Chromosome segregation in mediated by a hybrid DNA partition machine

Anne K. Kalliomaa-Sanforda, Fernando A. Rodriguez-Castañedaa, Brett N. McLeoda, Victor Latorre-Rosellóa, Jasmine H. Smitha, Julia Reimannb, Sonja V. Albersb, and Daniela Barillàa,1

aDepartment of Biology, University of York, Wentworth Way, York YO10 5DD, United Kingdom; and bMax Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse 10, D-35043 Marburg, Germany

Edited by Stanley N. Cohen, Stanford University School of Medicine, Stanford, CA, and approved January 10, 2012 (received for review August 15, 2011)

Eukarya and, more recently, some bacteria have been shown to rely actin-like, or tubulin-like (3). Although dissimilar in primary on a cytoskeleton-based apparatus to drive chromosome segrega- sequence and structure, upon nucleotide binding many of these tion. In contrast, the factors and mechanisms underpinning this NTPases polymerize into extensive filaments that move apart the fundamental process are underexplored in archaea, the third do- newly replicated plasmids to opposite cell poles. Walker-type parti- main of life. Here we establish that the archaeon solfa- tion cassettes are the most widespread. They encode an ATPase, taricus harbors a hybrid segrosome consisting of two interacting ParA, harboring a Walker motif, and a DNA-binding , , SegA and SegB, that play a key role in genome segrega- ParB. ParA, specified by several plasmids, assembles into filaments tion in this organism. SegA is an ortholog of bacterial, Walker-type and has been proposed as the dynamic component of a mitotic ParA proteins, whereas SegB is an archaea-specific factor lacking spindle-like apparatus in bacteria (4–7). Segregation cassettes of sequence identity to either eukaryotic or bacterial proteins, but the parAB class are found also on most bacterial chromosomes (8). sharing homology with a cluster of uncharacterized factors con- Mechanistic insights into how ParAB proteins mediate chromo- served in both crenarchaea and euryarchaea, the two major archae- some segregation have been provided by studies on Vibrio cholerae al sub-phyla. We show that SegA is an ATPase that polymerizes in and Caulobacter crescentus. Both exhibit an asymmetric chromo- vitro and that SegB is a site-specific DNA-binding protein contact- some segregation pattern: in newborn cells the origin of replication ing palindromic sequences located upstream of the segAB cassette. is located close to the old pole and, after replication, one of the SegB interacts with SegA in the presence of nucleotides and origins is translocated to the opposite, new pole (9, 10). V. c h o l e r a e dramatically affects its polymerization dynamics. Our data demon- has two chromosomes that replicate and segregate independently. strate that SegB strongly stimulates SegA polymerization, possibly Both contain a Walker-type cassette specifying ParA and ParB by promoting SegA nucleation and accelerating polymer growth. orthologs. Microscopy studies have shown that ParAI encoded by Increased expression levels of segAB resulted in severe growth chromosome I assembles into a comet-like structure that pulls the and chromosome segregation defects, including formation of an- origin of the chromosome from the old to the new cell pole. ParBI ucleate cells, compact nucleoids confined to one half of the cell bound to the centromere site of the chromosome (located in proxi- compartment and fragmented nucleoids. The overall picture emer- mity to the origin of replication) lags behind and is translocated ging from our findings indicates that the SegAB complex fulfills a across the cell by the shrinking ParAI structure (10). Cells in which crucial function in chromosome segregation and is the prototype of parAI is deleted display chromosome I segregation defects. These a DNA partition machine widespread across archaea. observations suggest a model in which the V. c h o l e r a e Par system mediates chromosome I segregation via a mitosis-like pulling me- nucleoid ∣ polymerization ∣ segrosome ∣ Sulfolobus ∣ Walker-type ParA chanism. Similarly, ParA and ParB of C. crescentus are components of a segrosome in which ParA polymerizes into an elongated band very organism, whether unicellular or multicellular, needs to that pulls the chromosome from the old to the new pole (11). Eensure an accurate distribution of its genetic material to the Deletion or overexpression of parA and parB causes filamentous progeny to preserve survival of the species. In both eukaryotic cell morphology and disrupts cell division and chromosome segre- and prokaryotic cells, the machine involved in genome segrega- gation (9). tion includes DNA-anchoring factors and polymeric proteins In contrast with bacteria, our knowledge concerning chromo- capable of driving DNA molecules apart in dividing cells. some segregation in archaea, the third of life, is very The events underpinning chromosome segregation in eukar- restricted. The nucleoid undergoes changes in shape and conden- yotes have been extensively investigated. During mitosis, the sation during the cell cycle in the model organism Sulfolobus microtubules of the mitotic spindle capture sister chromatids and solfataricus and a long interval occurs between termination of pull them to opposite spindle poles (1). The mechanisms and key chromosome replication and genome partitioning (12). Newly players behind chromosome segregation in prokaryotes are not replicated chromosomes are held together for a prolonged time yet fully elucidated. However considerable progress has been made during the G2 phase of the cell cycle (13). However, the mole- in deciphering this process in bacteria in the last two decades. An cular mechanisms and factors underpinning chromosome segre- important principle that has emerged is that chromosomes are gation in archaea are terra incognita. actively translocated to specific cellular addresses prior to cell divi- Here we have investigated an uncharacterized segregation lo- sion. The most detailed picture of how a segregation-designed cus harbored by the chromosome of S. solfataricus. The sso0034 apparatus drives DNA molecules to specific cellular locations derives from studies on low copy number plasmids. These mobile Author contributions: A.K.K.-S., F.A.R.-C., B.N.M., and D.B. designed research; A.K.K.-S., elements harbor a dedicated partition cassette consisting of three F.A.R.-C., B.N.M., V.L.-R., J.H.S., and J.R. performed research; J.R. and S.V.A. contributed components: a gene encoding a NTPase, a second gene encoding a new reagents/analytic tools; A.K.K.-S., F.A.R.-C., B.N.M., and D.B. analyzed data; and D.B. DNA-binding protein, and a cis-acting centromere-like site located wrote the paper. in proximity to the genes. The DNA-binding factor directly con- The authors declare no conflict of interest. tacts the centromere and recruits the NTPase. The resulting com- This article is a PNAS Direct Submission. plex drives the attached plasmids to specific subcellular locations 1To whom correspondence should be addressed. E-mail: [email protected]. (2). Partition cassettes belong to three main classes on the basis of This article contains supporting information online at www.pnas.org/lookup/suppl/ the NTPase that they encode, which can be either Walker-type, doi:10.1073/pnas.1113384109/-/DCSupplemental.

