Hindawi Publishing Corporation Archaea Volume 2011, Article ID 723604, 4 pages doi:10.1155/2011/723604 Research Article Characterization of Plasmid pPO1 from the Hyperacidophile Picrophilus oshimae Angel Angelov, 1 J¨ orn Voss, 2 and Wolfgang Liebl 1 1 Lehrstuhl f¨ ur Mikrobiologie, Technische Universit¨ at M¨ unchen, Emil-Ramann-Straße 4, Weihenstephan, 85354 Freising, Germany 2 Institut f¨ ur Mikrobiologie und Genetik, Georg-August-Universit¨ at G¨ ottingen, Grisebachstrasse 8, 37077 G¨ ottingen, Germany Correspondence should be addressed to Wolfgang Liebl, wliebl@wzw.tum.de Received 1 June 2011; Accepted 21 July 2011 Academic Editor: Matthias Hess Copyright © 2011 Angel Angelov et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Picrophilus oshimae and Picrophilus torridus are free-living, moderately thermophilic and acidophilic organisms from the lineage of Euryarchaeota. With a pH optimum of growth at pH 0.7 and the ability to even withstand molar concentrations of sulphuric acid, these organisms represent the most extreme acidophiles known. So far, nothing is known about plasmid biology in these hyperacidophiles. Also, there are no genetic tools available for this genus. We have mobilized the 7.6 Kbp plasmid from P. oshimae in E. coli by introducing origin-containing transposons and described the plasmid in terms of its nucleotide sequence, copy number in the native host, mode of replication, and transcriptional start sites of the encoded ORFs. Plasmid pPO1 may encode a restriction/modification system in addition to its replication functions. The information gained from the pPO1 plasmid may prove useful in developing a cloning system for this group of extreme acidophiles. 1. Introduction Picrophilus torridus and Picrophilus oshimae are the most extreme organisms with respect to acidophilic combined with thermophilic lifestyle known to date. These species rep- resent thermoacidophilic archaea, originally isolated from a dry solfataric field in Northern Japan [1]. Together with the genera Thermoplasma and Ferroplasma they form a phylogenetically distinct group of free-living, moderately thermophilic and acidophilic organisms within the Eur- yarchaeota. The two species of the Picrophilus genus are so far unsurpassed in their ability to grow at pH values around 0, with an optimum at pH 0.7. Also, P. oshimae has been shown to maintain an unusually low intracellular pH of 4.6, in contrast to other acidophilic organisms where this value is usually close to neutral [2]. P. torridus and P. oshimae share similar physiological properties and are morphologically indistinguishable. On the other hand, they differ in their DNA restriction fragment patterns, their 16S rDNA gene sequences, and the presence of extrachromosomal elements. Plasmids of 8.3 kb and 8.8 kb, which showed strong cross- hybridization in southern blot analysis, have been isolated from samples later assigned to P. oshimae but not from samples assigned to P. torridus [3]. Unlike the situation in the Sulfolobales order and especially in the genus Sulfolobus, where a large number of genetic elements have been char- acterized [4], little is known of extrachromosomal elements in the Thermoplasmatales. To date, the only sequenced and characterized plasmid from this phylogenetic order is pTA1 isolated from Thermoplasma acidophilum [5]. The analysis of plasmid pPO1 from P. oshimae reported here should prove useful in developing genetic tools for this group of organisms. 2. Materials and Methods 2.1. Strains and Plasmids. P. oshimae was obtained from DSMZ (DSM 9789) and was grown in a modified Brock’s medium with a pH of 0.7 at 55 ◦ C as described previously [3, 6]. The E. coli strain JM104 (pir + ) was kindly provided by Professor Ruth Schmitz-Streit (University of Kiel, Germany). pPO1 was isolated from exponentially growing P. oshimae cells using a QIAprep Miniprep Kit (Qiagen), and total DNA used in real time PCR was prepared by the alkaline lysis method [7].