The Archaeal Ced System Imports DNA

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The archaeal Ced system imports DNA

Marleen van Wolferena,1, Alexander Wagnera,1, Chris van der Doesa, and Sonja-Verena Albersa,2

aMolecular 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 species exchange chromosomal DNA between cells connected by in all domains of life and is a major driving force of evolution. bridges (11). This transfer is thought to occur in a bidirectional Upon UV-light treatment, cells of the crenarchaeal genus Sulfo- manner via cell fusion leading to the formation of diploid cells with lobus express Ups pili, which initiate cell aggregate formation. mixed chromosomes (12). Interestingly, this type of DNA transfer Within these aggregates, chromosomal DNA, which is used for was shown to occur between different Haloferax species and in- the repair of DNA double-strand breaks, is exchanged. Because volved DNA fragments of up to 500 kbp DNA (13). Nevertheless, so far no clear homologs of bacterial DNA transporters have the mechanism of DNA transfer is so far not understood. Other been identified among the genomes of Archaea, the mechanisms described archaeal conjugative systems include self-transmissible of archaeal DNA transport have remained a puzzling and under- plasmids, which have so far only been studied for Sulfolobus spe- saci_0568 saci_0748, investigated topic. Here we identify and cies. These plasmids are grouped into the so-called pKEF and Sulfolobus acidocaldarius two genes from that are highly in- pARN plasmids (14, 15) and only a few of their genes encode duced upon UV treatment, encoding a transmembrane protein homologs of bacterial conjugation proteins, including the so-far- and a membrane-bound VirB4/HerA homolog, respectively. DNA unstudied ATPases VirD4 and VirB4. It is unknown how cellular transfer assays showed that both proteins are essential for DNA Sulfolobus contact is initiated to achieve plasmid transfer. During plasmid transfer between cells and act downstream of the conjugation, Sulfolobus islandicus cells form aggregates, similar to Ups pili system. Our results moreover revealed that the system those observed upon UV stress (16). One could therefore imagine is involved in the import of DNA rather than the export. We that cells make use of the genomically encoded Ups system to therefore propose that both Saci_0568 and Saci_0748 are part initiate cell contact. Many other conjugation proteins such as of a previously unidentified DNA importer. Given the fact that relaxases can also not be identified in archaea based on homology, we found this transporter system to be widely spread among the MICROBIOLOGY Crenarchaeota, we propose to name it the Crenarchaeal system indicating that again distinct mechanisms must be present that for exchange of DNA (Ced). In this study we have for the first differ significantly from their bacterial counterparts. Hence, ar- time to our knowledge described an archaeal DNA transporter. chaeal DNA transfer remains a poorly investigated topic. Previously performed microarray studies on Sulfolobus solfataricus Archaea | DNA transport | conjugation | type IV pili | VirB4 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 pon UV treatment, Sulfolobales species induce the expression encoding helicase HerA, nuclease NurA, Rad50, and Mre11 (17, 18). of Ups pili (UV-inducible pili of Sulfolobus) (1–3). These are U Because we were interested in the mechanism of DNA transfer be- type-IV pili (T4P) that are essential for cellular aggregation and tween Sulfolobus cells, we searched for up-regulated genes putatively chromosomal DNA exchange (3, 4). The ability of Sulfolobales to involvedinDNAtransport.Wefocusedonthreeclusteredgenes exchange DNA was shown to increase cellular fitness under UV encoding one larger and two smaller membrane proteins. Addition- stress (4). Because other DNA-damaging agents such as bleomycin ally, we looked at a virB4/herA homolog. Homologs of these genes 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). Significance Not much is known about DNA transfer among archaea; only a few examples of