5088–5104 Nucleic Acids Research, 2010, Vol. 38, No. 15 doi:10.1093/nar/gkq236

Published online 18 April 2010

Two novel families of plasmids from hyperthermophilic archaea encoding new families of replication proteins Nicolas Soler1,*, Evelyne Marguet1, Diego Cortez2, Nicole Desnoues2, Jenny Keller3, Herman van Tilbeurgh3, Guennadi Sezonov2,4 and Patrick Forterre1,2,* 1

Institut de Ge´ne´tique et Microbiologie, Univ Paris-Sud, 91405 Orsay Cedex, CNRS UMR 8621, Institut Pasteur, 25 rue du Docteur Roux, 75015 Paris, 3Institut de Biochimie et de Biophysique Mole´culaire et Cellulaire, Universite´ Paris-Sud, IFR115, UMR8619-CNRS, 91405 Orsay and 4Universite´ Pierre et Marie Curie, 4 place Jussieu, 75005 Paris, France

2

Received November 21, 2009; Revised March 16, 2010; Accepted March 22, 2010

ABSTRACT

INTRODUCTION

Thermococcales (phylum Euryarchaeota) are model organisms for physiological and molecular studies of hyperthermophiles. Here we describe three new plasmids from Thermococcales that could provide new tools and model systems for genetic and molecular studies in Archaea. The plasmids pTN2 from Thermococcus nautilus sp. 30-1 and pP12-1 from Pyrococcus sp. 12-1 belong to the same family. They have similar size (12 kb) and share six genes, including homologues of genes encoded by the virus PAV1 from Pyrococcus abyssi. The plasmid pT26-2 from Thermococcus sp. 26-2 (21.5 kb), that corresponds to another plasmid family, encodes many proteins having homologues in virus-like elements integrated in several genomes of Thermococcales and Methanococcales. Our analyses confirm that viruses and plasmids are evolutionary related and co-evolve with their hosts. Whereas all plasmids previously isolated from Thermococcales replicate by the rolling circle mechanism, the three plasmids described here probably replicate by the theta mechanism. The plasmids pTN2 and pP12-1 encode a putative helicase of the SFI superfamily and a new family of DNA polymerase, whose activity was demonstrated in vitro, whereas pT26-2 encodes a putative new type of helicase. This strengthens the idea that plasmids and viruses are a reservoir of novel protein families involved in DNA replication.

Plasmids are especially abundant in the domains Archaea and Bacteria [for a recent monography, see ref. (1)]. Their size ranges from a very small plasmid with a single gene, such as the plasmid pRQ7 from the hyperthermophilic bacterium Thermotoga (2), to large megaplasmids whose size rival those of bacterial or archaeal chromosomes (3,4). Plasmids can use different replication strategies (mostly rolling-circle mode for the smallest ones and theta mode for the others) and a variety of replication origins (5). In Archaea, plasmids from Sulfolobales (thermoacidophilic members of the phylum Crenarchaea) have been especially well characterized (6,7). Two plasmid families have been identified. The first one is the family of pRN-type plasmids (with sizes ranging mainly from 5 up to 14 kb) (6,8,9). The second one corresponds to rather large conjugative plasmids of 24 to 36 kb, pNOB8 and its relatives (10,11). All pRN-type plasmids encode two DNA binding proteins (CopG and PlrA) that could be involved in the regulation of copy number and gene expression (12,13) and a large protein, dubbed RepA, involved in plasmid replication. The RepA protein of pRN1 is a multifunctional enzyme whose N-terminal domain harbours DNA primase/polymerase activities and the C-terminal domain corresponds to a DNA helicase of the SFIII superfamily (14,15). The primase/polymerase of pRN1 is the prototype of a new DNA polymerase family whose members are encoded by various archaeal and bacterial plasmids and some bacterial viruses (15). The structural characterization of the N-terminal domain of the pRN1 DNA polymerase revealed that its catalytic domain is evolutionary related to those of archaeal and eukaryotic DNA primases (16). Two plasmids of the pRN family lack homologue of

*To whom correspondence should be addressed. Tel: +33 1 40 51 65 76; Fax: +0033 140516570; Email: [email protected] Correspondence may also be addressed to Patrick Forterre. Tel: +33 1 69 15 74 89; Fax: +1 802 656 0747; Email: [email protected] ß The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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pRN1 RepA: one of them, pXZ1, encodes instead a large protein of similar size but without sequence similarity to pRN1 RepA (17), whereas another one, pTAU4, encodes a MCM helicase instead of a Rep protein (8). An interesting member of the pRN family is pSSVx, a virus–plasmid hybrid that coexists intracellularly with the fusellovirus SSV1 and can be packaged into viral particles (18,19). Besides typical pRN proteins, pSSVx encodes two SSV1 proteins probably involved in plasmid packaging. Several pRN-type plasmids have been found integrated into tRNA genes in the chromosomes of different Sulfolobales (18). The site-specific integrase that promotes their insertion is homologous to the integrases of the SSV1 and other fuselloviruses which can also integrate in the chromosomes of Sulfolobus species (20). The insertion site recognized by SSV-type integrases is located in the coding part of their own gene and the integration event provokes its disruption. The plasmid pXZ1 also encodes an SSV-type integrase and coexists in Sulfolobus cell with a fusellovirus (SSV4) but, unlike pSSVx, pXZ1 cannot be packaged into viral particle (17). The SSV1 virus, as well as pSSVx and pRN1 has been used to develop genetic tools for Sulfolobus species, including shuttle vectors to express foreign proteins in Sulfolobales (21,22). The Sulfolobus conjugative plasmids of the pNOB8-type encodes 40–50 proteins (10,11). Ten proteins are conserved in all these plasmids and 80% of the remaining proteins are common to at least two plasmids (11). Most of these proteins are of unknown function. Their conserved genes are grouped in two regions: one of them includes two genes encoding proteins with low similarity to TraG and TraE proteins involved in bacterial plasmids conjugation, as well as several proteins with membrane domains that are supposed to be involved in DNA transfer. The second region contains the genes encoding homologues of the pRN-type plasmids, CopG and PrlA proteins, and an integrase gene. This integrase represents a novel family of integrases, the pNOB8-type, that are widely distributed in archaeal genomes (10). Unlike the SSV-type, pNOB8-type integrases never bear their insertion site inside the sequence of their genes and consequently these genes are never disrupted after integration. Several pNOB8-type plasmids encode transposases, and one of them (pKEF9) also encodes a pRN-like RepA protein. A new member of the pNOB8 conjugative plasmid family, pAH1, has been recently discovered in an Acidianus species that belongs to the order Sulfolobales (20). Interestingly, the replication of pAH1 is inhibited by infection by the lipothrixvirus AFV1, revealing a new type of plasmid/virus functional interaction (20). All plasmids presently isolated from Sulfolobales are larger than 5 kb. Although their mode of replication has not yet been determined experimentally, they probably use the theta replication mode, since none of them encode the Rep protein typical for rolling-circle replication. In contrast, all known plasmids from Thermococcales (hyperthermophilic and anaerobic members of the phylum Euryarhaea) are small (