3754–3759 ∣ PNAS ∣ March 6, 2012 ∣ vol. 109 ∣ no. 10 www.pnas.org/cgi/doi/10.1073/pnas.1113384109 Downloaded by guest on October 2, 2021 gene specifies an ortholog of bacterial ParA partition proteins SegB is a Dimeric Protein Displaying a α-Helix-Rich Secondary Struc- (14). The 3′ end of sso0034 overlaps the start of a short gene, ture. To investigate the function of SegB, the protein was purified sso0035, encoding a hypothetical protein of unknown function. from E. coli. Dimethyl pimelimidate (DMP) crosslinking experi- We have defined the biochemical function of both proteins and ments clearly showed that SegB forms dimers (Fig. 1A). This re- established that increasing their concentration in the cell results sult was confirmed by size-exclusion chromatography/multiangle in severe defects in growth and chromosome segregation. Our laser light scattering (SEC-MALLS) analysis that detected dimers findings indicate an involvement of the two proteins in genome with a molecular weight of 34 kDa and no higher oligomers partitioning in S. solfataricus and, in view of this function, we have (Fig. 1B). Circular dichroism (CD) spectroscopy revealed that named them SegA (SSO0034) and SegB (SSO0035) for chromo- SegB is highly thermostable up to 90 °C and is mostly α-helical some segregation. with a low percentage of β-sheet and disordered regions (Fig. 1C).