competence and conjugation systems have Among bacteria, transfer of DNA has been studied in great detail. been described. Four archaeal species were shown to be natu- Several bacterial DNA transfer systems have been described on a rally competent: Pyrococcus furiosus, Thermococcus kodakarensis, molecular level including competence and conjugation systems. In Methanobacterium thermoautotrophicum,andMethanococcus voltae Archaea, DNA exchange has been observed for a number of or- (5–8). However, these natural transformation mechanisms have ganisms and its importance for horizontal gene transfer and DNA not been studied on a molecular level and in none of these ar- repair is greatly valued. However, for none of these organisms has chaeal species homologs from bacterial competence systems the mode of transport been studied on a molecular level. Here we could be identified. Distinct machineries must therefore be describe a set of genes directly involved in the transfer of chro- Sulfolobus acidocaldarius present in archaea. Because bacterial natural transformation mosomal DNA between cells. Homologs often involves T4P (9), one could hypothesize that Sulfolobales of these genes are widely distributed among the Crenarchaeota. also exchange DNA via an uptake and release mechanism in For the first time to our knowledge we give molecular insights into which the Ups pili play a vital role similar to that in bacterial intercellular transport of DNA between archaeal cells. competence systems. However, the exchange of DNA among Author contributions: M.v.W., A.W., C.v.d.D., and S.-V.A. designed research; M.v.W. and Sulfolobus species was shown to be insensitive to DNase treat- A.W. performed research; M.v.W., A.W., C.v.d.D., and S.-V.A. analyzed data; and M.v.W., ment, and recombinants could not be obtained by mixing the A.W., and S.-V.A. wrote the paper. cells with lysate or purified DNA (10). This demonstrates that The authors declare no conflict of interest. exchange of DNA requires cellular contact and transfer occurs This article is a PNAS Direct Submission. directly from one cell to another without passing through the Freely available online through the PNAS open access option. surrounding medium. A conjugation-like mechanism or cellular 1M.v.W. and A.W. contributed equally to this work. fusion therefore seems more likely. 2To whom correspondence should be addressed. Email: [email protected] DNA transfer among archaea via direct cellular contact was first freiburg.de. described for the euryarchaeon Haloferax volcanii. Similar to the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. UV-inducible transfer of DNA among Sulfolobales, Haloferax 1073/pnas.1513740113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1513740113 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 are present in the genomes of all Sulfolobales and several Desul- furococcales 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 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 Fig. 1. Schematic overview of the ced cluster and the predicted topology of transport systems are anchored to the membrane, we expected the the proteins. (A)Theced genes encode two small transmembrane proteins presence of at least one transmembrane protein essential for DNA (CedA1 and CedA2), a larger transmembrane protein (CedA), and a HerA/VirB4 homolog (CedB). Homologous genes from Sulfolobales, Desulfurococcales, and transport. The two genes that were found to be most highly up- Acidilobales are depicted (see also Table S1). Homology and synteny was regulated in S. solfataricus upon UV stress were sso0691 and sso3146 found using SyntTax (43) and is indicated by similar colors. (B)Schematic (1). Both genes encode predicted polytopic transmembrane proteins overview of the Ced proteins and their predicted topology. Depicted are and are homologous to each other. Given the fact that the genomic CedA1, CedA, CedA2, and CedB. Dashed lines indicate differences between the neighborhood of sso0691 is conserved among Sulfolobales but not species: CedA1 is absent in Ignicoccus hospitalis, CedA homologs from Desul- that of sso03146, sso3146 seems to be a paralog of sso0691 that arose furococcales and Acidilobales contain an extracellular loop between the last during recent gene duplication. Except for S. islandicus, which is two transmembrane domains, CedA2 is absent in certain species from the orders Desulfurococcales and Acidilobales, and some of the CedB homologs do highly similar to S. solfataricus, all other Sulfolobales contain not contain a transmembrane domain. 