The archaeal Ced system imports DNA Marleen van Wolferena,1, Alexander Wagnera,1, Chris van der Doesa, and Sonja-Verena Albersa,2 a

Molecular Biology of Archaea, Institute of Biology II – Microbiology, University of Freiburg, 79104 Freiburg, Germany

Edited by Norman R. Pace, University of Colorado at Boulder, Boulder, CO, and approved January 12, 2016 (received for review July 13, 2015)

The intercellular transfer of DNA is a phenomenon that occurs in all domains of life and is a major driving force of evolution. Upon UV-light treatment, cells of the crenarchaeal genus Sulfolobus express Ups pili, which initiate cell aggregate formation. Within these aggregates, chromosomal DNA, which is used for the repair of DNA double-strand breaks, is exchanged. Because so far no clear homologs of bacterial DNA transporters have been identified among the genomes of Archaea, the mechanisms of archaeal DNA transport have remained a puzzling and underinvestigated topic. Here we identify saci_0568 and saci_0748, two genes from Sulfolobus acidocaldarius that are highly induced upon UV treatment, encoding a transmembrane protein and a membrane-bound VirB4/HerA homolog, respectively. DNA transfer assays showed that both proteins are essential for DNA transfer between Sulfolobus cells and act downstream of the Ups pili system. Our results moreover revealed that the system is involved in the import of DNA rather than the export. We therefore propose that both Saci_0568 and Saci_0748 are part of a previously unidentified DNA importer. Given the fact that we found this transporter system to be widely spread among the Crenarchaeota, we propose to name it the Crenarchaeal system for exchange of DNA (Ced). In this study we have for the first time to our knowledge described an archaeal DNA transporter. Archaea

| DNA transport | conjugation | type IV pili | VirB4

U

pon UV treatment, Sulfolobales species induce the expression of Ups pili (UV-inducible pili of Sulfolobus) (1–3). These are type-IV pili (T4P) that are essential for cellular aggregation and chromosomal DNA exchange (3, 4). The ability of Sulfolobales to exchange DNA was shown to increase cellular fitness under UV stress (4). Because other DNA-damaging agents such as bleomycin and mitomycin C also induce Ups pili and cellular aggregation, the transfer of DNA is thought to play a role in repair of double-strand breaks via homologous recombination (4). Not much is known about DNA transfer among archaea; only a few examples of competence and conjugation systems have been described. Four archaeal species were shown to be naturally competent: Pyrococcus furiosus, Thermococcus kodakarensis, Methanobacterium thermoautotrophicum, and Methanococcus voltae (5–8). However, these natural transformation mechanisms have not been studied on a molecular level and in none of these archaeal species homologs from bacterial competence systems could be identified. Distinct machineries must therefore be present in archaea. Because bacterial natural transformation often involves T4P (9), one could hypothesize that Sulfolobales also exchange DNA via an uptake and release mechanism in which the Ups pili play a vital role similar to that in bacterial competence systems. However, the exchange of DNA among Sulfolobus species was shown to be insensitive to DNase treatment, and recombinants could not be obtained by mixing the cells with lysate or purified DNA (10). This demonstrates that exchange of DNA requires cellular contact and transfer occurs directly from one cell to another without passing through the surrounding medium. A conjugation-like mechanism or cellular fusion therefore seems more likely. DNA transfer among archaea via direct cellular contact was first described for the euryarchaeon Haloferax volcanii. Similar to the UV-inducible transfer of DNA among Sulfolobales, Haloferax

