Regulation Mechanism of Nicotine Catabolism in Sphingomonas melonis TY by a Dual Role Transcriptional Regulator NdpR

Abstract

A gene cluster <i>ndp</i>, responsible for nicotine degradation via a variant of the pyridine and pyrrolidine pathways, was previously identified in Sphingomonas melonis TY, but the regulation mechanism remains unknown. The gene <i>ndpR</i> within the cluster was predicted to encode a TetR family transcriptional regulator. Deletion of <i>ndpR</i> resulted in a notably shorter lag phase, higher maximum turbidity, and faster substrate degradation when cultivated in the presence of nicotine. Real-time quantitative PCR and promoter activity analysis in wild-type TY and TYΔ<i>ndpR</i> strains revealed that genes in the <i>ndp</i> cluster were negatively regulated by NdpR. However, complementation of <i>ndpR</i> to TYΔ<i>ndpR</i> did not restore transcription repression, but, instead, the complemented strain showed better growth than TYΔ<i>ndpR</i>. Promoter activity analysis indicates that NdpR also functions as an activator in the transcription regulation of <i>ndpHFEGD</i>. Further analysis through electrophoretic mobility shift assay and DNase I footprinting assay revealed that NdpR binds five DNA sequences within <i>ndp</i> and that NdpR has no autoregulation. These binding motifs overlap with the -35 or -10 box or are located distal upstream of the corresponding transcriptional start site. Multiple sequence alignment of these five NdpR-binding DNA sequences found a conserved motif, with two of the binding sequences being partially palindromic. 2,5-Dihydroxypyridine acted as a ligand of NdpR, preventing NdpR from binding to the promoter region of <i>ndpA_SA_L</i>, <i>ndpTB</i>, and <i>ndpHFEGD</i>. This study revealed that NdpR binds to three promoters in the <i>ndp</i> cluster and is a dual-role transcriptional regulator in nicotine metabolism. <b>IMPORTANCE</b> Gene regulation is critical for microorganisms in the environment in which they may encounter various kinds of organic pollutants. Our study revealed that transcription of <i>ndpA_SA_L</i>, <i>ndpTB</i>, and <i>ndpHFEGD</i> is negatively regulated by NdpR, and NdpR also exhibits a positive regulatory effect on P<i>_ndpHFEGD</i>. Furthermore, 2,5-dihydroxypyridine was identified as the effector molecular for NdpR and can both prevent the binding of free NdpR to the promoter and release NdpR from the promoters, which is different from previously reported NicR2. Additionally, NdpR was found to have both negative and positive transcription regulatory effects on the same target, P<i>_ndpHFEGD</i>, while only one binding site was identified, which is notably different from the previously reported TetR family regulators. Moreover, NdpR was revealed to be a global transcriptional regulator. This study provides new insight into the complex gene expression regulation of the TetR family.