Transcription of the Rhodobacter sphaeroides cycA P1 Promoter by Alternate RNA Polymerase Holoenzymes

In order to survive, all cells must continue to generate energy in response to a wide variety of environmental changes and signals. While viability in response to such challenges requires the coordinated induction and repression of numerous genes, relatively little is known about how the expression of energy-generating functions is altered by these stimuli. To analyze the control of proteins that participate in biological energy generation, we are studying transcriptional regulation of the cytochrome c2 gene (cycA) from the facultative phototroph Rhodobacter sphaeroides. We know that cycA contains several promoters (16, 21, 22). The downstream cycA promoter (cycA P1) provides sufficient cytochrome c2 to support growth, but its output is not altered by conditions that alter levels or demand function of this electron carrier (16, 21, 22). In contrast, activity of the upstream promoter (cycA P2) is increased ∼10-fold under photosynthetic conditions where cytochrome c2 function is required (16, 21, 22). Activity of a third promoter (cycA P3) increased dramatically in cells containing the trans-acting chr-4 mutation (21, 22). A key unresolved issue is the nature of the RNA polymerase holoenzyme that recognizes each of the three cycA promoters (16, 21, 22). A previous study noted the similarity of DNA sequences upstream of cycA P1 and P2 to promoters recognized by the major eubacterial sigma factor, ς70 (16). Thus, one possibility was that cycA P1 and P2 were each recognized by the previously described R. sphaeroides RNA polymerase holoenzyme (EςD) that transcribes the Escherichia coli ς70-dependent lacUV5 and ColE1 RNA promoters in vitro (10). Alternatively, cycA promoter recognition by minor RNA polymerase holoenzymes (5, 13, 14) could link cytochrome c2 synthesis to conditions that increase or decrease function of alternate sigma factors. This appears to be the case for cycA P3, since the chr-4 mutation appears to inactivate a negative regulator of an R. sphaeroides member of the ςE family of alternative eubacterial sigma factors (18a, 21, 22). These experiments sought to analyze function of cycA P1. We focused on cycA P1 because sequences within ∼60 bp of the transcription start site (+1) are sufficient for function under both respiratory and photosynthetic conditions (16, 21, 22). In vitro transcription assays indicated that R. sphaeroides EςD (10) did not recognize cycA P1. By analyzing a series of mutant cycA P1 promoters fused to a promoter-less E. coli β-galactosidase (lacZ) gene in vivo, we found that sequences within a presumed RNA polymerase binding site were sufficient for activity. Other promoter mutations identified elements related to the E. coli heat shock (Eς32) consensus sequence that were necessary for cycA P1 function. Reconstituted RNA polymerases were used to demonstrate cycA P1 transcription by a holoenzyme containing either a 37-kDa protein (ς37) related to E. coli ς32 or a 38-kDa protein (ς38) that also recognizes heat shock promoters from enteric bacteria (10, 11). We suggest that one reason why cycA P1 might be transcribed by alternate RNA polymerase is to ensure that cytochrome c2 levels can change when its function in energy generation is required to provide ATP for chaperone activity. Consistent with this suggestion, cycA P1 function is elevated after an increase in temperature when eubacterial chaperones are commonly needed for cells to mount a heat shock response (11).