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species exchange chromosomal DNA between cells connected by bridges (11). This transfer is thought to occur in a bidirectional manner via cell fusion leading to the formation of diploid cells with mixed chromosomes (12). Interestingly, this type of DNA transfer was shown to occur between different Haloferax species and involved DNA fragments of up to 500 kbp DNA (13). Nevertheless, the mechanism of DNA transfer is so far not understood. Other described archaeal conjugative systems include self-transmissible plasmids, which have so far only been studied for Sulfolobus species. These plasmids are grouped into the so-called pKEF and pARN plasmids (14, 15) and only a few of their genes encode homologs of bacterial conjugation proteins, including the so-farunstudied ATPases VirD4 and VirB4. It is unknown how cellular contact is initiated to achieve plasmid transfer. During plasmid conjugation, Sulfolobus islandicus cells form aggregates, similar to those observed upon UV stress (16). One could therefore imagine that cells make use of the genomically encoded Ups system to initiate cell contact. Many other conjugation proteins such as relaxases can also not be identified in archaea based on homology, indicating that again distinct mechanisms must be present that differ significantly from their bacterial counterparts. Hence, archaeal DNA transfer remains a poorly investigated topic. Previously performed microarray studies on Sulfolobus solfataricus and Sulfolobus acidocaldarius revealed in addition to an up-regulation of the ups operon several other up-regulated genes (1, 2), including genes involved in, for instance, DNA repair, such as the operon encoding helicase HerA, nuclease NurA, Rad50, and Mre11 (17, 18). Because we were interested in the mechanism of DNA transfer between Sulfolobus cells, we searched for up-regulated genes putatively involved in DNA transport. We focused on three clustered genes encoding one larger and two smaller membrane proteins. Additionally, we looked at a virB4/herA homolog. Homologs of these genes Significance Among bacteria, transfer of DNA has been studied in great detail. Several bacterial DNA transfer systems have been described on a molecular level including competence and conjugation systems. In Archaea, DNA exchange has been observed for a number of organisms and its importance for horizontal gene transfer and DNA repair is greatly valued. However, for none of these organisms has the mode of transport been studied on a molecular level. Here we describe a set of genes directly involved in the transfer of chromosomal DNA between Sulfolobus acidocaldarius cells. Homologs of these genes are widely distributed among the Crenarchaeota. For the first time to our knowledge we give molecular insights into intercellular transport of DNA between archaeal cells. Author contributions: M.v.W., A.W., C.v.d.D., and S.-V.A. designed research; M.v.W. and A.W. performed research; M.v.W., A.W., C.v.d.D., and S.-V.A. analyzed data; and M.v.W., A.W., and S.-V.A. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. 1

M.v.W. and A.W. contributed equally to this work.

2

To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1513740113/-/DCSupplemental.

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are present in the genomes of all Sulfolobales and several Desulfurococcales and Acidilobales, in which they are predicted to form an operon. In deletion mutants of either the larger membrane protein or the VirB4/HerA homolog, DNA transfer was completely abolished, showing that they are indeed involved in DNA transfer. Using PCR on genomic markers we could moreover show that, unlike other prokaryotic cell-to-cell contact-dependent DNA transfer systems, this system functions as a DNA importer. We have therefore for the first time to our knowledge given insights into an archaeal DNA transporter and showed that it functions very differently from bacterial conjugation systems. Because this system is present in many members of the Crenarchaeota, we propose the name Crenarchaeal system for exchange of DNA (Ced). Results Bioinformatics and Transcriptional Analysis of Putative DNA Transport Proteins. To find genes involved in DNA transfer between Sulfolobus

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Fig. 1. Schematic overview of the ced cluster and the predicted topology of the proteins. (A) The ced genes encode two small transmembrane proteins (CedA1 and CedA2), a larger transmembrane protein (CedA), and a HerA/VirB4 homolog (CedB). Homologous genes from Sulfolobales, Desulfurococcales, and Acidilobales are depicted (see also Table S1). Homology and synteny was found using SyntTax (43) and is indicated by similar colors. (B) Schematic overview of the Ced proteins and their predicted topology. Depicted are CedA1, CedA, CedA2, and CedB. Dashed lines indicate differences between the species: CedA1 is absent in Ignicoccus hospitalis, CedA homologs from Desulfurococcales and Acidilobales contain an extracellular loop between the last two transmembrane domains, CedA2 is absent in certain species from the orders Desulfurococcales and Acidilobales, and some of the CedB homologs do not contain a transmembrane domain.

