A majority of <i>Rhodobacter sphaeroides</i> promoters lack a crucial RNA polymerase recognition feature, enabling coordinated transcription activation.

Abstract

Using an in vitro transcription system with purified RNA polymerase (RNAP) to investigate rRNA synthesis in the photoheterotrophic α-proteobacterium <i>Rhodobacter sphaeroides</i>, we identified a surprising feature of promoters recognized by the major holoenzyme. Transcription from <i>R. sphaeroides</i> 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 <i>R. sphaeroides</i> RNAP can utilize -7T when present. rRNA promoters were activated by purified <i>R. sphaeroides</i> 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 <i>R. sphaeroides</i> 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 <i>R. sphaeroides</i> 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 <i>R. sphaeroides</i> 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.