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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 trans- porter 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
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