Abstract
Utilization of L-nicotine as growth substrate by Arthrobacter nicotinovorans pAO1 starts with hydroxylation of the pyridine ring at C6. Next, the pyrrolidine ring is oxidized by 6-hydroxy-L-nicotine oxidase, which acts strictly stereo-specific on the L-enantiomer. Surprisingly, L-nicotine also induces the synthesis of a 6-hydroxy-d-nicotine-specific oxidase in the bacteria. Genes of nicotine-degrading enzymes are located on the catabolic plasmid pAO1. The pAO1 sequence revealed that the 6-hydroxy-D-nicotine oxidase gene is flanked by two open reading frames with a similarity to amino acid permeases and a divergently transcribed open reading frame with a similarity to proteins of the tetracycline repressor TetR family. Reverse transcription PCR and primer extension analysis of RNA transcripts isolated from A. nicotinovorans pAO1 indicated that the 6-hydroxy-D-nicotine oxidase gene represents a transcriptional unit. DNA electromobility shift assays established that the purified TetR-similar protein represents the 6-hydroxy-D-nicotine oxidase gene repressor HdnoR and binds to the 6-hydroxy-D-nicotine oxidase gene operator with a Kd of 21 nM. The enantiomers 6-hydroxy-D- and 6-hydroxy-L-nicotine acted in vitro as inducers. In vivo analysis of 6-hydroxy-D-nicotine oxidase gene transcripts from bacteria grown with L- and D-nicotine confirmed this conclusion. The poor discrimination by HdnoR between the 6-hydroxy-L- and 6-hydroxy-D-nicotine enantiomers explains the presence of the 6-hydroxy-D-nicotine-specific enzyme in bacteria grown on L-nicotine.
Highlights
The Gram-positive soil bacterium Arthrobacter nicotinovorans pAO1, formerly known as Arthrobacter oxidans [1], has the metabolic ability to grow on the tobacco alkaloid nicotine [2]
It has been shown before that a protein present in A. nicotinovorans pAO1 extracts binds to an operator site consisting of two inverted repeats, namely IR1, covering the 6hdno promoter region, and IR2, situated upstream from the 6hdno promoter [10]
The experimental data presented in this paper demonstrate that the protein encoded by ORF114 of pAO1 represents the transcriptional repressor of 6hdno
Summary
Genes of nicotine-degrading enzymes are situated on the catabolic plasmid pAO1 [9]. It has been shown before that a protein present in A. nicotinovorans pAO1 extracts binds to an operator site consisting of two inverted repeats, namely IR1, covering the 6hdno promoter region, and IR2, situated upstream from the 6hdno promoter [10]. The position on pAO1 of genes of enzymes involved in nicotine catabolism by A. nicotinovorans has been determined [11]. In this work we performed a transcriptional analysis of the 6hdno gene cluster and present evidence that 6hdno represents a transcriptional unit. We cloned and expressed the DNA carrying ORF114, purified the TetR-similar transcriptional regulator, and show that it represents the 6hdno repressor. Evidence is presented that 6-hydroxy-D-nicotine and 6-hydroxy-Lnicotine act as inducers of 6hdno expression. Induction of 6hdno expression by both the L- and the D-nicotine enantiomer can be explained by the poor discrimination by HdnoR between 6-hydroxy-L- and 6-hydroxy-D-nicotine
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
