A PROMOTER-SCREENING PLASMID AND XYLOSE-INDUCIBLE, GLUCOSE-REPRESSIBLE EXPRESSION VECTORS FOR STAPHYLOCOCCUS-CARNOSUS

We describe a series of plasmid vectors for DNA cloning in staphylococ ci. pPS11 is a promoter probe plasmid containing a promoterless lipase (Lip)-encoding gene (lip). Insertion of a promoter-bearing DNA fragme nt at the single BamHI site turns on lip expression. Lip activity can be easily determined to estimate the strength of the inserted promoter . pPS11 served also as a basis for the construction of vectors which a llow xylose-inducible gene expression in Staphylococcus carnosus (Sc). Using plasmid pCX15, we studied xylose-inducible lip expression in Sc . The lip expression is under transcriptional control of the repressor , XylR. The xylR gene, the XylR target sequence and the xylA promoter/ operator sequence with the cis-acting catabolite-responsive element (c re) are derived from the xyl operon of S. xylosus. The single BamHI si te in front of the lip ribosome-binding site (RBS) also makes it possi ble to put other promoterless genes under transcriptional control of X ylR. To facilitate the controlled expression of genes which merely sta rt with the start codon and have no RBS, or to insert genes with their own RBS, pCX26 and pCX26 Delta lip were constructed. The influence of xylose and glucose on lip expression was studied both in a batch cult ure and in a fermenter under controlled pH conditions. With pCX15, the presence of xylose led to a 40-fold increase in extracellular Lip act ivity, while the presence of glucose caused a repression of lip expres sion. The results suggest that the xylA promoter is subject to two dif ferent regulatory mechanisms, one of which involves the repression of the xylA promoter by XylR in the absence of xylose, and the other invo lves a glucose-mediated catabolite repression which dominates over the xylose induction.