The Expression of the Escherichia coli lacZ Gene in Streptomyces

Abstract

The Escherichia coli lacZ gene was stably introduced into .vphi.C31-based phage cloning vectors in Streptomyces lividans. However, lacZ could not be stably introduced into S. lividans on the plasmid vectors pIJ702 or pIJ41. Studies of the expression of lacZ in S. lividans and S. coelicolor were facilitated by the use of mutants and/or growth conditions in which endogenous .beta.-galactosidase activity was low or absent. Plaques and lysogens of .vphi.C31::lacZ constructs involving transcriptional fusions to the pBR322 tet gene contained .beta.-galactosidase activity. Activities were marked higher in S. lividans than in S. coelicolor. Insertion of the major transcriptional terminator of coliphage fd between the tet promoter and lacZ reduced lacZ expression more in lysogens than in lytically infected cultures, suggesting the existence of a phage-specified anti-termination function. Several .vphi.C31 derivatives suitable for the construction of different kinds of transcriptional and translational fusions in Streptomyces were derived. The expression of lacZ in one transcriptional fusion vector (KC659) was increased both in plaques and in lysogens by the appropriately positioned insertion of a previously characterized promoter from the aph gene of S. fradiae. When .vphi.C31 DNA fragments were inserted into KC659, at least one recombinant phage gave high .beta.-galactosidase activity in lytic infections, but not in lysogens. The potential usefulness of lacZ fusions in Streptomyces is discussed.