Two Functionally Distinct Regions Upstream of the cbb(I) Operon of Rhodobacter sphaeroides Regulate Gene Expression

A number of cbbF(I)::lacZ translational fusion plasmids containing various lengths of sequence 5′ to the form I (cbb(I)) Calvin-Benson-Bassham cycle operon (cbbF(I)cbbP(I)cbbA(I)cbbL(I)cbbS(I)) of Rhodobacter sphaeroides were constructed. Expression of β-galactosidase was monitored under a variety of growth conditions. It was found that 103 bp of sequence upstream of the cbbF(I) transcription start was sufficient to confer low levels of regulated cbb(I) promoter expression; this activity was dependent on the presence of an intact cbbR gene. Additionally, 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 additional upstream sequence, from 280 to 636 bp 5′ to cbbF(I), resulted in a significant increase in regulated cbb(I) promoter expression under all growth conditions tested. A 50-bp region responsible for the majority of this increase occurs between 280 and 330 bp 5′ to cbbF(I). The additional 306 bp of upstream sequence from 330 to 636 bp also appears to 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 regulatory pattern was retained. These studies provide evidence for the presence of two functionally distinct regions of the cbb(I) promoter, with the distal domain providing significant regulated promoter activity that adheres to the normal pattern of expression.