1A). In these organisms, cedB is predicted to be part of the above suggested operon (20). To confirm the up-regulation of the ced genes in S. acidocaldarius, quantitative RT-PCR (qRT-PCR) experiments were performed. As observed for S. solfataricus in microarray studies (1, 2), cedA, cedB, and upsE, which encodes the ATPase of the Ups pilus, were found to be highly up-regulated in S. acidocaldarius after induction with UV stress. Both cedA1 and cedA2 were also up-regulated upon UV stress (Fig. S1). To illustrate the presence of the ced genes among different archaeal species, we created a phylogenetic tree based on 16S rRNA sequences (Fig. S2). The presence of both CedA and CedB is designated as the Ced system (green circles in Fig. S2). Additionally, we indicated the cooccurrence of the Ups system (orange circles in Fig. S2). The Ced system is specific for the Crenarchaeota and can be found in most species from the orders Sulfolobales, Acidilobales, and Desulfurococcales. However, it cannot be found among the Thermoproteales. We know that DNA exchange among Sulfolobales is dependent on the Ups system, because it is responsible for the formation of cellular interactions (3). Interestingly, no Ups system can be found among the other species harboring a Ced system. These species therefore probably have another mode of initiating cellular contact. Localization of the Ced Proteins. To confirm the predicted membrane localization of the Ced proteins, we overexpressed CedA and CedB in S. acidocaldarius. Subsequently, we performed Western blotting on the membrane and cytosol fractions from cells treated with or without UV light. For the CedA proteins, we cloned the complete cluster (saci_0569-0567) into an expression plasmid with a tag only on CedA2. We obtained a His-tag-specific signal of around 50 kDa exclusively in membranes of UV-treated cells, suggesting that CedA1 (8.6 kDa), CedA (28.4 kDa), and CedA2-His/Strep PNAS | March 1, 2016 | vol. 113 | no. 9 | 2497

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cells, we searched among the highest up-regulated genes upon UV stress as observed in previous microarray studies (1, 2). Archaeal DNA transport systems have not been studied in detail and they moreover seem to differ greatly from their bacterial counterparts, hence no obvious candidates could directly be identified. Because transport systems are anchored to the membrane, we expected the presence of at least one transmembrane protein essential for DNA transport. The two genes that were found to be most highly upregulated in S. solfataricus upon UV stress were sso0691 and sso3146 (1). Both genes encode predicted polytopic transmembrane proteins and are homologous to each other. Given the fact that the genomic neighborhood of sso0691 is conserved among Sulfolobales but not that of sso03146, sso3146 seems to be a paralog of sso0691 that arose during recent gene duplication. Except for S. islandicus, which is highly similar to S. solfataricus, all other Sulfolobales contain only one sso0691 homolog, now named cedA (saci_0568 in S. acidocaldarius) (Fig. 1A). In addition, homologs can be found in species from the orders Desulfurococcales and Acidilobales. In all species, CedA is predicted to contain six or seven transmembrane domains (Fig. 1B). In species from the orders Desulfurococcales and Acidilobales, cedA is significantly larger due to an additional region that encodes a predicted extracellular loop between the two C-terminal membrane domains (Fig. 1). Synteny analysis revealed that in all Sulfolobales cedA is flanked by two small genes: cedA1 and cedA2 (upstream and downstream, respectively), encoding proteins that contain two predicted transmembrane domains (Fig. 1). Owing to their small size (150–200 bp), these genes are not annotated in many of the analyzed genomes and were therefore annotated by hand (“+” in Table S1). Most Desulfurococcales also contain these two small neighboring genes; in Acidilobales only cedA1 can be found (Fig. 1A and Table S1). CedA1 shows homology to the two most N-terminal membrane domains of CedA. It therefore seems that cedA1 emerged from a duplication event. Deep sequencing results and operon predictions suggest the cedA cluster to be present as an operon with a single promotor (19, 20). Another highly up-regulated gene is sso0152 (saci_0748 in S. acidocaldarius), encoding a VirB4/HerA homolog that we now name CedB (Table S1). VirB4-ATPases are associated with conjugative type-IV secretion systems in both gram-negative and grampositive bacteria, where they are essential for DNA transfer (21). Interestingly, CedB could be modeled on a HerA crystal structure from S. solfataricus. The latter forms a hexameric DNA translocase and functions in DNA end resection, creating 3′ overhang templates for homologous recombination (22). Unlike HerA, most CedB homologs have a predicted N-terminal transmembrane domain (Fig. 1B). BLAST analysis revealed that all species containing CedA also possess a CedB homolog. Synteny analysis revealed that in many species from the orders Desulfurococcales and Acidilobales cedB is located directly downstream to the cedA cluster (Table S1 and Fig.