Results SegB is a Site-specific DNA-binding Protein: Identification and Fine- SegA is an ATPase Belonging to the ParA Family of Walker-type DNA Mapping of its Binding Site. SegB was likely to be a DNA-binding Partition Proteins. SegA encoded by the chromosome of the ar- protein based on the location of its gene downstream of a parA chaeon S. solfataricus shares substantial sequence identity with gene. The second gene of a DNA segregation cassette typically bacterial ParA proteins involved in genome segregation. These encodes a DNA-binding protein. In bacterial partition cassettes, factors harbor a divergent Walker A ATP-binding motif, whose the centromere-like sites are located upstream or downstream of consensus is KGG-gKt/s (15). SegA is a 220 residue protein ex- the genes and clustered around the origin of replication of the hibiting the deviant Walker A box, KGGVGKT. It shows signifi- chromosome. Assuming an analogous arrangement in S. solfatar- cant identity to chromosomally encoded bacterial icus, we amplified DNA regions upstream and downstream of ParA proteins: 34% to B. subtilis Soj, 29% to V. cholerae ParAI segAB. The fragments were incubated with SegB and subjected and 27% to C. crescentus ParA (Fig. S1). The Walker A motifs in to electrophoretic mobility shift assay (EMSA). A 1 kb fragment these homologs are identical and share a more extended patch of spanning the region upstream of segA was clearly shifted by SegB invariant residues compared to the A box of other ParA family members. It appears that orthologs of SegA are widespread in archaea. A phylogenetic tree of selected archaeal orthologs and bacterial ParAs shows that SegA proteins encoded by crenarch- aea cluster together forming a branch distinct from that including some euryarchaeal SegA orthologs (Fig. S2A). The presence of Walker motifs in SegA suggests that the protein binds ATP. To test this, the protein was purified from Escherichia coli and assayed by fluorescence anisotropy in the presence of 20∕30-O-(N- Methyl-anthraniloyl) adenosine-5′-O-tri- phosphate (MANT-ATP). SegA avidly binds MANT-ATP,exhibit- ing a hyperbolic increase in anisotropy values and a Kd of approximately 170 nM (Fig. S2B). The ATPase activity of SegA was assessed by thin-layer chromatography (Fig. S2C). The values obtained show that SegA is a weak ATPase, as observed for bac- terial ParAs (4, 16, 17).

SegB is a Conserved, Archaea-specific Protein. The segA gene is followed by a short gene, segB (sso0035), encoding a 109 residue

protein of unknown function. SegB lacks homology to either eu- BIOCHEMISTRY karyotic or bacterial proteins, but displays high sequence identity to a group of conserved, uncharacterized proteins widespread in both crenarchaea (approximately 80% identity) and euryarchaea (32 to 46% identity) (Fig. S3). The level of conservation is more pronounced within crenarchaea, whereas the similarity with eur- yarchaea orthologs is confined mostly to the C-terminal domain. This region contains invariant residues, many of which are char- acterized by side chains harboring cyclic structures (P51, W56, Y66, P72, F74, Y92, and P93). All the proteins of this conserved cluster have similar size (approximately 100 residues) with the exception of STS240 (67 amino acids), Sulfo- lobus acidocaldarius Saci_0203 (151 amino acids) and two larger polypeptides, Metallosphera sedula Msed_0557 (272 residues) and Methanobrevibacter ruminantium Mru_2161 (248 residues), which are not included in the alignment. Interestingly, most genes encoding SegB-like proteins are located downstream of segA homologs (18, this work). The 3′ Fig. 1. SegB is a dimeric, helical protein. (A) 15% SDS-gel showing the pro- end of S. solfataricus segA overlaps with the 5′ end of segB: this ducts of a time-course crosslinking experiment in which SegB (10 μM) was arrangement suggests that the genes may be part of a single tran- incubated with DMP (10 mM). (B) SEC-MALLS analysis of SegB. The protein μ scriptional unit implying that SegA and SegB are involved in the (10 M) was injected into a Superdex 200 HR10/30 column and the elution profile monitored by differential refractive index (dashed line) and Rayleigh same biological process. Supporting evidence derives from a tran- light scattering (continuous line). The brown line at the top of the peak is the scription profiling study showing that the S. acidocaldarius homo- derived molar mass and the green tract is the portion used for the peak molar logs of segA and segB (Saci_0204 and Saci_0203, respectively) are mass calculation. (C) CD spectrum of SegB (10 μM) recorded at 30 °C and cal- coordinately expressed in a cell cycle-regulated fashion (19). culated percentages of secondary structure elements.

