Identification of the binding site of the σ 54 hetero-oligomeric FleQ/FleT activator in the flagellar promoters of Rhodobacter sphaeroides

Abstract

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. INTRODUCTIONPromoter recognition in bacteria is mediated by the RNA polymerase core (E) associated with a sigma factor. There are two families of sigma factors, the s 70 family and that of s 54 . These two families differ not only in their sequence but also in the mechanism that brings about transcriptional initiation. Whereas Es 70 is capable of forming open complex by itself, Es 54 requires an activator protein that, through ATP hydrolysis, allows open complex formation (for reviews, see Buck et al., 2000;Wigneshweraraj et al., 2005). Activator proteins of Es 54 usually bind approximately 100 bp upstream of the promoter sequence, and a DNA loop favours the interaction of the activator with the RNA polymerase holoenzyme bound to the promoter (Reitzer & Magasanik, 1986;Ninfa et al., 1987;Buck et al., 1986;Hoover et al., 1990;Su et al., 1990;Wedel et al., 1990). The mechanism that allows open complex formation is still unknown, but the current model suggests that the activator protein remodels the nucleoprotein complex formed by Es 54 and DNA in order to initiate transcription Cannon et al., 2000;Burrows et al., 2004;Rappas et al., 2007). The DNA region to which the activator proteins bind is known as the upstream activation sequence or UAS (Buck et al., 1986;Morett et al., 1988). It has been shown for the glnA promoter that this region can be moved more than 1000 bp without losing its ability to stimulate transcription (Ninfa et al., 1987). Based on this property, the UASs are also known as enhancers, and the activator proteins as enhancer binding proteins, or EBPs.The s 54 -dependent promoters show a highly conserved consensus sequence, and their main characteristics are the dinucleotides GG ...

The branched CcsA/CckA‐ChpT‐CtrA phosphorelay of Sphingomonas melonis controls motility and biofilm formation

“…fleQ, a class I gene, encodes a σ 54 -dependent regulator that activates the expression of class II genes; fleT is itself a class II gene and encodes a polypeptide which consists of a AAA +ATPase domain but no DNA-binding domain. FleQ and FleT activate the expression of class III genes as a heterodimer (Poggio et al, 2005;Pena-Sanchez et al, 2009). In S. melonis, despite the fact that fleT is encoded upstream of fliE and is a class II gene, similar to fleT of R. sphaeroides, it contains a DNA-binding domain and might thus directly regulate class III genes.…”

A Small RNA, UdsC, Interacts with the RpoHII mRNA and Affects the Motility and Stress Resistance of Rhodobacter sphaeroides

“…RpoHII mRNA was suggested as a putative target and ranked at position seven in the IntaRNA search based on the IntaRNA energy value, whereas TorF was ranked at position nineteen [ 25 ]. RpoHII is known as main regulator in the oxidative stress response of R. sphaeroides , and TorF (FleT, RSP_0051) is known as regulator of class III flagellar genes together with RpoN2 and FleQ [ 26 , 27 ]. TorF was among the 50 most increased mRNAs in the UdsC-overexpression strain after 15 min of IPTG treatment ( Table 1 ).…”

Interactions between paralogous bacterial enhancer‐binding proteins enable metal‐dependent regulation of alternative nitrogenases in Azotobacter vinelandii

“…It is possible that VnfZ might perform a similar role in promoting the formation of heteromeric VnfA1‐VnfA3 complexes, in line with the observation that VnfZ potentially interacts with both VnfA1 and VnfA3 as revealed by our bacterial two‐hybrid analysis. Further rare examples of hetero‐oligomeric interactions between bEBPs include FleQ and FleT, which act in concert to activate σ 54 ‐dependent promoters controlling flagella gene expression in Rhodobacter sphaeroides (Peña‐Sánchez et al, 2009; Poggio et al, 2005) and the co‐dependent RedR1 and RedR2 bEBPs that activate expression of the resorcinol degradation pathway in Azoarcus anaerobius (Pacheco‐Sánchez et al, 2017).…”