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 staphylococci. pPS11 is a promoter probe plasmid containing a promoterless lipase (Lip)-encoding gene (lip). Insertion of a promoter-bearing DNA fragment 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 allow 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 (cre) are derived from the xyl operon of S. xylosus. The single BamHI site in front of the lip ribosome-binding site (RBS) also makes it possible to put other promoterless genes under transcriptional control of XylR. To facilitate the controlled expression of genes which merely start 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 culture and in a fermentor under controlled pH conditions. With pCX15, the presence of xylose led to a 40-fold increase in extracellular Lip activity, while the presence of glucose caused a repression of lip expression. The results suggest that the xylA promoter is subject to two different regulatory mechanisms, one of which involves the repression of the xylA promoter by XylR in the absence of xylose, and the other involves a glucose-mediated catabolite repression which dominates over the xylose induction.
Wieland
K P
KP
Universität Tübingen, Germany.
Wieland
B
B
Götz
F
F
D001483:Base Sequence; D017931:DNA Primers; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology*; D008049:Lipase / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013210:Staphylococcus / Q000235:genetics*; D014994:Xylose / Q000235:genetics / Q000494:pharmacology*
D017931:DNA Primers; D014994:Xylose; D008049:Lipase; D005947:Glucose