Kalliomaa-Sanford et al. PNAS ∣ March 6, 2012 ∣ vol. 109 ∣ no. 10 ∣ 3755 Downloaded by guest on October 2, 2021 in the presence of competitor DNA indicating that it contains one may be centromeric sites. However, we cannot rule out whether or more specific binding sites for the protein (Fig. 2A). This result some might act as regulatory sites to which SegB binds to control established that SegB is a DNA-binding protein. We narrowed expression of the divergent segAB cassette and sso0033 gene. the region down to 500 and then 250 bp upstream of segA Bacterial centromere-like sites consist of repeat motifs. An in- (Fig. 2A). An unrelated DNA fragment was not shifted by SegB spection of the region protected by SegB did not disclose obvious (Fig. 2B). The binding site was characterized by DNase I foot- repeats, however MEME software (20) highlighted an imperfect printing on the region spanning approximately 200 bp upstream palindromic motif (Fig. 2D) located at two sites, one immediately of segA including the translational start site of the gene. The pat- upstream of the segA start codon (site 1) and one further up- tern indicated a first, well-defined region of protection beginning stream centered at position −59 with respect to the same codon a few bases downstream of the segA ATG codon (Fig. 2C, first (site 2). SegB binds to site 1 with high affinity, as protection of this box). At higher SegB concentrations, the protection became region is clearly visible at the lowest concentration of SegB more extended. In addition, a second region of protection was (50 nM) (Fig. 2C). To crosscheck that the identified motif is in- observed further upstream: it covered the start of the upstream deed a SegB binding site, we performed fluorescence anisotropy gene sso0033 (Fig. 2C, second box). This gene is located on the experiments with SegB and a fluorescently labeled oligonucleo- strand opposite to that of segAB and encodes a 165 residue hy- tide harboring site 1, in the presence of polydIdC. SegB bound pothetical protein of unknown function, which is highly conserved the oligonucleotide with high affinity (Kd of approximately in two genera of (Sulfolobus and ). 55 nM). No binding was observed when an unrelated oligonucleo- In a number of these organisms sso0033 homologs systematically tide was used (Fig. 2E). These findings establish that the identi- are found approximately 100 bp upstream of parA genes (Fig. S4). fied motif is a bona fide SegB binding site. The extended DNA This observation and the DNase I footprinting data suggest a protection observed in the footprint spans beyond site 1 and 2: functional link between sso0033 and the segAB cassette. The pat- this pattern is consistent either with the presence of additional tern of DNA protection mediated by SegB indicates that the re- motifs yet to be identified or with the spreading of SegB along gion upstream of segA harbors multiple SegB binding sites, which the DNA. Multiple SegB binding is also supported by the EMSA results, which show gradually more pronounced retardation with increasing protein concentration.

SegA Assembles into Polymers. By using CD spectroscopy we showed that SegA is highly thermostable and contains a large percentage of β-strands followed by a lower proportion of turns, disordered regions and α-helices (Fig. 3A). Several ParA proteins assemble into filaments involved in plasmid and chromosome partitioning. DMP crosslinking experiments in the presence of ATP indicated that SegA forms dimers, but also higher oligomers, some of which are too large to enter the gel (Fig. 3B). This result signalled that SegA may be able to polymerize and thus we per- formed sedimentation assays. SegA was incubated at 30 °C with or without nucleotides, separated by centrifugation in pellet and supernatant fractions and analyzed by SDS-PAGE. In the absence of nucleotides, most of SegA was in the supernatant (Fig. 3C). However, when either ATP or ADP was added, a larger portion of SegA was in the pellet, indicating that the binding of these nucleotides promotes SegA self-assembly into polymers. The nonhydrolysable analog adenosine-5-O-(3-thiotriphosphate) (ATPγS) did not elicit significant polymerization, as the SegA amount in the pellet is comparable to that present in the nucleo- tide free reaction (Fig. 3C). These results show that the binding of specific nucleotides mediates SegA assembly into polymers. Indeed short SegA needles (>100 nm) were observed with nega- tive-stain electron microscopy (EM) (Fig. S5). We further inves- tigated the real time polymerization kinetics of SegA by dynamic light scattering (DLS). Without nucleotides, no polymerization was detected (Fig. 3D). Upon addition of ADP (1 mM), the light scattering intensity increased reaching a value of approximately 1;000 kilocounts/second ðkct∕sÞ within a few minutes (Fig. 3D, Bottom). In parallel, the size of the particles increased from approximately 4 to approximately 300 nm (Fig. 3D, Top). When ATP (1 mM) was added to SegA, it elicited an immediate and Fig. 2. SegB is a site-specific DNA-binding protein. (A) EMSAs in which bio- more dramatic surge in scattering intensity that then increased tinylated DNA fragments (1–5 nM) spanning the region upstream of segA steadily to approximately 4;000 kct∕s. Interestingly, the polymer were incubated with SegB in the presence of competitor polydIdC DNA size (approximately 30 nm) was more modest than that observed (1 μg). Arrow: free DNA; bracket: SegB•DNA complexes. (B) EMSA control ex- upon ADP binding. This is consistent with the recovery of a larger periment including an unrelated DNA fragment. (C) Top, DNase I footprinting amount of SegA in the pellet of the sedimentation reaction with showing the regions protected by SegB; Bottom, diagram illustrating the ADP (Fig. 3C). ATPγS did not trigger polymerization (Fig. 3D). SegB protection regions and corresponding DNA sequence. Site 1 and 2 These findings mirror the outcome of sedimentation assays and are underlined. (D) Logo of the palindromic motif identified through MEME (20). (E) Fluorescence anisotropy assay measuring binding of SegB to a dou- indicate that both ATP and ADP stimulate SegA polymerization. ble-stranded [Cy3]-labeled oligonucleotide (23 bp) (5 nM) containing the mo- The different patterns observed for the two nucleotides suggest tif (site 1) (blue line) and to an unrelated oligonucleotide (5 nM) (red line) in that ATP is more proficient than ADP at nucleating polymers. the presence of competitor polydIdC DNA (50 μg∕mL). However, ADP binding appears to mediate a conformational

