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Applied and Environmental Microbiology, November 1998, p. 4363-4367, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Characterization of the Basic Replicon of Rhodococcus Plasmid pSOX and Development of a Rhodococcus-Escherichia coli Shuttle Vectordagger

Claude Denis-Larose,1 Hélène Bergeron,1 Diane Labbé,1 Charles W. Greer,1 Jalal Hawari,1 Matthew J. Grossman,2 Bruce M. Sankey,3 and Peter C. K. Lau1,*

Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada H4P 2R21; Exxon Research and Engineering Company, Corporate Research, Annandale, New Jersey 088012; and Imperial Oil Resources Ltd., Calgary, Alberta, Canada T2L 2K83

Received 14 May 1998/Accepted 24 August 1998

The replication region of a 100-kb desulfurization plasmid (pSOX) from Rhodococcus sp. strain X309 was localized to a 4-kb KpnI fragment, and its sequence was determined. The amino acid sequence of one of the predicted open reading frames (ORFs) was related to the putative replication (Rep) protein sequences of the mycobacterial pLR7 family of plasmids. Three of the five predicted ORF products were identified by radiolabelling with the Escherichia coli T7 polymerase/promoter system. In E. coli, the Rep protein of pSOX was apparently synthesized in a shortened form, 21.3 kDa instead of the predicted 41.3 kDa, as a result of an internal initiation. This situation is reminescent of that for some bacterial Rep proteins. A shuttle plasmid was constructed with the pSOX origin, pBluescript II KS-, and the chloramphenicol resistance (Cmr) gene from pRF29. This new shuttle plasmid was used to demonstrate expression of the Bacillus subtilis sacB gene in a strain of Rhodococcus, rendering it sensitive to the presence of sucrose.

* Corresponding author. Mailing address: Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Ave., Montreal, Quebec, Canada H4P 2R2. Phone: (514) 496 6325. Fax: (514) 496 6265. E-mail: peter.lau{at}nrc.ca.

dagger NRCC publication 41774.

Applied and Environmental Microbiology, November 1998, p. 4363-4367, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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