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Disruption of the gene encoding restriction endonuclease SuaI and development of a host–vector system for the thermoacidophilic archaeon Sulfolobus acidocaldarius

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Abstract

Sulfolobus acidocaldarius is a useful model organism for the genetic study of thermophilic archaea due to its ease of cultivation. Here we describe the development of a host–vector system for S. acidocaldarius consisting of SuaI restriction system-deficient strain SK-1 and shuttle vector pSAV2. The new host strain SK-1 was constructed by pop-out recombination based on the pyrE marker gene. Plasmid pSAV2 was constructed from the S. islandicus native plasmid pRN1, in which selectable markers and functional genes were inserted in suitable locations and orientations followed by the deletion of non-essential open reading frames. SK-1 allowed direct transformation without N4-methylation at SuaI restriction sites, so unmethylated vector pSAV2 could be introduced directly into SK-1 by electroporation. The transformants were selected by pyrEF complementation on xyrose–tryptone solid medium without prior liquid culturing. The transformation efficiency was approximately 1.0 × 103/μg DNA. After replication in S. acidocaldarius, pSAV2 was successfully recovered from transformant cultures by the standard alkaline lysis method. Plasmid yield was approximately 40–50 ng/ml from late-log through stationary phase cultures. In addition, pSAV2 was maintained stably and at relatively high copy number in S. acidocaldarius.

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Acknowledgments

We thank Dennis Grogan for providing a viable culture of S. acidocaldarius MR31 and for valuable suggestions to improve the manuscript. We also thank Kenneth M. Stedman for providing a viable culture of S. islandicus REN1H1, and Yoko Kawasaki for valuable technical assistance.

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  1. Department of Science and Engineering for Sustainable Innovation, Faculty of Science and Engineering, Soka University, Tokyo, Japan

    Shoji Suzuki & Norio Kurosawa

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  1. Shoji Suzuki
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  2. Norio Kurosawa
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Correspondence to Norio Kurosawa.

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Communicated by L. Huang.

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Suzuki, S., Kurosawa, N. Disruption of the gene encoding restriction endonuclease SuaI and development of a host–vector system for the thermoacidophilic archaeon Sulfolobus acidocaldarius . Extremophiles 20, 139–148 (2016). https://doi.org/10.1007/s00792-016-0807-0

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