3756 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1113384109 Kalliomaa-Sanford et al. Downloaded by guest on October 2, 2021 the effect of SegB on the polymerization kinetics of SegA, we performed DLS experiments including both proteins. First, SegA alone was monitored, then ATP/ATPγS or ADP was added and finally SegB (Fig. 3E). In the absence of nucleotides, SegA dis- played a flat baseline; when either ATP or ADP were added, this triggered an increase in light scattering, whereas ATPγS elicited no polymerization. However, subsequent addition of SegB induced a very abrupt surge in scattering intensity in reactions including either ATP (up to 45;000 kct∕s) or ATPγS (up to 30;000 kct∕s). A less dramatic increase was observed for the ADP-containing reaction (Fig. 3E, Bottom). The rise in intensity was accompanied by a striking increase in particle size (up to 2 μm) (Fig. 3E, Top). These observations show that SegB stimu- lates SegA polymerization, possibly by facilitating SegA nuclea- tion, and vigorously accelerates polymer growth. The DLS results confirm the outcome of the pelleting assays: both lines of evidence establish that SegB interacts with SegA only in the pre- sence of ATP, ATPγS, or ADP, although ATP and ATPγS are the preferred nucleotide configurations to drive SegB-induced poly- merization.

Increased Expression of segA and segB Results in Growth and Chromo- some Segregation Defects. The in vitro data point to a plausible scenario in which SegA and SegB are involved in chromosome segregation. To test this hypothesis, the cellular concentration of both SegA and SegB or SegA alone was increased by transform- ing S. solfataricus with a virus-based vector harboring the genes under control of an arabinose-inducible promoter. The rationale was that increased protein levels would impinge on the natural role of these factors and the phenotype would provide clues about their function. When grown in selective medium, the transfor- Fig. 3. SegA assembles into extensive polymers, whose growth and dy- mants expressing higher levels of segAB exhibited growth impair- namics are affected by SegB. (A) CD spectrum of SegA (10 μM) recorded at ment, which was visible both in the absence (leaky expression) 30 °C and percentages of secondary structure elements. (B) 15% SDS-gel and presence (overexpression) of arabinose and slightly less in μ showing a DMP crosslinking time-course of SegA (7 M) in the presence of the presence of (repression) (Fig. S6). No substantial ATP (2 mM). The bracket at the top indicates higher oligomers. (C) Sedimen- tation assay in which SegA (18 μM) was incubated with and without nucleo- growth defect was observed for transformants overexpressing tides (4 mM) at 30 °C and then centrifuged. 100% of the pellet (P) and 20% of segA alone, which displayed a growth rate similar to that of the the supernatant (S) fractions were resolved on a 15% SDS-gel. (D) SegA poly- untransformed strain (Fig. S6). This suggests that overexpression merization followed by DLS. Bottom illustrates the increase in light scattering of both genes affects the fitness of the strain, whereas SegA over- intensity, expressed as kct∕s; Top shows the corresponding augmentation in production is better tolerated. Although the presence of the polymer average size (nm). SegA (5 μM) was incubated at 30 °C with and virus-based vector in S. solfataricus has been reported to cause without nucleotides (ADP/ATP/ATPγS) (1 mM). The arrows indicate the point growth retardation (21), we did not observe any significant differ- at which MgCl2 (1 mM) (gray) and nucleotides (black) were added. (E) SegB μ ence in growth rate between the untransformed strain and trans- promotes SegA polymerization. DLS kinetics showing the effect SegB (5 M) formants expressing segA alone. BIOCHEMISTRY in the presence and absence of nucleotides. The green arrow indicates the time of SegB addition. Note the substantial difference in vertical scale If SegA and SegB are chromosome segregation factors, then in panel D and E.