Isolation and Characterization of Transcriptional Elements from Corynebacterium glutamicum

Abstract

A promoter-probe shuttle vector pSK1Cat was constructed for the isolation of transcriptional signal sequences from Corynebacterium glutamicum. Besides conferring resistance to kanamycin in Escherichia coli and C. glutamicum, the vector carried a promoterless cat gene to confer resistance to chloramphenicol upon insertion of the appropriate transcriptional signals in the multiple cloning site. By utilizing the vector, a series of transcriptionally active fragments were isolated from the genome of C. glutamicum. The clones, ranging from 200 bp to 1 kb in size, were grouped into 3 classes of strong, medium, and weak, based on the chloramphenicol acetyltransferase (CAT) activity and sensitivity to the chloramphenicol of the clone-carrying C. glutamicum cells. C. glutamicum cells carrying the P 19 clone, a representative in the strong class, were able to grow on minimal agar plates containing over 40 mg/ml chloramphenicol, and showed CAT activity of 10 mmol/mg.min, performing slightly better than the cells carrying P tac , a strong E. coli promoter. Subcloning analysis of the P 19 clone identified a 180 bp intergenic fragment (P 180 ), which was located upstream of a gene encoding a hypothetical membrane protein. The expression conferred by P 180 was not affected by either the kinds of carbon sources or changes in temperature. These properties make the P 180 clone useful for the deregulated expression of biosynthetic genes in C. glutamicum during amino acid fermentation.