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Molecular Characterization of the Minimal Replicon and the Unidirectional Theta Replication of pSCM201 in Extremely Halophilic Archaea

Chaomin Sun, Meixian Zhou, Yun Li, and Hua Xiang^*

State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China

Received 6 July 2006/ Accepted 13 August 2006

A 3,463-bp plasmid, pSCM201, was isolated from a halophilic archaeon, Haloarcula sp. strain AS7094. The minimal replicon that is essential and sufficient for autonomous replication and stable maintenance in Haloarcula hispanica was determined by deletion analysis of the plasmid. This minimal replicon (1.8 kb) consisted of only two functionally related segments: (i) a putative origin (ori201) containing an AT-rich region and sets of repeats and (ii) an adjacent gene encoding a putative replication initiation protein (Rep201). Electron microscopic observation and Southern blotting analysis demonstrated that pSCM201 replicates via a theta mechanism. Precise mapping of the putative origin suggested that the replication initiated from a fixed site close to the AT-rich region and proceeded unidirectionally toward the downstream rep201 gene, which was further confirmed by electron microscopic analysis of the ClaI-digested replication intermediates. To our knowledge, this is the first unidirectional theta replication plasmid experimentally identified in the domain of archaea. It provides a novel plasmid system to conduct research on archaeal DNA replication.

Published ahead of print on 22 September 2006.

Present address: Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405.

Present address: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148.

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