(F) Sedimentation assay in which SegA (12 μM) and SegB disruption of their cellular concentration should cause chromo- (12 μM) were coincubated with and without nucleotides. some partition defects. Cells were stained with 4′,6-diamidino-2- phenylindole (DAPI) and chromosome morphology examined change in SegA that allows the nucleation of longer, but less by fluorescence microscopy. As defective growth was obvious abundant polymers. in any of the conditions tested irrespective of presence/absence of arabinose, we began by examining cultures without arabinose. SegB Associates with SegA and Affects Its Polymerization Dynamics. The strain harboring the segAB construct showed a high percen- The interplay between SegA and the putative partner SegB was tage of anucleate cells: out of 697 cells examined, approximately investigated. Sedimentation assays including both proteins (1∶1 18.0% were nucleoid-free (Fig. 4 A and B). These cells had a cor- ratio, monomer) were performed to assess whether SegB binds rugated surface. We observed several pairs of conjoined cells un- to SegA (Fig. 3F). In the absence of nucleotides, the amounts dergoing division, in which only one of the cells harbored the of SegA and SegB in supernatant and pellet fractions did not dif- chromosome due to aberrant segregation, as well as early predi- fer from those observed when the proteins were centrifuged se- visional cells displaying a nucleoid confined to one half of the cell parately. This suggests that the proteins do not interact in the (Fig. 4B and Fig. S7A). DAPI intensity quantitations provided absence of nucleotides or that under this condition SegB does further evidence in support of a segregation rather than replica- not stimulate SegA polymerization, thus no proteins are recov- tion defect: values obtained for conjoined cells, one of which is ered in the pellet. However, in the presence of ATP, a significant anucleate, are consistent with the nucleate cell containing two fraction of SegB cosedimented with SegA polymers. To a lesser chromosome equivalents. This also applies to predivisional cells extent, the same pattern was observed with ATPγS and ADP. harboring a compact nucleoid squeezed into one half of the cell. When SegB was tested alone, virtually no protein was recovered Overexpression of segA resulted in approximately 11.0% of an- in the pellet either in the absence or presence of nucleotides. ucleate cells (out of 653): this strain also exhibited cells with mul- These findings indicate that SegB associates with SegA polymers tilobbed chromosomes (Fig. 4A and Fig. S7B). To obtain further and that the interaction requires ATP,ATPγS, or ADP.Toaddress evidence for the role of SegA in chromosome segregation, we

Kalliomaa-Sanford et al. PNAS ∣ March 6, 2012 ∣ vol. 109 ∣ no. 10 ∣ 3757 Downloaded by guest on October 2, 2021 Fig. 4. Increased gene dosage of segA and segB re- sults in a high rate of anucleate cells and anomalous nucleoid morphology in S. solfataricus. Phase con- trast and fluorescence microscopy of DAPI-stained cells expressing higher levels of segAB and segA (A)orsegA-K14Q (C). The arrows point to anucleate cells. Bar ¼ 2 μm. (B) Examples of aberrant chromo- some segregation phenotypes observed for the strain with increased levels of SegAB. Bar ¼ 1 μm.

