College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, China.
MOE Laboratory of Biosystem Homeostasis and Protection, College of Life Sciences, Zhejiang University, Hangzhou, China.
Appl Environ Microbiol. 2023 May 31;89(5):e0032423. doi: 10.1128/aem.00324-23. Epub 2023 Apr 18.
A gene cluster , 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 within the cluster was predicted to encode a TetR family transcriptional regulator. Deletion of 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 strains revealed that genes in the cluster were negatively regulated by NdpR. However, complementation of to TYΔ did not restore transcription repression, but, instead, the complemented strain showed better growth than TYΔ. Promoter activity analysis indicates that NdpR also functions as an activator in the transcription regulation of . Further analysis through electrophoretic mobility shift assay and DNase I footprinting assay revealed that NdpR binds five DNA sequences within 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 , , and . This study revealed that NdpR binds to three promoters in the cluster and is a dual-role transcriptional regulator in nicotine metabolism. 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 , , and is negatively regulated by NdpR, and NdpR also exhibits a positive regulatory effect on P. 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, 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.
一个基因簇，负责通过吡啶和吡咯烷途径的变体降解尼古丁，先前在 Sphingomonas melonis TY 中被鉴定，但调控机制仍不清楚。簇内的基因预测编码一个 TetR 家族转录调节因子。当在尼古丁存在的情况下培养时，缺失导致明显更短的潜伏期、更高的最大浊度和更快的底物降解。在野生型 TY 和 TYΔ菌株中进行的实时定量 PCR 和启动子活性分析表明，簇内的基因受 NdpR 负调控。然而，将补充到 TYΔ中并没有恢复转录抑制，而是补充后的菌株表现出比 TYΔ更好的生长。启动子活性分析表明，NdpR 也作为转录调节的激活剂在发挥作用。通过电泳迁移率变动分析和 DNase I 足迹分析的进一步分析表明，NdpR 结合到 内的五个 DNA 序列，并且 NdpR 没有自身调控。这些结合基序与 -35 或 -10 框重叠或位于相应转录起始位点的上游。对这五个 NdpR 结合 DNA 序列的多重序列比对发现了一个保守的基序，其中两个结合序列部分是回文的。2,5-二羟基吡啶作为 NdpR 的配体，阻止 NdpR 结合到 、 、和 的启动子区域。这项研究表明，NdpR 结合到簇内的三个启动子，并且是尼古丁代谢中的双角色转录调节因子。基因调控对于它们可能遇到各种有机污染物的环境中的微生物至关重要。我们的研究表明， 、 、和 的转录受 NdpR 负调控，NdpR 对 P 也表现出正调控效应。此外，鉴定出 2,5-二羟基吡啶是 NdpR 的效应分子，既能阻止游离 NdpR 与启动子结合，又能从启动子上释放 NdpR，这与先前报道的 NicR2 不同。此外，发现 NdpR 对同一靶标 P 既有负转录调控效应又有正转录调控效应，而只鉴定出一个结合位点，这与先前报道的 TetR 家族调节因子明显不同。此外，NdpR 被揭示为一个全局转录调节因子。这项研究为 TetR 家族的复杂基因表达调控提供了新的见解。
