A majority of Rhodobacter sphaeroides promoters lack a crucial RNA polymerase recognition feature, enabling coordinated transcription activation

Significance Bacterial promoters are often predicted by similarity to the Escherichia coli −10 and −35 consensus elements. Although these elements are highly conserved in diverse bacterial phyla, only 30 to 43% of promoters we analyzed from Rhodobacter sphaeroides and two other α-proteobacteria contained −7T, a base in the −10 element present in 90 to 99% of promoters from non–α-proteobacteria. Expression from 15 of 16 R. sphaeroides promoters tested in vitro that lacked −7T was very weak, but we identified an essential transcription factor, CarD, that activated all 15 of these promoters. We suggest that promoters lacking a critical base in a consensus element can shape the transcriptome by coordinating expression of large numbers of genes by a single transcription factor.
Using an in vitro transcription system with purified RNA polymerase (RNAP) to investigate rRNA synthesis in the photoheterotrophic α-proteobacterium Rhodobacter sphaeroides, we identified a surprising feature of promoters recognized by the major holoenzyme. Transcription from R. sphaeroides rRNA promoters was unexpectedly weak, correlating with absence of −7T, the very highly conserved thymine found at the last position in −10 elements of promoters in most bacterial species. Thymine substitutions for adenine at position −7 in the three rRNA promoters strongly increased intrinsic promoter activity, indicating that R. sphaeroides RNAP can utilize −7T when present. rRNA promoters were activated by purified R. sphaeroides CarD, a transcription factor found in many bacterial species but not in β- and γ-proteobacteria. Overall, CarD increased the activity of 15 of 16 native R. sphaeroides promoters tested in vitro that lacked −7T, whereas it had no effect on three of the four native promoters that contained −7T. Genome-wide bioinformatic analysis of promoters from R. sphaeroides and two other α-proteobacterial species indicated that 30 to 43% contained −7T, whereas 90 to 99% of promoters from non–α-proteobacteria contained −7T. Thus, promoters lacking −7T appear to be widespread in α-proteobacteria and may have evolved away from consensus to enable their coordinated regulation by transcription factors like CarD. We observed a strong reduction in R. sphaeroides CarD levels when cells enter stationary phase, suggesting that reduced activation by CarD may contribute to inhibition of rRNA transcription when cells enter stationary phase, the stage of growth when bacterial ribosome synthesis declines.