Expression in Escherichia coli of streptococcal plasmid-determined erythromycin resistance directed by the cat gene promoter of pACYC 184.

Abstract

The streptococcal erythromycin resistance (Emr) plasmid pSM7 (6.4 kb) and the E. coli vector pACYC184 (4.0 kb) were fused at their single EcoRI sites to form the bifunctional chimeric plasmid pSM7184 (10.4 kb) in which the Emr determinant was placed under control of the chloramphenicol acetyl transferase (cat) promoter of pACYC184. In the sense orientation (orientation I) of pSM7, the cat promoter directed expression of Emr in the E. coli host strains 294 and DB11 more efficiently than did the indigenous transcription signals of pSM7, which were functional in the opposite orientation II. In Streptococcus sanguis (Challis), the level of Emr was independent of the orientation of pSM7 in pACYC184, showing that the cat promoter was not recognized in the gram-positive host. The growth of E. coli (pSM7184I) in a defined medium containing glycerol as carbon source, or containing glucose plus extraneous cyclic 3'-5' adenosine monophosphate (cAMP) led to an Emr level which was 15-30 times higher than that of cultures grown on glucose. These results showed that under control of the cat promoter, Emr is subject to cAMP-mediated catabolite repression and provided conclusive evidence that the enhancement of Emr expression in E. coli carrying pSM7184I is controlled at the transcriptional level. Besides enabling us to determine the orientation of transcription of the Emr gene in pSM7 and related vectors, this work also made available new bifunctional cloning vehicles able to replicate in both E. coli and S. sanguis.