Genome-wide analysis of the nicotine degradation mechanism in Agrobacterium tumefaciens strain At_42

To address the issue of high nicotine content in upper tobacco leaves and the remediation of environmental nicotine pollution, this study employed genomic approaches to investigate the nicotine degradation mechanism of Agrobacterium tumefaciens At_42, a highly efficient nicotine-degrading strain previously isolated in our laboratory.

Classification of At_42 and its nicotine metabolic pathway at the genetic level were studied using Average Nucleotide Identity (ANI) analysis, synteny alignment, pan-genome analysis, molecular docking, and RT-qPCR. Concurrently, metabolites resulting from nicotine degradation by At_42 were analyzed using MS/MS technology.

The results demonstrated that ANI analysis confirmed At_42 as A. tumefaciens. Its complete genome size is 5.86 Mb, with a GC content of 59.24%, encoding 5770 predicted genes. Pan-genome analysis revealed that At_42 possesses 13296 gene families, of which 1301 are core gene families. Synteny analysis demonstrated a high degree of homology between the nicotine degradation gene cluster of At_42 and that of A. tumefacien S33, with 10 homologous genes identified. Molecular docking results indicated that the NdhA and NdhB proteins in At_42 can bind to nicotine, while the Hsh protein can bind to 6-Hydroxy-3-succinoylpyridine (Hsp). RT-qPCR detection revealed significant upregulation of key nicotine metabolic genes (NdhA, NdhB, Pno, Nfo, Hsh, Iso, Ami, Hpo, Hno) in At_42 in response to nicotine induction. MS/MS analysis identified metabolites including 6-Hydroxy-N-methylmyosmine, 2, 5-Dihydroxypyridine, 6-Hydroxy-pseudo-oxynicotine, and Maleate. Among these, 6-Hydroxy-N-methylmyosmine and 6-Hydroxy-pseudo-oxynicotine are generated via the pyridine pathway, while 6-Hydroxy-pseudo-oxynicotine and Maleate are derived from the pyrrolidine pathway.

Based on the metabolite detection and genomic bioinformatics analysis results, At_42 is predicted to degrade nicotine through a pyrrolidine-pyridine pathway. This study evaluated the application potential of strain At_42 for the remediation of environmental nicotine pollution, demonstrating considerable value in the fields of tobacco agriculture and environmental management.