Plasmid pGS5 from the Hyperthermophilic Archaeon Archaeoglobus profundus Is Negatively Supercoiled

Abstract

We present evidence that, in contrast to plasmids from other hyperthermophilic archaea, which are in the relaxed to positively supercoiled state, plasmid pGS5 (2.8 kb) from Archaeoglobus profundus is negatively supercoiled. This might be due to the presence of a gyrase introducing negative supercoils, since gyrase genes are present in the genome of its close relative A. fulgidus, and suggests that gyrase activity predominates over reverse gyrase whenever the two topoisomerases coexist in cells.Previous topological analyses of native plasmids from hyperthermophiles belonging to the two major phylogenetic lineages within the domain Archaea revealed a relaxed to positively supercoiled state of DNA (1, 2, 10). This was in contrast to the negatively supercoiled state of DNA in all mesophiles, including mesophilic archaea (1). The strains investigated in those studies were members of the orders Sulfolobales and Thermococcales, representing the two major archaeal kingdoms, Crenarchaeota and Euryarchaeota, respectively. All of them possessed reverse gyrase, a topoisomerase specific for hyperthermophiles that is able to introduce positive DNA supercoils (5). Therefore, reverse gyrase appeared responsible for this peculiar topological state. Accordingly, the idea that DNA was stabilized in organisms living at very high temperatures by an overall linking excess seemed to be supported (6). However, this hypothesis was called into question when plasmid pRQ7 from the hyperthermophilic bacterium Thermotoga maritima was found to be negatively supercoiled (7). In addition to reverse gyrase, T. maritima possesses DNA gyrase, a typical bacterial topoisomerase that introduces negative supercoils into DNA and is responsible for the negative supercoiling of pRQ7 (7). Two alternative possibilities could be then considered to explain the different plasmid topologies in hyperthermophiles. Either gyrase activity dominates whenever both reverse gyrase and gyrase are present in the cell, maintaining an overall DNA negative supercoiling, or, alternatively, the relaxed to positively supercoiled plasmid topology could be a taxonomic characteristic specific to hyperthermophilic archaea. The discovery of pGS5, a plasmid of 2,802 bp in the sulfate-reducing hyperthermophilic archaeon Archaeoglobus profundus (G. Erauso et al., unpublished data), allowed us to test which possibility was correct since the complete genome sequence of the closely related species Archaeoglobus fulgidus revealed the existence of genes encoding both gyrase and reverse gyrase (9).A. profundus strain AV18 was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM 5631) and cultivated under strict anaerobic conditions in a standard medium (Erauso et al., unpublished) consisting of a basal salt solution (containing, per liter, 20 g of NaCl, 3 g of MgCl 2 ⅐ 6H 2 O, 4 g of Na 2 SO 4 , 0.5 g of KCl, 0.25 g of NH 4 Cl, 0.15 g of CaCl 2 ⅐ 2H 2 O, and 0.14 g of K 2 HPO 4 ) plus 1 ml of a trace element solution (containing, per liter, 100 mg of Na 2 WO 4 ⅐ 2H...

Living Side by Side with a Virus: Characterization of Two Novel Plasmids from Thermococcus prieurii, a Host for the Spindle-Shaped Virus TPV1

“…Nevertheless, similar to pTP1, pTP2 is likely to replicate using the RC mechanism. ORF3 of pTP2 encodes a putative protein (RepTP2) of 252 aa which shares significant sequence similarity with RCReps encoded by diverse archaeal MGE (Table 2), including Archaeoglobus profundus plasmid pGS5 (24), putative Methanococcus voltae A3 provirus (MVV) (46), and a group of pleomorphic haloarchaeal viruses (47). Sequence analysis of RepTP2 showed that, unlike RepTP1, it possesses two conserved Tyr resi- dues in motif 3 ( Fig.…”

“…In this phylum, plasmids have been isolated from hyperthermophilic organisms belonging to three different orders, the Methanococcales (genus Methanocaldococcus) (23), Archaeo-globales (24), and Thermococcales (25). However, viruses have been isolated only from members of the latter order (26,27).…”

Bipartite Network Analysis of the Archaeal Virosphere: Evolutionary Connections between Viruses and Capsidless Mobile Elements

“…Finally, module 10 includes two small rolling-circle plasmids, Archaeoglobus profundus plasmid pGS5 (71) and Thermococcus prieurii plasmid pTP2 (72), which connect to members of the genus Alphapleolipovirus (Halorubrum pleomorphic viruses 1, 2, and 6) through an RCRE. The latter protein is also shared with the putative provirus MVV, which is related to Turriviridae from module 6, as well as with the Sulfolobus monocaudavirus 2 (SMV2) from module 9.…”

Separate and Combined Biochemical Activities of the Subunits of a Naturally Split Reverse Gyrase

“…It has not been established precisely how this enzyme can protect against DNA thermal instability. DNA positive supercoiling per se may not be the direct cause, because supercoiling in hyperthermophiles is highly variable ranging from positively supercoiled to relaxed or even negatively supercoiled (11). The biochemical activity of reverse gyrase as a renaturase for single-stranded DNA may have a critical role in maintaining genome stability at high temperature (12).…”