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

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Abstract
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.