Further Insights into the Architecture of the PN Promoter That Controls the Expression of the bzd Genes in Azoarcus.

The anaerobic degradation of benzoate in bacteria involves the benzoyl-CoA central pathway. Azoarcus/Aromatoleum strains are a major group of anaerobic benzoate degraders, and the transcriptional regulation of the bzd genes was extensively studied in Azoarcus sp. CIB. In this work, we show that the bzdR regulatory gene and the PN promoter can also be identified upstream of the catabolic bzd operon in all benzoate-degrader Azoarcus/Aromatoleum strains whose genome sequences are currently available. All the PN promoters from Azoarcus/Aromatoleum strains described here show a conserved architecture including three operator regions (ORs), i.e., OR1 to OR3, for binding to the BzdR transcriptional repressor. Here, we demonstrate that, whereas OR1 is sufficient for the BzdR-mediated repression of the PN promoter, the presence of OR2 and OR3 is required for de-repression promoted by the benzoyl-CoA inducer molecule. Our results reveal that BzdR binds to the PN promoter in the form of four dimers, two of them binding to OR1. The BzdR/PN complex formed induces a DNA loop that wraps around the BzdR dimers and generates a superstructure that was observed by atomic force microscopy. This work provides further insights into the existence of a conserved BzdR-dependent mechanism to control the expression of the bzd genes in Azoarcus strains.

Two levels of cooperativeness in the binding of TodT to the tod operon promoter.

The TodS/TodT two-component system controls the expression of tod genes for toluene degradation in Pseudomonas putida. TodT binds to two pseudopalindromes at -106 (Box-1) and -85 (Box-2), as well as to a half-palindrome (Box-3), with respect to the main transcription initiation site in the PtodX promoter. TodT recognizes each half-palindrome in Boxes-1 and -2, but affinities for these sequences are lower than those for the pseudopalindromes, pointing towards positive cooperativeness in intrabox recognition. TodT's affinity for DNA fragments containing two vicinal boxes (either Boxes-1 and -2 or Boxes-2 and -3) is higher than its affinity for individual boxes, suggesting interbox cooperativeness. Similar patterns of cooperativeness were observed for the recombinant TodT DNA-binding domain [C-terminal TodT fragment (aa 154-206) (C-TodT)], suggesting important cooperativeness determinants in this domain. Occupation of PtodX by TodT is initiated at Box-1, and optimization of its palindromic order increases affinity in vitro; however, this does not result in enhanced in vivo gene expression. Mutations at either half of the Box-1 palindrome have no significant effects on transcriptional activity, whereas mutations in the entire Box-1 cause a 12-fold reduction. Using atomic force microscopy, we show that TodT induces a DNA hairpin bend at PtodX between Boxes-2 and -3, as supported by footprint studies showing a hyperreactive nucleotide at G -68. The N-terminal part of TodT seems to play a central role in hairpin formation, since C-TodT neither induces a bend nor causes G -68 hyperreactivity in footprints. This hairpin seems important for transcriptional activation, since C-TodT binding to PtodX does not stimulate transcription.

The transcriptional repressor TtgV recognizes a complex operator as a tetramer and induces convex DNA bending.

The TtgV repressor belongs to the large but infrequently investigated IclR family of transcriptional regulators. Although members of this family usually exhibit high effector specificity, TtgV possesses multidrug binding properties. The TtgV protein regulates the expression of the ttgGHI operon encoding the main solvent extrusion pump of the extremophile Pseudomonas putida DOT-T1E strain. Here we used a multidisciplinary approach to study the functional oligomeric state of TtgV during repression and derepression events, as well as the molecular basis of TtgV-DNA operator interactions. Analytical ultracentrifugation studies (AUC) show that TtgV is a tetramer in solution and that this oligomeric state does not change in the presence of effectors. We also show that the binding of effectors leads to the dissociation of TtgV as a tetramer from the DNA-TtgV complex. Previous dimethyl sulfate and DNase I footprints revealed that TtgV protected a 42 bp region. Based on AUC, electrophorectic mobility shift assays and isothermal titration calorimetry analyses we show that TtgV recognition specificity is restricted within this operator to a 34-nucleotide stretch and that TtgV may interact with intercalated inverted repeats that share no significant DNA sequence similarities within this short 34-nucleotide segment. Binding stoichiometry is one TtgV tetramer per operator, and affinity for its target DNA is around 200 nM. Circular dichroism analysis reveals that TtgV binding causes DNA distortion and atomic force microscopy imaging of TtgV-DNA operator complexes shows that TtgV induces a 57 degrees convex bend in its operator DNA. We propose that the mechanism of TtgV repression is based on the steric occlusion of the RNA polymerase binding site reinforced by DNA-bending of the ttgV-ttgG promoter region.