An unusual repressor controls the expression of a crucial nicotine-degrading gene cluster in Pseudomonas putida S16.

Transcriptional factors that contain helix-turn-helix (HTH) DNA-binding domains are widespread in bacteria for regulating gene expression on demand, and function as homodimers that bind a palindromic DNA segment. Here, we show that an HTH-containing transcriptional regulator, NicR2, in Pseudomonas putida S16 plays a critical role in controlling the expression of a crucial gene cluster (nic2) in nicotine degradation, and NicR2 binds DNA in a manner different from most other DNA-binding proteins that use HTHs for recognition. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting indicate that NicR2 directly interacts with a 28 bp inverted repeat (IR) in the nic2 promoter region. Using EMSA with synthetic DNA fragments, we found that both NicR2 dimer and tetramer can bind to the half-site of the IR. This is confirmed independently by biolayer interferometry and cross-linking experiments. Our results indicate that two NicR2 dimers bind to the IR cooperatively through protein-protein interactions, with each dimer binding the half-site of the IR. Thus, NicR2 appears to be an unusual regulator, which uses HTH for recognition and displays the binding characteristics of some regulators that use β-sheets. The transcriptional regulation of nicotine degradation in Pseudomonas highlights a new level of complexity in prokaryotic transcriptional regulation.