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 1A). In these organisms, cedB is predicted to be part of the above species, CedA is predicted to contain six or seven transmembrane suggested operon (20). domains (Fig. 1B). In species from the orders Desulfurococcales To confirm the up-regulation of the ced genes in S. acidocaldarius, and Acidilobales, cedA is significantly larger due to an additional quantitative RT-PCR (qRT-PCR) experiments were performed. As region that encodes a predicted extracellular loop between the two observed for S. solfataricus in microarray studies (1, 2), cedA, cedB, C-terminal membrane domains (Fig. 1). and upsE, which encodes the ATPase of the Ups pilus, were found Synteny analysis revealed that in all Sulfolobales cedA is flanked to be highly up-regulated in S. acidocaldarius after induction with by two small genes: cedA1 and cedA2 (upstream and downstream, UV stress. Both cedA1 and cedA2 were also up-regulated upon UV respectively), encoding proteins that contain two predicted trans- stress (Fig. S1). membrane domains (Fig. 1). Owing to their small size (150–200 bp), To illustrate the presence of the ced genes among different ar- these genes are not annotated in many of the analyzed genomes chaeal species, we created a phylogenetic tree based on 16S rRNA and were therefore annotated by hand (“+” in Table S1). Most sequences (Fig. S2). The presence of both CedA and CedB is Desulfurococcales also contain these two small neighboring genes; designated as the Ced system (green circles in Fig. S2). Addition- in Acidilobales only cedA1 can be found (Fig. 1A and Table S1). ally, we indicated the cooccurrence of the Ups system (orange CedA1 shows homology to the two most N-terminal membrane circles in Fig. S2). The Ced system is specific for the Crenarchaeota domains of CedA. It therefore seems that cedA1 emerged from a and can be found in most species from the orders Sulfolobales, duplication event. Deep sequencing results and operon predictions Acidilobales, and Desulfurococcales. However, it cannot be found suggest the cedA cluster to be present as an operon with a single among the Thermoproteales. We know that DNA exchange among promotor (19, 20). Sulfolobales is dependent on the Ups system, because it is re- Another highly up-regulated gene is sso0152 (saci_0748 in sponsible for the formation of cellular interactions (3). Interestingly, S. acidocaldarius), encoding a VirB4/HerA homolog that we now no Ups system can be found among the other species harboring a name CedB (Table S1). VirB4-ATPases are associated with con- Ced system. These species therefore probably have another mode jugative type-IV secretion systems in both gram-negative and gram- of initiating cellular contact. positive bacteria, where they are essential for DNA transfer (21). Interestingly, CedB could be modeled on a HerA crystal structure Localization of the Ced Proteins. To confirm the predicted mem- from S. solfataricus. The latter forms a hexameric DNA translocase brane localization of the Ced proteins, we overexpressed CedA and and functions in DNA end resection, creating 3′ overhang templates CedB in S. acidocaldarius. Subsequently, we performed Western for homologous recombination (22). Unlike HerA, most CedB ho- blotting on the membrane and cytosol fractions from cells treated mologs have a predicted N-terminal transmembrane domain (Fig. with or without UV light. For the CedA proteins, we cloned the 1B). BLAST analysis revealed that all species containing CedA also complete cluster (saci_0569-0567)intoanexpressionplasmidwitha possess a CedB homolog. Synteny analysis revealed that in many tag only on CedA2. We obtained a His-tag-specific signal of around species from the orders Desulfurococcales and Acidilobales cedB is 50 kDa exclusively in membranes of UV-treated cells, suggesting located directly downstream to the cedA cluster (Table S1 and Fig. that CedA1 (8.6 kDa), CedA (28.4 kDa), and CedA2-His/Strep