Fig. 2. Localization of CedA1, A, A2, and CedB in S. acidocaldarius. Membrane (M) and soluble (S) fractions from S. acidocaldarius overexpressing CedA1, CedA, and CedA2-His or CedB-His treated with or without UV light (75 J/m2). Samples were taken 3 h after induction with UV and separated by SDS/PAGE. As a negative control, we used S. acidocaldarius transformed with an empty vector (pSVA1551). Predicted protein sizes are 8.6 kDa (CedA1), 28.4 kDa (CedA), 8.7 kDa (CedA2-His), and 71.5 kDa (CedB-His). Proteins were visualized by Western blotting using α-His conjugated antibodies. The asterisk indicates an unspecific band.

(8.7 kDa) together form an SDS stable protein complex (Fig. 2). In addition, we could show that CedB also localizes to the membrane (Fig. 2), which is in agreement with the predicted transmembrane domains of CedB (Fig. 1B). An increased amount was observed upon induction with UV light. Together these data suggest that CedA proteins and CedB are more abundant and/or more stable after UV treatment. Cellular Aggregation and Chromosomal Marker Exchange of ced Deletion Mutants. To study the putative roles of cedA and cedB

in DNA transfer, markerless deletion mutants of both genes were made in two different S. acidocaldarius pyrE mutant backgrounds (MW001 and JDS22). Additionally, a Walker A mutation (K292A) in CedB was genomically inserted into both background strains (Table S2). PyrE (orotate phosphoribosyltransferase) is an enzyme involved in the de novo uracil biosynthesis. The two pyrE mutant background strains contain mutations in different regions of the pyrE locus and can, upon DNA exchange, recombine to a wild-type pyrE locus. Growth experiments and microscopy revealed wild-type growth and a normal cellular phenotype for all mutants. In addition, UV-induced cellular aggregation of the mutants was similar to

that of wild-type cells, indicating that neither CedA nor CedB functions in Ups pili-mediated cellular recognition and interaction (Fig. S3). To exclude effects of the deletion of cedA and cedB on other UV-inducible genes, qRT-PCR experiments were performed. We compared the transcription levels of upsX, upsE, upsA, cedA, cedB, and herA between the mutants and S. acidocaldarius MW001 and could not observe any significant differences (except for the respective mutant genes) (Fig. S4). DNA transfer assays were performed using the auxotrophic saci_0568/pyrE, saci_0748/pyrE, and Saci_0748 K292A/pyrE double mutants (from now on referred to as ΔcedA, ΔcedB, and CedB K292A, respectively) (Table S2). Two strains were mixed together and upon exchange of chromosomal DNA pyrE mutations could be restored via homologous recombination, resulting in prototrophic colonies as described previously (4). A mixture of the two background strains resulted in the formation of recombinants (Fig. 3, MW001*JDS22, green bar), which was found to be increased up to 10 times upon induction of one or both strains with UV light (Fig. 3, MW001*JDS22, second, third, and fourth bars), confirming previously observed results (4). The cedA deletion mutant did not contribute to increased DNA transfer when treated with UV light. Only when the wild-type strain was induced with UV light in these mixtures, a significant increase of DNA exchange was observed (Fig. 3, second mixture and Fig. S5). The latter suggests that only one ced+ mating partner is sufficient for DNA exchange. When mating two ΔcedA strains, no recombinants were formed (Fig. 3, third mixture). Similar results were obtained when mating two cedB deletion mutants or two CedB Walker A mutants (Fig. 3, fifth and eighth mixtures). These results thereby indicate that both membrane protein CedA and CedB and its ATPase activity are essential for transfer of DNA between cells. Importantly, a mixture of ΔcedA with ΔcedB also did not result in the formation of colonies (Fig. 3, sixth mixture and Fig. S5), suggesting that both genes function in the same pathway. In contrast, mixtures of ΔupsE, a mutant that does not assemble pili and therefore is deficient in aggregation (3), with ΔcedA or ΔcedB did result in the formation of recombinants (Fig. 3, last two mixtures), even though both systems are essential for DNA transport. The latter can be explained by the fact that the cells can still form mating pairs using the Ups system present in the Δced mutants and still exchange DNA using the Ced system present in the ΔupsE strain. We have thereby shown that the Ups system and the Ced system are essential for successful DNA exchange, with the Ced system most likely acting downstream of the Ups system.

