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research-article

Construction and application of plasmid- and transposon-based promoter-probe vectors for Streptomyces spp. that employ a Vibrio harveyi luciferase reporter cassette.

Department of Microbiology, University of Texas, Austin 78712-1095.

ABSTRACT

Several versatile promoter-probe vectors have been constructed for Streptomyces strains which utilize the production of blue-green light as a measure of transcription activity. Three plasmid vectors (two high and one low copy number) and two transposons are described. The multicopy plasmids pRS1106 and pRS1108 contain a transcription terminator and multiple-cloning polylinker upstream of promoterless luciferase (lux) and neomycin resistance reporter genes. Plasmid pHI90 is similar in structure to the pRS vectors except that its single copy number is an advantage for regulation studies or situations in which overexpression is otherwise toxic to the cell. The two transposons carry a promoterless lux cassette cloned such that transposition into a target DNA and fusion to the target's transcription unit occur simultaneously. Tn5351 was created by inserting the luciferase genes near the right end of the viomycin resistance transposon Tn4563. Tn5353 carries the luciferase genes near the right end of a neomycin resistance transposon derived from Tn4556. The size of Tn5353 was minimized by deleting nonessential transposon sequences, making this element small enough to be cloned into phi C31 bacteriophages for efficient transposon delivery to target cells of Streptomyces strains. The two Tnlux transposons have been used to generate Streptomyces coelicolor morphological mutants and to monitor transcription from chromosomal promoters during development.