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1513740113 van Wolferen et al. Downloaded by guest on September 24, 2021 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,andherA 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 Fig. 2. Localization of CedA1, A, A2, and CedB in S. acidocaldarius. Mem- restored via homologous recombination, resulting in prototrophic brane (M) and soluble (S) fractions from S. acidocaldarius overexpressing colonies as described previously (4). A mixture of the two back- CedA1, CedA, and CedA2-His or CedB-His treated with or without UV light ground strains resulted in the formation of recombinants (Fig. 3, (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 MW001*JDS22, green bar), which was found to be increased up to an empty vector (pSVA1551). Predicted protein sizes are 8.6 kDa (CedA1), 10 times upon induction of one or both strains with UV light (Fig. 28.4 kDa (CedA), 8.7 kDa (CedA2-His), and 71.5 kDa (CedB-His). Proteins were 3, MW001*JDS22, second, third, and fourth bars), confirming visualized by Western blotting using α-His conjugated antibodies. The as- previously observed results (4). The cedA deletion mutant did not terisk indicates an unspecific band. 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 (8.7 kDa) together form an SDS stable protein complex (Fig. 2). In (Fig. 3, second mixture and Fig. S5). The latter suggests that only addition, we could show that CedB also localizes to the membrane one ced+ mating partner is sufficient for DNA exchange. When (Fig. 2), which is in agreement with the predicted transmembrane mating two ΔcedA strains, no recombinants were formed (Fig. 3, domains of CedB (Fig. 1B). An increased amount was observed third mixture). Similar results were obtained when mating two cedB upon induction with UV light. Together these data suggest that deletion mutants or two CedB Walker A mutants (Fig. 3, fifth and MICROBIOLOGY CedA proteins and CedB are more abundant and/or more stable eighth mixtures). These results thereby indicate that both mem- after UV treatment. brane protein CedA and CedB and its ATPase activity are essential for transfer of DNA between cells. Importantly, a mixture of ΔcedA ced Cellular Aggregation and Chromosomal Marker Exchange of with ΔcedB also did not result in the formation of colonies (Fig. 3, Deletion Mutants. To study the putative roles of cedA and cedB sixth mixture and Fig. S5), suggesting that both genes function in in DNA transfer, markerless deletion mutants of both genes were the same pathway. In contrast, mixtures of ΔupsE,amutantthat made in two different S. acidocaldarius pyrE mutant backgrounds does not assemble pili and therefore is deficient in aggregation (3), (MW001 and JDS22). Additionally, a Walker A mutation (K292A) with ΔcedA or ΔcedB did result in the formation of recombinants in CedB was genomically inserted into both background strains (Fig. 3, last two mixtures), even though both systems are essential (Table S2). PyrE (orotate phosphoribosyltransferase) is an enzyme for DNA transport. The latter can be explained by the fact that the involved in the de novo uracil biosynthesis. The two pyrE mutant cells can still form mating pairs using the Ups system present in background strains contain mutations in different regions of the the Δced mutants and still exchange DNA using the Ced system pyrE locus and can, upon DNA exchange, recombine to a wild-type present in the ΔupsE strain. We have thereby shown that the Ups pyrE locus. Growth experiments and microscopy revealed wild-type system and the Ced system are essential for successful DNA ex- growth and a normal cellular phenotype for all mutants. In addi- change, with the Ced system most likely acting downstream of the tion, UV-induced cellular aggregation of the mutants was similar to 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%.