Fig. 3. DNA exchange assays using cedA, cedB, and upsE deletion mutants as well as a genomic Walker A mutated cedB strain (K292A). Two different strains (JDS22 or MW001 background) treated with (UV) or without (C) UV light were mixed in different combinations and plated on selective media. Both background strains contained mutations in the pyrE gene (involved in de novo uracil biosynthesis) located at different positions, such that recombination between the strains can restore the pyrE wild-type phenotype. Bars represent the average of at least three independent mating experiments each; every experiment was normalized to JDS22 (UV) * MW001 (UV) as 100%.

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Mixture ΔupsE*ΔcedA upsE cedA ΔupsE*ΔcedB upsE cedB

Colonies + 1 30 + 3 30

Gene present in Δ 29 0 Δ 27 0

3% 100% 10% 100%

To determine the directionality of DNA exchange, colony PCR was performed on conjugants obtained in UV*UV mixtures of ΔupsE *Δced strains. We determined the genotype (presence or deletion of the respective gene) of 30 recombinants per mixture. For the ΔupsE*ΔcedA mixture we obtained 100% cedA+ background and 10% upsE+ background. Similar results were obtained for the ΔupsE*ΔcedB mixture, with 100% cedB+ background and 3% upsE+ background (Table 1). The transfer of the marker gene pyrE therefore probably occurred from the Δced (upsE+) strains to the ΔupsE (ced+) strain. This means that in these mixtures the Δced strains functioned as donor strains and the ΔupsE strain as recipient strain. From this observation we can conclude that the Ced system (present in ΔupsE) functions as an importer. The small percentage of upsE+ background can be explained by the fact that besides the pyrE region the genomic region of upsE was probably cotransferred to the recipient cell. Discussion DNA transport between cells from the same or different species occurs throughout all domains of life via diverse mechanisms. Purposes of DNA transfer include DNA repair and horizontal gene transfer (23). Among bacteria and archaea, DNA transfer via natural transformation as well as conjugation has been described for several species, although bacterial DNA transfer mechanisms have been studied in far greater detail. Also, for eukaryal cells and organelles mechanisms for DNA/RNA transfer have been described, including vesicle-mediated DNA/RNA transfer between cells and the import or export of DNA/RNA by mitochondria and the nucleus (24–26). Because archaeal DNA transport systems seem to differ greatly from their bacterial and eukaryal counterparts, it was difficult to identify putative transporter proteins based on homology. On a molecular level, transport of DNA among archaea therefore remained far from well understood. The previously described Ups system (3, 4) enables the exchange of chromosomal DNA between Sulfolobus cells upon UV stress by mediating mating-pair formation. However, because the ups operon only encodes proteins involved in T4P formation, it seems likely that other, unknown proteins function in the actual transport of DNA. In this study, we therefore sought to find proteins for the UV-induced DNA transfer among Sulfolobales. One of the highest up-regulated genes upon UV stress encodes a transmembrane protein, now named CedA. Homologs are found among all Sulfolobales and additionally in several Desulfurococcales and Acidilobales, where it contains six or seven transmembrane domains. A deletion of cedA in S. acidocaldarius did not result in a reduction of cellular aggregation but showed an abolishment of DNA transfer. This indicates that the transmembrane protein is truly involved in DNA transfer between cells. One could therefore envision that, similar to ComEC of competence systems or VirB6 of conjugation systems, this protein forms a pore in the membrane to transfer the DNA in or out of the cell (27–29). Among the Desulfurococcales and Acidilobales, CedA is significantly longer due to a predicted extracellular loop between the two C-terminal transmembrane domains. This loop might function in protein interactions that differ from those in the Sulfolobales system; one could, for instance, imagine an interaction with distinct pilin subunits functioning van Wolferen et al.

