2 FUNCTIONALLY DISTINCT REGIONS UPSTREAM OF THE CBB(I) OPERON OF RHODOBACTER-SPHAEROIDES REGULATE GENE-EXPRESSION

A number of c6bF(I)::lacZ translational fusion plasmids containing var ious lengths of sequence 5' to the form I (cbb(I)) Calvin-Benson-Bassh am cycle operon (cbbF(I)cbbP(I)cbbA(I)cbbL(I)cbbS(I)) of Rhodobacter s phaeroides were constructed. Expression of beta-galactosidase,vas moni tored under a variety of growth conditions. It was found that 103 bp o f sequence upstream of the cbbF(I), transcription start was sufficient to confer low levels of regulated cbb(I) promoter expression; this ac tivity was dependent on the presence of an intact cbbR gene. Additiona lly, R. sphaeroides CbbR was shown to bind to the region between 9 and 100 bp 5' to the cbbF(I) transcription start. Inclusion of an additio nal upstream sequence, from 280 to 636 bp 5' to cbbF(I), resulted in a significant increase in regulated cbb(I) promoter expression under al l growth conditions tested. A 50-bp region responsible for the majorit y of this increase occurs between 280 and 330 bp 5' to cbbF(I). The ad ditional 306 bp of upstream sequence from 330 to 636 bp also appears t o play a positive regulatory role. A 4-bp deletion 281 to 284 bp 5' to cbbF(I) significantly reduced cbb(I) expression while the proper regu latory pattern aas retained. These studies provide evidence for the pr esence of two functionally distinct regions of the cbb(I) promoter, wi th the distal domain providing significant regulated promoter activity that adheres to the normal pattern of expression.