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.
State Key Laboratory of Microbial Metabolism, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
Lijuan
L
Wang
Hongzhi
H
Tang
Hao
H
Yu
Yuxiang
Y
Yao
Ping
P
Xu
D001711:Biotransformation; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial; D053858:Metabolic Networks and Pathways; D005810:Multigene Family; D009538:Nicotine / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D055503:Protein Multimerization; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*
D004269:DNA, Bacterial; D012097:Repressor Proteins; D009538:Nicotine