in the formation of cellular connections, because no ups operon is present in these species. In bacterial T4SSs, the same has been observed between a loop of membrane protein VirB6 and pilin subunits VirB2 (30). In most of the species carrying a gene encoding CedA this gene is flanked by two small genes (cedA1 and cedA2); together, these genes form an operon. Both genes encode very small proteins with two predicted transmembrane domains. Interestingly, when coexpressing CedA1, CedA, and CedA2, a highly stable complex of all three proteins is formed that localizes at the membrane. This suggests that the proteins physically function together in DNA transport. Some species from the order Desulfurococcales lack CedA1 and CedA2; members of the order Acidilobales only contain CedA1. In bacterial T4SS gene clusters, small membrane proteins such as VirB3 are often encoded directly upstream of a VirB4 encoding gene (14). In some cases virB3/virB4 fusion genes can even be found (31). The exact role of VirB3 proteins is unknown but they also have two membrane domains and are known to interact with VirB4, where they are essential for both substrate transformation and pili formation (32). The two small archaeal membrane proteins might therefore have functions similar to those of VirB3. Interestingly, a virB4 homolog, now named cedB, was also found to be highly up-regulated upon induction with UV and essential for DNA transfer. VirB4 proteins are AAA ATPases energizing substrate transfer that are associated with all so-far-described type-IV secretion systems (21, 33). CedB can additionally be modeled onto a crystal structure of helicase HerA from S. solfataricus. The latter forms a hexameric ATPase that, together with nuclease NurA, functions in DNA end processing in archaea. For this, the N-terminal domain of HerA binds to NurA and the C-terminal domain of HerA binds to DNA. The HerA–NurA complex subsequently translocates along the DNA upon ATP hydrolysis (17, 22, 34, 35). Notably, CedB does not show homology to the N-terminal domain of HerA, which binds to NurA. Instead, it atypically contains an N-terminal membrane domain and localizes to the membrane. It is therefore likely that CedB, similar to HerA, binds and translocates DNA but unlike HerA translocates across the membrane. Homologs of CedB are present among all species encoding CedA and, intriguingly, cedB often lies directly downstream of cedA as part of the predicted operon. This suggests a functional link between the two proteins. Importantly, no DNA transfer was observed between cedA and cedB deletion strains, showing that both proteins function together, either in the donor or the recipient cell. We were highly interested in determining the direction of DNA transfer between Sulfolobus cells. Previous data showed that 62–81% of the recombinants (all pyrE+) in a upsE+*ΔupsE mixture were upsE+ (4). Based on this result it was suggested that the transfer of DNA involves an active recruitment of DNA by a UV-damaged cell that produces pili. Alternatively, the transfer of DNA was thought to be bidirectional, with one partner being UV-activated, resulting in the mutual transfer of two markers (4). Here, however, we show that the Ups system functions separately from the Ced system, even though both systems are essential for DNA transport. We therefore propose that the Ups system acts in mating-pair formation, whereas the Ced system subsequently functions actively in DNA import. In nature, the transfer of DNA between Sulfolobus cells presumably occurs in both directions because all cells are genotypically similar. To determine the direction of Ced-mediated DNA transport, we determined the genotypes of recombinants obtained in Δced*ΔupsE mixtures and found that all recombinants were ced+ and only 3–10% were upsE+. The fact that we only obtained ced+ recombinants indicated that the pyrE locus was imported by the ΔupsE (ced+) strain, suggesting that the Ced system functions as an importer. Haloferax species exchange large pieces of chromosomal DNA of up to 500 kbp (13). The genomic distance between the pyrE locus and the upsE locus is around 90 kbp, whereas the genomic PNAS | March 1, 2016 | vol. 113 | no. 9 | 2499

