Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator

Abstract

Several versatile, multi-copy, promoter-probe plasmid vectors have been constructed that replicate in a wide range of Streptomyces species. Transcriptional activity is detected by the expression of a promoter-less aminoglycoside phosphotransferase gene (neo) derived from the transposon Tn5; expression of this gene confers kanamycin and neomycin resistance on Streptomyces lividans. An efficient transcriptional terminator from E. coli phage fd has been inserted upstream of the neo coding region to prevent significant transcriptional read-through from vector promoters. A translational stop codon situated downstream from the site(s) used for cloning and preceding and in frame with the ATG start codon of the neo gene ensures the detection of transcriptional, rather than translational, fusions. Relative promoter strengths can be determined by gradient plate assays of kanamycin resistance, by measuring the amount of aminoglycoside phosphotransferase produced or by estimating neo mRNA synthesised. The high copy number of the vectors facilitates the rapid isolation and characterisation of promoter-active fragments and convenient restriction sites are available for DNA sequencing and S1 mapping of cloned inserts. Some derivatives contain a polylinker that facilitates the insertion, excision and analysis of cloned fragments and which enhances the use of these plasmids as general cloning vectors.

Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2) is controlled by malR, a member of the lacI–galR family of regulatory genes

“…pIJ486 (Ward et al, 1986) was used as a high-copy-number vector (Ϸ50-100 copies per chromosome; T. Kieser, personal communication) in S. coelicolor. The 2.2 kb Fok I fragment ( Fig.…”

Construction of thiostrepton-inducible, high-copy-number expression vectors for use in Streptomyces spp.

“…This fragment was inserted into a derivative of the hcn plasmid pIJ487 (50-100 copies per chromosome; T. Kieser, personal communication; Ward et al, 1986) to give pIJ6021 (Fig. 1).…”

“…(Hayes and Szybalski, 1973;Zukowski and Miller, 1986) occurs upstream from Ptip.4 to prevent transcriptional read-through from the vector and to maximise the induction ratio upon addition of Th. A transcriptional terminator from phage fd (tfd; Gentz et al, 1981;Ward et al, 1986) is located downstream from the MCS to prevent potentially deleterious transcription of vector sequences from PapA. A Km R gene (kan) from Micromonospora echinospora (C. Nojiri, personal communication) allows selection of transformants, and the Th R gene (tsr) from S. azureus provides resistance to Th upon induction of PtipA with the antibiotic.…”

Design-based re-engineering of biosynthetic gene clusters: plug-and-play in practice

“…To facilitate reliable transcription termination, terminators derived from various phages have been used and shown to function in E. coli and Streptomyces [36][37][38]. A list of terminators for various hosts can be found in the catalogue of the registry of standardized biological parts.…”