van Wolferen et al. PNAS Early Edition | 3of6 Downloaded by guest on September 24, 2021 Table 1. Genotypes of recombinants from UV-treated in the formation of cellular connections, because no ups operon ΔupsE*Δced mixtures determined by colony PCR is present in these species. In bacterial T4SSs, the same has been Mixture Colonies Gene present in observed between a loop of membrane protein VirB6 and pilin subunits VirB2 (30). ΔupsE*ΔcedA + Δ In most of the species carrying a gene encoding CedA this gene is upsE 129 3%flanked by two small genes (cedA1 and cedA2); together, these genes cedA 30 0 100% form an operon. Both genes encode very small proteins with two ΔupsE*ΔcedB + Δ predicted transmembrane domains. Interestingly, when coexpressing upsE 327 10%CedA1, CedA, and CedA2, a highly stable complex of all three cedB 30 0 100% 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; To determine the directionality of DNA exchange, colony members of the order Acidilobales only contain CedA1. In bacterial PCR was performed on conjugants obtained in UV*UV mixtures T4SS gene clusters, small membrane proteins such as VirB3 are of ΔupsE *Δced strains. We determined the genotype (presence often encoded directly upstream of a VirB4 encoding gene (14). In or deletion of the respective gene) of 30 recombinants per mix- some cases virB3/virB4 fusion genes can even be found (31). The ture. For the ΔupsE*ΔcedA mixture we obtained 100% cedA+ exact role of VirB3 proteins is unknown but they also have two background and 10% upsE+ background. Similar results were membrane domains and are known to interact with VirB4, where obtained for the ΔupsE*ΔcedB mixture, with 100% cedB+ back- they are essential for both substrate transformation and pili forma- ground and 3% upsE+ background (Table 1). The transfer of the tion (32). The two small archaeal membrane proteins might there- marker gene pyrE therefore probably occurred from the Δced fore have functions similar to those of VirB3. (upsE+) strains to the ΔupsE (ced+) strain. This means that in Interestingly, a virB4 homolog, now named cedB, was also found these mixtures the Δced strains functioned as donor strains and the to be highly up-regulated upon induction with UV and essential for ΔupsE strain as recipient strain. From this observation we can DNA transfer. VirB4 proteins are AAA ATPases energizing sub- conclude that the Ced system (present in ΔupsE) functions as an strate transfer that are associated with all so-far-described type-IV importer. The small percentage of upsE+ background can be secretion systems (21, 33). CedB can additionally be modeled onto a crystal structure of helicase HerA from S. solfataricus.Thelatter explained by the fact that besides the pyrE region the genomic forms a hexameric ATPase that, together with nuclease NurA, region of upsE was probably cotransferred to the recipient cell. functions in DNA end processing in archaea. For this, the N-ter- Discussion minal domain of HerA binds to NurA and the C-terminal domain of HerA binds to DNA. The HerA–NurA complex subsequently DNA transport between cells from the same or different species translocates along the DNA upon ATP hydrolysis (17, 22, 34, 35). occurs throughout all domains of life via diverse mechanisms. Pur- Notably, CedB does not show homology to the N-terminal domain poses of DNA transfer include DNA repair and horizontal gene of HerA, which binds to NurA. Instead, it atypically contains an transfer (23). Among bacteria and archaea, DNA transfer via natural N-terminal membrane domain and localizes to the membrane. It is transformation as well as conjugation has been described for several therefore likely that CedB, similar to HerA, binds and translocates species, although bacterial DNA transfer mechanisms have been DNA but unlike HerA translocates across the membrane. studied in far greater detail. Also, for eukaryal cells and organelles Homologs of CedB are present among all species encoding mechanisms for DNA/RNA transfer have been described, including CedA and, intriguingly, cedB often lies directly downstream of vesicle-mediated DNA/RNA transfer between cells and the import or – cedA as part of the predicted operon. This suggests a functional export of DNA/RNA by mitochondria and the nucleus (24 26). link between the two proteins. Importantly, no DNA transfer Because archaeal DNA transport systems seem to differ greatly from was observed between cedA and cedB deletion strains, showing their bacterial and eukaryal counterparts, it was difficult to identify that both proteins function together, either in the donor or the putative transporter proteins based on homology. On a molecular recipient cell. level, transport of DNA among archaea therefore remained far from We were highly interested in determining the direction of well understood. The previously described Ups system (3, 4) enables DNA transfer between Sulfolobus cells. Previous data showed the exchange of chromosomal DNA between Sulfolobus cells upon that 62–81% of the recombinants (all pyrE+)inaupsE+*ΔupsE UV stress by mediating mating-pair formation. However, because the mixture were upsE+ (4). Based on this result it was suggested ups operon only encodes proteins involved in T4P formation, it seems that the transfer of DNA involves an active recruitment of DNA likely that other, unknown proteins function in the actual transport by a UV-damaged cell that produces pili. Alternatively, the transfer of DNA. In this study, we therefore sought to find proteins for the of DNA was thought to be bidirectional, with one partner being UV-induced DNA transfer among Sulfolobales. UV-activated, resulting in the mutual transfer of two markers (4). One of the highest up-regulated genes upon UV stress encodes a Here, however, we show that the Ups system functions separately transmembrane protein, now named CedA. Homologs are found from the Ced system, even though both systems are essential for among all Sulfolobales and additionally in several Desulfurococcales DNA transport. We therefore propose that the Ups system acts in and Acidilobales, where it contains six or seven transmembrane mating-pair formation, whereas the Ced system subsequently domains. A deletion of cedA in S. acidocaldarius did not result in a functions actively in DNA import. reduction of cellular aggregation but showed an abolishment of In nature, the transfer of DNA between Sulfolobus cells pre- DNA transfer. This indicates that the transmembrane protein is truly sumably occurs in both directions because all cells are genotypically involved in DNA transfer between cells. One could therefore envi- similar. To determine the direction of Ced-mediated DNA trans- sion that, similar to ComEC of competence systems or VirB6 of port, we determined the genotypes of recombinants obtained in conjugation systems, this protein forms a pore in the membrane to Δced*ΔupsE mixtures and found that all recombinants were ced+ transfer the DNA in or out of the cell (27–29). Among the Desul- and only 3–10% were upsE+. The fact that we only obtained ced+ furococcales and Acidilobales, CedA is significantly longer due to recombinants indicated that the pyrE locus was imported by the a predicted extracellular loop between the two C-terminal trans- ΔupsE (ced+) strain, suggesting that the Ced system functions as an membrane domains. This loop might function in protein interactions importer. Haloferax species exchange large pieces of chromosomal that differ from those in the Sulfolobales system; one could, for in- DNA of up to 500 kbp (13). The genomic distance between the pyrE stance, imagine an interaction with distinct pilin subunits functioning locus and the upsE locus is around 90 kbp, whereas the genomic

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1513740113 van Wolferen et al. Downloaded by guest on September 24, 2021 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 genesisatleast760kbp. performed by the Ced system. CedA proteins are thought to build If Sulfolobus cells would also exchange pieces of up to 500 kbp DNA, a membrane pore through which the DNA can be transferred. it would be likely that next to the pyrE locus also the upsE locus Membrane-bound ATPase CedB presumably binds the DNA and would frequently be transferred. Because we only obtained the energizes the translocation. We assume that CedA and CedB form ced+/upsE+ genotype in 3–10% of the recombinants, this does not one complex to perform this process, but this is not yet proven. In seem to be the case. We therefore assume that transferred pieces of addition, it is unclear whether single- or double-stranded DNA is chromosomal DNA are mostly smaller than 90 kbp. Of course, the transferred (Fig. 4). Once the DNA is imported, it can be used for pyrE locus and the upsE locus do not necessarily need to lie on the homologous recombination and thereby it can rescue the cells from MICROBIOLOGY same piece of DNA to be incorporated in the same chromosome; extensive DNA damage induced by UV light. The importance of this an alternative explanation for the transfer of both markers would mechanism is illustrated by the fact that cells that cannot exchange be the occurrence of a second DNA transfer event between the cells. DNA show significantly lower survival rates upon DNA damage (4). The finding that the Ced system functions in the import of Summarizing, we identified for the first time to our knowledge DNA was initially unexpected; other known DNA exchange an archaeal DNA transporter that differs from any other previously systems involving direct cellular contact (such as conjugation characterized system. This so-called Ced system is essential for systems) are based on the active export of DNA. The