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Table 1. Genotypes of recombinants from UV-treated ΔupsE*Δced mixtures determined by colony PCR

Fig. 4. Current model of DNA transport in Sulfolobales using the Ced system. (Left) Sulfolobus cells form aggregates upon UV stress using the Ups pili system (4). (Right) cedA and cedB are up-regulated upon UV treatment and encode a polytopic membrane protein CedA and a membrane-bound ATPase CedB, respectively. Both CedA and CedB are essential for DNA exchange and probably form a DNA importer in the recipient cell. It is unclear whether double-stranded or single-stranded DNA is transported. Indicated are Ups pili (yellow), membrane (M), S layer, CedA (green), and CedB (green). Question marks indicate parts of the system that are unknown.

distance between the pyrE locus and the ced genes is at least 760 kbp. If Sulfolobus cells would also exchange pieces of up to 500 kbp DNA, it would be likely that next to the pyrE locus also the upsE locus would frequently be transferred. Because we only obtained the ced+/upsE+ genotype in 3–10% of the recombinants, this does not seem to be the case. We therefore assume that transferred pieces of chromosomal DNA are mostly smaller than 90 kbp. Of course, the pyrE locus and the upsE locus do not necessarily need to lie on the same piece of DNA to be incorporated in the same chromosome; an alternative explanation for the transfer of both markers would be the occurrence of a second DNA transfer event between the cells. The finding that the Ced system functions in the import of DNA was initially unexpected; other known DNA exchange systems involving direct cellular contact (such as conjugation systems) are based on the active export of DNA. The fact that this system actively imports DNA emphasizes the unique and social nature of the transfer of chromosomal DNA between Sulfolobus cells. Other known DNA uptake systems such as competence systems only take up DNA from the environment, but not from other cells (36, 37). How DNA is exported from the donor cell is still unclear. As mentioned above, we initially expected a bacterial-like conjugation system; however, except for VirB4 homologs, no other genomically encoded homologs of bacterial DNA transport systems can be found among the Crenarchaeota. This suggests the presence of a DNA export mechanism that is evolutionarily distinct from the bacterial conjugation system. Among the highest up-regulated genes upon UV stress, sso0283 was found (saci_0667 in S. acidocaldarius) (2). Similar to cedB, this gene encodes a membrane-bound HerA homolog. All organisms harboring the Ced system also encode a homolog of this protein. We therefore hypothesize this protein either to be an additional ATPase functioning in the Ced system or alternatively it might function on the donor side of DNA transfer events. This protein might therefore be the start of our study on the putative export machinery. With this study we propose a previously unidentified Crenarchaeal DNA exchange mechanism in which chromosomal DNA is imported from other cells using the Ced system (Fig. 4). In Sulfolobales, the initiation of aggregation upon UV stress is mediated by the Ups system (3). Thereby, species-specific mating pairs are formed (Fig. 4, Left) (4). Because the Ups system is absent in other Crenarchaeota containing the Ced system, it is unclear how mating pairs are formed in these species. We speculate that the extracellular loop in CedA, which is not present in the Sulfolobales, might be involved in this process. Subsequent to the initiation of cellular contact, DNA is exported from the donor cell via a so-far-unknown mechanism (Fig. 4, Right). The active import of DNA by the recipient cell is then 2500 | www.pnas.org/cgi/doi/10.1073/pnas.1513740113

