Abstract
Sphingomonas melonis TY utilizes nicotine as a sole source of carbon, nitrogen, and energy through a variant of the pyridine and pyrrolidine pathways (VPP). A 31-kb novel nicotine-degrading gene cluster, ndp, in strain TY exhibited a different genetic organization with the vpp cluster in strains Ochrobactrum rhizosphaerae SJY1 and Agrobacterium tumefaciens S33. Genes in vpp were separated by a 20-kb interval sequence, while genes in ndp were localized together. Half of the homolog genes were in different locus in ndp and vpp. Moreover, there was a gene encoding putative transporter of nicotine or other critical metabolite in ndp. Among the putative nicotine-degrading related genes, the nicotine hydroxylase, 6-hydroxy-L-nicotine oxidase, 6-hydroxypseudooxynicotine oxidase, and 6-hydroxy-3-succinyl-pyridine monooxygenase responsible for catalyzing the transformation of nicotine to 2, 5-dihydropyridine in the initial four steps of the VPP were characterized. Hydroxylation at C6 of the pyridine ring and dehydrogenation at the C2–C3 bond of the pyrrolidine ring were the key common reactions in the VPP, pyrrolidine and pyridine pathways. Besides, VPP and pyrrolidine pathway shared the same latter part of metabolic pathway. After analysis of metabolic genes in the pyridine, pyrrolidine, and VPP pathways, we found that both the evolutionary features and metabolic mechanisms of the VPP were more similar to the pyrrolidine pathway. The linked ndpHFEG genes shared by the VPP and pyrrolidine pathways indicated that these two pathways might share the same origin, but variants were observed in some bacteria. And we speculated that the pyridine pathway was distributed in Gram-positive bacteria and the VPP and pyrrolidine pathways were distributed in Gram-negative bacteria by using comprehensive homologs searching and phylogenetic tree construction.
Highlights
Nicotine is the most abundant alkaloid in tobacco plants, and it maintains a high content (15.680–32.536 mg/g dry weight according to the particle size of the solid powdery waste) in tobacco waste, which accumulates in large amounts (Civilini et al, 1997; Novotny and Zhao, 1999; Cosic et al, 2012)
After performing a BLAST analysis against the genome of strain TY using previously known nicotine metabolism genes, we found that the genes including ndhLSM and 6hlno in A. nicotinovorans pAO1, hspB, hpo, nfo, ami, and iso in Pseudomonas putida S16 all had hits in one 31-kb scaffold of the genome of strain TY, demonstrating a compact arrangement in this scaffold (Figure 1)
The underlying molecular mechanism has been the most well-studied in strain SJY1, excluding the enzyme that catalyzes the reaction from 6HPON to HSP (Yu et al, 2014, 2015); this enzyme was recently found in strain S33 (Li et al, 2016)
Summary
Nicotine is the most abundant alkaloid in tobacco plants, and it maintains a high content (15.680–32.536 mg/g dry weight according to the particle size of the solid powdery waste) in tobacco waste, which accumulates in large amounts (Civilini et al, 1997; Novotny and Zhao, 1999; Cosic et al, 2012). It was anticipated that more new pathways would be found Among these published pathways, the most well-established were the PD in A. nicotinovorans pAO1 (Dang et al, 1968; Brühmüller et al, 1972; Grether-Beck et al, 1994; Schenk et al, 1998; Baitsch et al, 2001; Chiribau et al, 2004, 2006; Sachelaru et al, 2005, 2006; Mihasan et al, 2007), the PRL in Pseudomonas putida S16 (Tang et al, 2008, 2009, 2011, 2012, 2013; Jiang et al, 2015), and the VPP in O. rhizosphaerae SJY1 (Yu et al, 2014, 2015). The nicotine-degrading gene clusters, for example, the nic-genes in A. nicotinovorans pAO1, nic and nic in strain S16, and the vpp cluster in strain SJY1, have been comprehensively studied
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