fact that DNA import and functions in the intriguing community-based this system actively imports DNA emphasizes the unique and DNA repair system of Sulfolobales. How DNA is exported from social nature of the transfer of chromosomal DNA between the donor cells and how incoming DNA finds the Ced system are Sulfolobus cells. Other known DNA uptake systems such as topics for future studies. competence systems only take up DNA from the environment, but not from other cells (36, 37). How DNA is exported from Materials and Methods the donor cell is still unclear. As mentioned above, we initially Construction of Deletion Mutants of S. acidocaldarius. To construct deletion strains expected a bacterial-like conjugation system; however, except of saci_0568 and saci_0748 in MW001 and JDS22, up- and downstream flanking ∼ for VirB4 homologs, no other genomically encoded homologs 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 of bacterial DNA transport systems can be found among the PCR products were subsequently cloned into pSVA406 or pSVA431, resulting in Crenarchaeota. This suggests the presence of a DNA export pSVA1833 and the pSVA3506, respectively (Table S2). To reintroduce cedB with a mechanism that is evolutionarily distinct from the bacterial Walker A mutation, plasmid pSVA1837 was constructed. Methylation and trans- conjugation system. Among the highest up-regulated genes upon formation of the plasmids, selection of integrants, and screening for mutants UV stress, sso0283 was found (saci_0667 in S. acidocaldarius) (2). were performed as described previously (38). Correctness of strains was confirmed Similar to cedB, this gene encodes a membrane-bound HerA by DNA sequencing (listed in Table S2). homolog. All organisms harboring the Ced system also encode a homolog of this protein. We therefore hypothesize this protein 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 either to be an additional ATPase functioning in the Ced system 2 or alternatively it might function on the donor side of DNA UV dose of 75 J/m (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 transfer events. This protein might therefore be the start of our after induction with UV, 5 μL of cell culture (diluted to OD 0.2) were spotted on study on the putative export machinery. a microscope slide covered with a thin layer of 1% agarose in Brock minimal With this study we propose a previously unidentified Crenarchaeal medium. Free and aggregated cells (n ≥ 3) were counted for at least 1,000 cells DNA exchange mechanism in which chromosomal DNA is imported per strain per replicate using ImageJ cell counter. Percentages of cells in ag- from other cells using the Ced system (Fig. 4). In Sulfolobales, the gregates were subsequently calculated from three different experiments. initiation of aggregation upon UV stress is mediated by the Ups system (3). Thereby, species-specific mating pairs are formed (Fig. 4, DNA Transfer Assays. DNA transfer between S. acidocaldarius cells was Left) (4). Because the Ups system is absent in other Crenarchaeota assayed by selecting prototrophic (pyr+) recombinants of two pyrE mutant – containing the Ced system, it is unclear how mating pairs are formed 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 in these species. We speculate that the extracellular loop in CedA, saci_0568 (cedA)andsaci_0748 (cedB) were made in these backgrounds as which is not present in the Sulfolobales, might be involved in this described above. Liquid cultures were grown at 75 °C and harvested at

process. Subsequent to the initiation of cellular contact, DNA is OD600 0.4–0.6. Pellets were concentrated to an OD600 of 1. UV irradiation exported from the donor cell via a so-far-unknown mechanism (Fig. was performed as described above and mixtures (1 mL per mating partner) 4, Right). The active import of DNA by the recipient cell is then were further incubated for 3 h at 75 °C in 24-well plates while shaking.

van Wolferen et al. PNAS Early Edition | 5of6 Downloaded by guest on September 24, 2021 Recombination was assayed by spreading 200 μL of each mixture, after ACKNOWLEDGMENTS. We thank Dennis Grogan for kindly providing us vigorously vortexing, on selective plates without uracil. Plates were incubated with strain JDS22. This work was supported by German Research Foundation for 5–6 d at 75 °C as was described previously (4). To determine the genotype (DFG) Grant AL1206/3-1 and European Research Council Starting Grant of the recombinants, colony PCRs were performed on UV*UV mixtures of ARCHAELLUM 311523 (to M.v.W.) and Max Planck Society Grant CRC987 and ΔupsE.1*ΔcedA.2 and ΔupsE.1*ΔcedB.2 (for strains see Table S2) using primers DFG Grant AL1206/4-1 (to A.W.). S.-V.A. and C.v.d.D. received intramural listed in Table S3. funds from the Max Planck Society.

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