CHARACTERIZATION OF THE BASIC REPLICON OF RHODOCOCCUS PLASMID PSOX AND DEVELOPMENT OF A RHODOCOCCUS ESCHERICHIA-COLI SHUTTLE VECTOR

The replication region of a 100-kb desulfurization plasmid (pSOX) from Rhodococcus sp, strain X309 was localized to a 4-kb KpnI fragment, an d its sequence was determined. The amino acid sequence of one of the p redicted open reading frames (ORFs) was related to the putative replic ation (Rep) protein sequences of the mycobacterial pLR7 family of plas mids, Three of the five predicted ORF products were identified by radi olabelling with the Escherichia coli T7 polymerase/promoter system. In E. coli, the Rep protein of pSOX was apparently synthesized in a shor tened 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 som e bacterial Rep proteins. A shuttle plasmid was constructed with the p SOX origin, pBluescript II KS-, and the chloramphenicol resistance (Cm -r) gene from pRF29, This new shuttle plasmid was used to demonstrate expression of the Bacillus subtilis sacB gene in a strain of Rhodococc us, rendering it sensitive to the presence of sucrose,