constructed a segA mutant allele encoding a protein with a lysine- ment of SegB proteins (Fig. S3) shows a high level of conservation to-glutamine substitution at position 14. This lysine residue of the throughout the sequence within crenarchaea, whereas the simi- Walker A motif is invariant in ParA proteins (4, 15). Overexpres- larity shared with euryarchaea is confined to the C-terminal re- sion of segA-K14Q resulted in a more severe phenotype than gion. The conservation of this domain across the two subphyla wild type segA with approximately 21% of anucleate cells (out suggests that it plays a key role in the interactions entertained of 748) (Fig. 4C and Fig. S7C). The untransformed strain showed by SegB with either DNA and/or SegA. The C-terminus harbors approximately 3.8% of anucleate cells. This percentage is rather several invariant residues, many of which are prolines and aro- high, however, S. solfataricus PH1-16 is a uracil auxotrophic strain matic amino acids. Residues containing ring structures in their and its genome hosts numerous transposons (22), whose insertion side chain are not surprising in thermophilic proteins, as they at multiple loci may have generated polar mutations and gene enhance thermostability by reducing backbone flexibility and low- inactivations, resulting in reduced fitness of the strain. Neverthe- ering the entropy of protein unfolding (24). The CD analysis in- less, the divide observed between the control and the strains har- dicated that SegB consists almost entirely of α-helices (79%) boring the segA/segB expression constructs remains significant. (Fig. 1C) and the secondary structure prediction is consistent with These results indicate that SegA and SegB are factors crucial the experimental data. On this ground, it is tempting to speculate for chromosome segregation in S. solfataricus. that SegB proteins may recognize DNA through a helix-turn-helix (HTH) DNA-binding motif, which is likely to map onto the con- Discussion served C-terminal domain. Bacterial centromere-binding pro- Archaea are the third branch of the tree of life (23). Despite the teins fall into two structural classes, containing either a HTH progress made in decoding molecular mechanisms in these organ- or a ribbon-helix-helix (RHH) DNA-binding fold (3). HTH cen- isms in the last three decades, virtually no information is available tromere-binding proteins have been shown to be able to spread on the fundamental process of chromosome segregation. Here we along the DNA. SegB is a dimer (Fig. 1) specifically recognizing have investigated the segAB locus harbored by the chromosome an imperfect palindromic motif positioned immediately upstream of S. solfataricus and established that the encoded proteins play a of the segA start codon (site 1) and at position −59 with respect to crucial role in mediating chromosome segregation in this archae- the same codon (site 2) (Fig. 2). This discovery raises intriguing on. While SegA is an ortholog of bacterial ParA proteins, SegB is questions: first, are the identified sequences centromeric sites? an archaea-specific invention. This “hybrid” system appears to be The motifs bear no resemblance to bacterial chromosomal parti- widespread in both crenarchaea and euryarchaea (Fig. S3) and tion sites, but this is not surprising, as SegB is unrelated to ParB. likely underpins an evolutionarily conserved mechanism of DNA A bioinformatic study found that the sequence of chromosome segregation in archaea. centromeric sites is extremely conserved throughout bacteria, but SegB is a site-specific DNA-binding protein (Fig. 2) and a absent from the primary chromosomes of 45 archaea spp., includ- member of a novel family of archaea-specific factors conserved ing S. solfataricus (8). Archaea using a par-based system for chro- in both crenarchaea and euryarchaea (Figs. S3 and S4), which mosome dynamics must harbor distinct partition sites recognized do not share homology with either bacterial or eukaryotic pro- by an archaea-specific set of proteins. We hypothesize that the teins. The arrangement of segB genes downstream of (21) and SegB boxes might be centromeric sites, although this remains partially overlapping with segA genes suggested a functional as- to be proven. The S. solfataricus chromosome exhibits three ori- sociation between the encoded products and provided clues to gins of replication (25) and the segAB cassette is positioned in the the function of SegB. In numerous bacterial plasmid partition oriC1-proximal 7% region. The location of site 1, positioned be- cassettes, parA is accompanied by a gene unrelated to parB and tween the putative TATA box and the start site of segA and site 2, belonging to a collection of short loci encoding diverse DNA- positioned just upstream of the putative TATA box for sso0033 binding proteins that behave as functional analogs of ParB (2). also suggests that they may act as regulatory sites to which SegB Thus the archaeal SegB cluster provides a further example of binds to control expression of the divergent segAB cassette and how evolution has crafted different prokaryotic DNA segregation sso0033 gene. The pattern of DNA protection mediated by SegB factors that associate with canonical ParA proteins. The align- (Fig. 2C) and the observation that a number of strains of Sulfo-

