Identification of the binding site of the σ54 hetero-oligomeric FleQ/FleT activator in the flagellar promoters of Rhodobacter sphaeroides

摘要

Expression of the flagellar genes in Rhodobacter sphaeroides is dependent on one of the four sigma-54 factors present in this bacterium and on the enhancer binding proteins (EBPs) FleQ and FleT. These proteins, in contrast to other well-characterized EBPs, carry out activation as a hetero-oligomeric complex. To further characterize the molecular properties of this complex we mapped the binding sites or upstream activation sequences (UASs) of six different flagellar promoters. In most cases the UASs were identified at approximately 100?bp upstream from the promoter. However, the activity of the divergent promoters flhAp-flgAp, which are separated by only 53?bp, is mainly dependent on a UAS located approximately 200?bp downstream from each promoter. Interestingly, a significant amount of activation mediated by the upstream or contralateral UAS was also detected, suggesting that the architecture of this region is important for the correct regulation of these promoters. Sequence analysis of the regions carrying the potential FleQ/FleT binding sites revealed a conserved motif. In vivo footprinting experiments with the motAp promoter allowed us to identify a protected region that overlaps with this motif. These results allow us to propose a consensus sequence that represents the binding site of the FleQ/FleT activating complex.