performed by the Ced system. CedA proteins are thought to build a membrane pore through which the DNA can be transferred. Membrane-bound ATPase CedB presumably binds the DNA and energizes the translocation. We assume that CedA and CedB form one complex to perform this process, but this is not yet proven. In addition, it is unclear whether single- or double-stranded DNA is transferred (Fig. 4). Once the DNA is imported, it can be used for homologous recombination and thereby it can rescue the cells from extensive DNA damage induced by UV light. The importance of this mechanism is illustrated by the fact that cells that cannot exchange DNA show significantly lower survival rates upon DNA damage (4). Summarizing, we identified for the first time to our knowledge an archaeal DNA transporter that differs from any other previously characterized system. This so-called Ced system is essential for DNA import and functions in the intriguing community-based DNA repair system of Sulfolobales. How DNA is exported from the donor cells and how incoming DNA finds the Ced system are topics for future studies. Materials and Methods Construction of Deletion Mutants of S. acidocaldarius. To construct deletion strains of saci_0568 and saci_0748 in MW001 and JDS22, up- and downstream flanking regions of both genes (∼600 bp) were amplified with primers listed in Table S3. Overlap PCR was performed to connect the up- and downstream fragments. The PCR products were subsequently cloned into pSVA406 or pSVA431, resulting in pSVA1833 and the pSVA3506, respectively (Table S2). To reintroduce cedB with a Walker A mutation, plasmid pSVA1837 was constructed. Methylation and transformation of the plasmids, selection of integrants, and screening for mutants were performed as described previously (38). Correctness of strains was confirmed by DNA sequencing (listed in Table S2). UV Treatment and Aggregation Assays. UV-light treatment was performed as described in ref. 3. Ten milliliters of culture (OD600 0.2–0.3) was treated with a UV dose of 75 J/m2 (254 nm, Spectroline UV cross-linker) in a plastic Petri dish. Afterward cultures were put back at 75 °C for 3 h. To quantify aggregated cells after induction with UV, 5 μL of cell culture (diluted to OD 0.2) were spotted on a microscope slide covered with a thin layer of 1% agarose in Brock minimal medium. Free and aggregated cells (n ≥ 3) were counted for at least 1,000 cells per strain per replicate using ImageJ cell counter. Percentages of cells in aggregates were subsequently calculated from three different experiments. DNA Transfer Assays. DNA transfer between S. acidocaldarius cells was assayed by selecting prototrophic (pyr+) recombinants of two pyrE mutant strains: MW001 and JDS22. The latter contain a 311-bp deletion (nt 91– 412) or a 22-bp deletion (nt 16–38) (38, 39) (Table S2). Deletion mutants of saci_0568 (cedA) and saci_0748 (cedB) were made in these backgrounds as described above. Liquid cultures were grown at 75 °C and harvested at OD600 0.4–0.6. Pellets were concentrated to an OD600 of 1. UV irradiation was performed as described above and mixtures (1 mL per mating partner) were further incubated for 3 h at 75 °C in 24-well plates while shaking.

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ACKNOWLEDGMENTS. We thank Dennis Grogan for kindly providing us with strain JDS22. This work was supported by German Research Foundation (DFG) Grant AL1206/3-1 and European Research Council Starting Grant ARCHAELLUM 311523 (to M.v.W.) and Max Planck Society Grant CRC987 and DFG Grant AL1206/4-1 (to A.W.). S.-V.A. and C.v.d.D. received intramural funds from the Max Planck Society.

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PNAS | March 1, 2016 | vol. 113 | no. 9 | 2501

MICROBIOLOGY

Recombination was assayed by spreading 200 μL of each mixture, after vigorously vortexing, on selective plates without uracil. Plates were incubated for 5–6 d at 75 °C as was described previously (4). To determine the genotype of the recombinants, colony PCRs were performed on UV*UV mixtures of ΔupsE.1*ΔcedA.2 and ΔupsE.1*ΔcedB.2 (for strains see Table S2) using primers listed in Table S3.