DEPENDENCE ON REPORTER GENE OF APPARENT ACTIVITY IN GENE FUSIONS OF ASTREPTOMYCES-GRISEUS STREPTOMYCIN BIOSYNTHESIS PROMOTER

The adjacent genes strR-strA-strB1 lie within the large cluster of gen es of streptomycin biosynthesis and resistance in Streptomyces griseus . strR encodes a pathway-specific activator StrR, suggested by previou s work to be either an antiterminator or a conventional activator, bin ding to its DNA target via a helix-turn-helix motif, strB1 is transcri bed in an StrR-dependent fashion from a promoter (PstrB1) that lies do wnstream from strA; between PstrB1 and strB1 there is a 300-bp leader region containing numerous inverted repeats that could represent modul atable transcription termination sites. Hybrid plasmids were construct ed in vitro with transcriptional fusions in which fragments containing PstrB1 and either the entire leader region (''long'' fragments) or a small part of it (the ''short'' fragment) were cloned upstream of (i) aph as reporter gene, in a high copy number plasmid background, or (ii ) xylE as reporter gene, in a low copy number plasmid background. The short fragment directed high levels of APH (aminoglycoside 3'-phosphot ransferase) whether StrR was present or not, while the long fragments did not do so in the absence of StrR; one long fragment directed high levels in wild-type S. griseus, in which StrR would be present. Insert ion of an extraneous fragment into PstrB1 in the short fragment constr uct led to loss of APH activity, demonstrating that no adventitious pr omoter had been formed in the short construct. Ln vitro deletion of pa rt of the leader region in a long fragment construct led to high APH e xpression with or without StrR present. Although these results are con sistent with the target of StrR being within the leader region, and th us with an antiterminator role, it was found that both long and short fragments in the low copy number background failed to direct high expr ession of catechol oxygenase (the product of xylE) unless strR was als o present on a compatible plasmid. Transfer of PstrBI-xylE fragments t o the high copy number vector did not increase catechol oxygenase expr ession. We interpret these results in terms of an effect, in the hybri d constructs, of one of the reporter genes on promoter function, possi bly by affecting local DNA topology.