3758 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1113384109 Kalliomaa-Sanford et al. Downloaded by guest on October 2, 2021 lobaceae display a conserved genomic arrangement (Fig. S4) hint and indicative of failed segregation. The strain overexpressing at a functional connection between sso0033 and the segAB mod- segA showed no growth defect and approximately 11.0% of nu- ule. The SegB footprint spans beyond the identified site 1 and 2, cleoid-free cells with this percentage increasing to approximately which is consistent either with the presence of additional motifs 21%, when segA-K14Q was expressed. Overall the phenotypes yet to be determined or with the possibility that SegB spreads observed indicate that SegA and SegB play a key role in chromo- along the DNA from defined nucleation points. some segregation dynamics. The levels of anucleate cells are com- SegA assembles into polymers upon binding ATP and to a parable to those reported for C. crescentus, wherein increased lesser extent ADP (Fig. 3). The ability to form filamentous struc- levels of ParA or ParAB resulted in 5% and 10% of anucleate – tures is a distinctive feature of Walker-type ParA proteins (4 7, cells, respectively (9). Interestingly, segAB are highly repressed 11). The finding that archaeal SegA polymerizes is exciting, as it upon UV irradiation (27). This is consistent with a role in chro- provides experimental evidence establishing that assembly of mosome segregation, as genome replication, partition, and cell DNA segregation proteins into polymers constitutes a feature division are put on hold until DNA damage is repaired. conserved across prokaryotes. SegB interacts with SegA in the As no other segregation apparatus has been investigated in ar- presence of ATP/ATPγS/ADP and profoundly affects SegA poly- chaea, we are unable to draw comparisons with other archaeal merization (Fig. 3 E and F). SegB addition instantly stimulates SegA polymerization, possibly by promoting SegA nucleation, species. However, it is interesting that inactivation of the gene and vigorously accelerates polymer growth. ATP and ATPγS encoding the Structural Maintenance of Chromosome protein appear to be the preferred catalysts to drive SegB-induced poly- caused approximately 20% of anucleate cells in the archaeon merization. Although SegB shows no sequence identity to known Methanococcus voltae (28). proteins, structural homology searches have revealed that it The picture emerging from our findings indicates that SegA shares similarity with the Drosophila Msps (mini-spindles) protein and SegB fulfill a crucial role in chromosome segregation in that associates with and modulates the assembly dynamics of and that they are the prototype of a DNA the plus end of mitotic spindle microtubules. Plus-end-tracking partition machine widespread across archaea. Lastly, as both eu- proteins (þTIPs) act as polymerization chaperones by facilitating karyotes and, more recently, bacteria have been shown to rely on tubulin subunit addition (26). This observation suggests a tanta- a cytoskeleton-based chromosome segregation system, the dis- lizing scenario in which SegB promotes SegA polymerization by covery of a polymer-involving apparatus in archaea is an exciting adopting a strategy analogous to that of þTIPs. We speculate that milestone bridging the gap between bacteria and eukarya. SegA polymers form a cytoskeletal framework in vivo involved in delivering chromosomes into the two halves of a dividing cell. It Materials and Methods remains to be elucidated whether another factor anchors the Detailed descriptions can be found in Supporting Information. The segA and SegA polymers to the membrane at a pole-like site. In C. crescen- segB genes were amplified from S. solfataricus P2, cloned in pET22b and tus a pole-localized protein, TipN, tethers the ParA polymer to proteins purified by affinity chromatography. Strains, conditions used for the new pole (11). fluorescence anisotropy, ATPase assays, CD, crosslinking, SEC-MALLS, EMSA, DNaseI footprinting, DLS, EM, sedimentation assays, and microscopy are pro- Increased expression of either segA alone or segAB in S. solfa- vided in figure legends and Supporting Information. taricus results in severe chromosome segregation defects (Fig. 4 and Fig. S7). The strain harboring the segAB construct exhibited ACKNOWLEDGMENTS. Supported by grants from the Biotechnology and approximately 18.0% of anucleate cells and a drastic growth Biological Sciences Research Council (BB/F012004/1) and The Leverhulme impairment. Predivisional cells with a nucleoid consisting of two Trust (RPG-245) (to D.B.). We thank Finbarr Hayes for valuable comments genomes strictly confined to one half of the cell were common on the manuscript.

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