes, orf45 and orf46, whose products show homology, respectively, to ParB and ParA families of bacterial partition proteins. The 36 kDa polypeptide encoded by orf46 is a 315-residue protein with similarity (33–37%) to bacterial ParAs. orf45 encodes a 470-amino acid protein (55 kDa), whose homology to bacterial ParBs is confined to the N-terminal domain (residues 1–190) (42–58% similarity), whereas the C-terminus shares homology with eukaryotic proteins, including kinesin-like motor proteins. Interestingly, a closer inspection of the region immediately upstream of parB revealed a small gene, orf44, which encodes a 93-amino acid protein of 10.7 kDa with no sequence homology to any characterized segregation protein []. The 3′ end of this gene overlaps with the 5′ end of parB, and similarly, the 3′ end of parB overlaps with 5′ end of parA ( Figure 3 A) . This arrangement suggests that orf44, parB, and parA may be part of a single transcriptional unit. A tricistronic partition cassette is an interesting feature that is not common in the bacterial domain, whose typical segregation modules are bicistronic []. Furthermore, there is evidence suggesting that the orf44–parBA cassette of this plasmid encodes a bona fide partition system: when pNOB8 is transferred by conjugation into a different Sulfolobus strain, the plasmid undergoes a genetic rearrangement due to a single recombination event, which produces the deletion variant pNOB8-33 []. This plasmid presents a deletion of a ∼8 kbp region resulting in the loss of the orf44–parBA cassette and is not stably maintained [].

55 Schumacher M.A.

et al. Structures of archaeal DNA segregation machinery reveal bacterial and eukaryotic linkages. a rchaeal s egregation p rotein A ), is a dimeric, site-specific DNA-binding protein that recognizes a 23 bp palindromic motif located upstream of its gene. DNase I footprints have shown that AspA binds to the 23 bp putative centromere and, at higher concentrations, spreads on the DNA in the 5′ direction, protecting over 200 bp from the initial nucleation site [ 55 Schumacher M.A.

et al. Structures of archaeal DNA segregation machinery reveal bacterial and eukaryotic linkages. A very recent study has provided structural and mechanistic insights into this novel DNA segregation machinery []. Orf44, renamed AspA (forrchaealegregationrotein), is a dimeric, site-specific DNA-binding protein that recognizes a 23 bp palindromic motif located upstream of its gene. DNase I footprints have shown that AspA binds to the 23 bp putative centromere and, at higher concentrations, spreads on the DNA in the 5′ direction, protecting over 200 bp from the initial nucleation site []. In contrast, ParB binds DNA nonspecifically only at high concentrations, which represents a departure from the bacterial paradigm. The structure of AspA discloses an elongated dimer containing a winged helix-turn-helix DNA binding fold. Remarkably, the AspA–DNA structures exhibit multiple AspA dimers ( Figure 3 B) that, when extended by packing, lead to the assembly of a continuous left-handed helix.

55 Schumacher M.A.

et al. Structures of archaeal DNA segregation machinery reveal bacterial and eukaryotic linkages. 55 Schumacher M.A.

et al. Structures of archaeal DNA segregation machinery reveal bacterial and eukaryotic linkages. 56 Leonard T.A.

et al. Structural analysis of the chromosome segregation protein Spo0J from Thermus thermophilus. Interaction investigations established that pNOB8 ParB binds to AspA and ParA. However, AspA does not associate with ParA, suggesting that ParB might act as an adaptor protein within the complex. With 470 residues, pNOB8 ParB is larger than canonical ParB proteins found in bacteria, and consists of two distinct domains, ParB-N (residues 1–320) and ParB-C (residues 370–470) connected by a flexible linker. AspA interacts with ParB-N only, whereas ParA does not bind to either the N- or C-terminus of ParB, suggesting that the extended linker region contains the ParA contacting interface []. ParB-C was found to mediate nonspecific DNA binding. The determination of the three-dimensional structure revealed that ParB-N shares weak similarity with the N-terminus of the chromosome segregation ParB, Spo0J, of Thermus thermophilus ( Figure 3 D, left) [].

55 Schumacher M.A.

et al. Structures of archaeal DNA segregation machinery reveal bacterial and eukaryotic linkages. 57 Tachiwana H.

et al. Crystal structure of the human centromeric nucleosome containing Cenp-A. Further small-angle X-ray scattering (SAXS) studies on the ParB-N-AspA complex showed that a ParB-N dimer encases the sides and top of the AspA dimer. Interestingly, in the SAXS model, ParB-N dimers can be docked onto each AspA dimer in the AspA-DNA helix, fitting in a lock-and-key fashion into the helix grooves and generating a multiprotein superhelical structure []. In this assembly the ParB-C protrudes into the solvent and is connected to the ParB-N domain through the long flexible linker. As ParB-C binds nonspecific DNA, this domain is free to associate with random DNA sequences on either the plasmid or chromosome, or both. Interestingly, this activity indicates that ParB is not simply an adaptor ‘cushion’ sitting between AspA and ParA, but is involved in additional aspects of the segregation process. Surprisingly, the structure of ParB C-terminus exhibits a fold similar to that of the CenpA histone variant which replaces histone H3 on centromere sequences and is involved in assembly of the kinetochore segregation machinery in eukaryotic cells ( Figure 3 D, right) []. This unforeseen observation draws a parallel between DNA segregation in archaea and eukaryotes. A further significance of the finding lies in that, to date, histone homologs have been identified in Euryarchaea; however, they are an exception in Crenarchaea.

58 Leonard T.A.

et al. Bacterial chromosome segregation: structure and DNA binding of the Soj dimer – a conserved biological switch. 59 Schumacher M.A.

et al. Structural mechanism of ATP induced polymerization of the partition factor ParF: implications for DNA segregation. 55 Schumacher M.A.

et al. Structures of archaeal DNA segregation machinery reveal bacterial and eukaryotic linkages. The structure of pNOB8 ParA shows strong resemblance to bacterial Walker-type segregation proteins, such as the chromosome ParA homolog, Soj, from Thermus thermophilus [] and multidrug resistant plasmid TP228 ParA homolog, ParF ( Figure 3 C) []. Similar to bacterial ParA proteins, pNOB8 ParA shows nonspecific DNA-binding activity []. This finding suggests that ParA might bind the nucleoid in Sulfolobus NOB8H2 and thereby might allow anchoring and transport of the plasmid with the aid of ParB. However, the mechanism underlying pNOB8 segregation remains to be elucidated.

Altogether, the AspA–ParB–ParA complex is a novel three-component segregation machine, encoded on both crenarchaeal plasmids and chromosomes, that merges building blocks from bacteria and eukaryotes and opens exciting fresh perspectives on genome segregation in archaea.