Regulatory cascade of NdpR and NdpR2 in controlling nicotine degradation operons in Sphingomonas melonis TY

In Sphingomonas melonis TY, the TetR-family NdpR and AraC-family NdpR2 coregulate nicotine catabolism. Despite overlapping target promoters, their hierarchical relationship remained unclear. Here, we elucidate a multi-layered cascade regulatory network where NdpR directly binds to the ndpR2 promoter and represses its transcription. Structural and mutational analyses elucidate the structure-function relationship of NdpR, featuring a wide HTH span that supports a strong steric barrier mechanism on core promoters. The intermediate 2,5-dihydroxypyridine (2,5-DHP) acts as an effector, preventing NdpR from binding the ndpR2 promoter. Furthermore, molecular simulations demonstrate a direct NdpR-NdpR2 physical interaction. We propose a double-lock regulatory mechanism operating synergistically at the transcriptional and protein levels. This precise dynamic regulatory balance enables cells to sensitively perceive environmental signals via basal metabolism and rapidly activate target gene expression upon signal confirmation, thereby achieving a balance between energy economy and efficient substrate degradation. Based on this cascade, we constructed a multi-factor system in Pseudomonas sp. JY-Q exhibiting bidirectional, reversible responses to nicotine, 2,5-DHP, and IPTG (isopropyl β-D-1-thiogalactopyranoside). This study uncovers microbial adaptive strategies and provides programmable, tunable regulatory tools for synthetic biology.IMPORTANCEDuring microbial degradation of complex pollutants like nicotine, substrate uptake, transport, and catabolism are typically coregulated by multiple factors. Understanding how this complex regulatory network prevents metabolic energy waste, rapidly responds to substance changes, and thereby achieves temporal control over related gene expression is crucial for the development of metabolic control elements. This study reveals the cascade regulatory logic of two regulatory proteins within the same metabolic pathway and the regulatory balance during metabolism. Through structural and functional analyses of NdpR, we elucidated the molecular mechanisms underlying its target sequence recognition, binding of the effector 2,5-DHP, and interaction with NdpR2, thereby clarifying the dynamic regulatory process by which this system responds to environmental changes. Finally, we applied this system in a heterologous Pseudomonas host to develop a programmable multi-factor gene circuit. This study deepens our mechanistic understanding of microbial adaptive strategies and provides a dynamically tunable regulatory tool for synthetic biology and metabolic engineering.