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

Extremophiles : Life Under Extreme Conditions
Shoji Suzuki, N Kurosawa

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 N(4)-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 × 10(3)/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 ...Continue Reading

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Citations

Feb 24, 2018·Frontiers in Microbiology·Mohea Couturier, Ann-Christin Lindås
Jun 27, 2018·FEMS Microbiology Reviews·Christopher T StraubRobert M Kelly
Dec 12, 2017·Archaea : an International Microbiological Journal·Shoji Suzuki, Norio Kurosawa
Jan 22, 2021·FEMS Microbiology Reviews·April M LewisRobert M Kelly

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