The Expression of the Escherichia coli lacZ Gene in Streptomyces

Abstract

The Escherichia coli lacZ gene was stably introduced into phi 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 phi C31::lacZ constructs involving transcriptional fusions to the pBR322 tet gene contained beta-galactosidase activity. Activities were markedly 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 phi 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 phi 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.

TTA codons in some genes prevent their expression in a class of developmental, antibiotic-negative, Streptomyces mutants.

“…1). Although lacZ could not be stably maintained in those Streptomyces plasmids tested (12), it could be introduced into 4C31 vectors, giving phages such as KC659 which expressed P-galactosidase activity in plaques on S. lividans 1326-9 (12). No B-galactosidase activity was seen in KC659 plaques on bldA16 and bldA39 mutants of 1326-9 (J1726 and J1725, respectively).…”

“…For f4-lactamase detection, cultures of R2YE plates were sprayed with a fresh solution of the chromogenic cephalosporin nitrocefin (20) to give a red-brown diffusible pigment in positive tests. For detecting B-galatosidase in plaques, 5-bromo-4-chloro-3-indolyl 8-D-galactoside (X-Gal) was incorporated into top layers (12). Spectrophotometric assay of hygromycin phosphotransferase was as in ref.…”

β-Glucuronidase as a Sensitive and Versatile Reporter in Actinomycetes

“…For such purposes, transcriptional and translational fusions with reporter genes are often used. Unfortunately, the most reliable, convenient, and widely used lacZ reporter gene has strong limitations in streptomyces due to their significant endogenous ␤-galactosidase activities (32). The current streptomyces reporter toolkit includes neo and cat resistance genes (43,44,51), catechol 2,3-dioxygenase (55), green fluorescent protein (49), luciferase (48), and others, but none of them is nearly as good as lacZ.…”

Identification of methanol-regulated promoter sequences from the facultative methylotrophic bacterium Methylobacterium organophilum XX

“…A number of vectors employing xylE as the reporter gene have been developed in systems where well-established reporter genes such as lac2 were not very effective (Zukowski et al, 1983;King & Chater, 1986;Ray et al, 1988Ray et al, , 1985Winstanley et al, 1989). In contrast, very little has been reported on the application of reporter genes in methylotrophs.…”