Characterization of Nicotine Catabolism through a Novel Pyrrolidine

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Biotechnology and Biological Transformations

Characterization of Nicotine Catabolism through a Novel Pyrrolidine Pathway in Pseudomonas sp. S-1 Dandan Pan, Mengmeng Sun, Yawen Wang, Pei Lv, Xiangwei Wu, Qing X. Li, Haiqun Cao, and Rimao Hua J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01868 • Publication Date (Web): 22 Jun 2018 Downloaded from http://pubs.acs.org on June 25, 2018

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Journal of Agricultural and Food Chemistry

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Characterization of Nicotine Catabolism through a Novel Pyrrolidine

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Pathway in Pseudomonas sp. S-1

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Dandan Pan,† Mengmeng Sun,† Yawen Wang,† Pei Lv,† Xiangwei Wu,†, * Qing X. Li,‡

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Haiqun Cao,† Rimao Hua†

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†

College of Resources and Environment, Anhui Agricultural University, Key Laboratory of

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Agri-food Safety of Anhui Province, Hefei 230036, China

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‡

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1955 East–West Road, Honolulu, HI 96822, USA

Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa,

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*Corresponding author (Tel/Fax: +86-551-65786296; E-mail: [email protected];

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[email protected]) 1

ACS Paragon Plus Environment


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ABSTRACT Nicotine is a major toxic alkaloid in wastes generated from tobacco production and cigarette

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manufacturing. In the present work, a nicotine-degrading bacterial strain was isolated from

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tobacco powdery waste. The isolate was identified as Pseudomonas sp. S-1 based on

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morphology, physiology, and 16S rRNA gene sequence. Suitable conditions of isolate S-1 for

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nicotine degradation were pH 7.0 and 30 °C. Catabolic intermediates of nicotine were isolated

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with preparative-HPLC and characterized with LC-HRMS and NMR. The catabolic pathways of

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nicotine were involved in dehydrogenation, oxidation, hydrolysis, and hydroxylation.

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Interestingly, nicotine catabolism in strain S-1 undergoes a new pyrrolidine pathway that differs

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from the other three catabolic pathways in bacterial species. This work sheds light on catabolic

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diversity of nicotine and heteroaromatics.

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KEYWORDS: Nicotine; Biodegradation; Pseudomonas sp.; Pollution; Bioremediation

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INTRODUCTION

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Nicotine [3-(1-methylpyrrolidin-2-yl)pyridine, C10H14N2] (1) is a principal toxic alkaloid in

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tobacco waste.1 Tobacco (Nicotiana tabacum L.) has many applications in agriculture, cigarette

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industry, plant research and vaccine production.2 A large amount of nicotine wastes are produced

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in cigarette manufacturing processes.3 It is estimated that about one million tons of solid wastes

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are generated per year, with a mean nicotine content of 18 g/kg dry weight (dw).4 Nicotine

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comprises pyridine and pyrrole heterocyclic rings and is thus quite water soluble. Nicotine is

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however quite persistent and highly toxic.5 Tabaco waste is considered as “toxic and hazardous

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waste’’ when the content of nicotine is greater than 0.5 g/kg dw.5 Nicotine could pollute the

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environment, destroy the ecological balance, disrupt the physiology and behavior of hydrobiose,6

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and be associated with cerebrovascular disease,7 emphysema,5 lung cancer,2 and strokes8 in

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humans. Therefore, an effective technology is needed to remove nicotine from tobacco wastes

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and contaminated sites.

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Numerous methods including physical and chemical means have been investigated to

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degrade nicotine in tobacco wastes.2-3,5 Bacterial transformation can effectively detoxify tobacco

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wastes and reduce the amount of nicotine in the contaminated environments.9 When compared

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with other treatments, microbial treatment is eco-compatible, cost-effective, and reliable to

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remove hazardous compounds.2,10 An increasing interest has therefore been received in using

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microorganisms to degrade nicotine. 3



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Heteroaromatic compounds such as carbazole, dibenzofuran and dibenzothiophene are

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generally toxic and mutagenic.11 For example, the N-heterocyclic aromatic compound nicotine

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can be readily converted into cotinine in mammalians and the latter is genotoxic and cytotoxic.12

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Nicotine, containing pyridine and pyrrole moieties, represents a model compound to investigate

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bacterial catabolism of heteroaromatics in the environment.13 Numerous nicotine-degrading

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bacterial strains have been isolated and documented for their degradation characteristics. Those

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include Pseudomonas plecoglossicida TND35,5 Pseudomonas putida S16,14 Pseudomonas sp.

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HZN6,15 Aspergillus oryzae 112822,16 Agrobacterium tumefaciens S33,17 Arthrobacter

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nicotinovorans pAO1,18 Ochrobactrum sp. SJY1,19 and Shinella sp. HZN7.20 Four distinct

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catabolic pathways, namely demethylation, pyrrolidine and pyridine pathways as well as a

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variant of pyridine and pyrrolidine pathways (VPP pathway), have been elucidated for nicotine

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degradation. The demethylation pathway has been studied in A. oryzae 112822. The main

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intermediates of this pathway include nornicotine, myosmine, N-methylnicotinamide,

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2-hydroxy-N-methylnicotinamide, and 2,3-dihydroxypyridine.16 The pyrrolidine pathway and its

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catabolic mechanism have been documented in Pseudomonas sp. HZN6 and P. putida S16.14-15

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This pathway initiated by formation of a double bond in the pyrrolidine moiety to produce

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N-methylmyosmine (10).14 Metabolite 10 is hydroxylated to yield pseudooxynicotine (11) that is

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then oxidized to form 3-succinoyl pyridine (6).15 Metabolite 6 is hydroxylated to produce

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6-hydroxy-3-succinoyl pyridine (7) followed by further degradation to 2,5-dihydroxypyridine (8) 4


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and succinic acid (9).21 The nicA, hspA, hspB, pao, and sap genes encoding enzymes are

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responsible for catalyzing the conversion of nicotine to metabolite 8 via intermediate

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products.15,22-24 Four enzymes responsible for last steps of nicotine catabolism, namely

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N-formylmaleamate deformylase, 2,5-dihydroxypyridine dioxygenase, maleate isomerase, and

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maleamate amidase, are well studied in P. putida S16.25 The pyridine pathway is found in A.

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nicotinovorans. Nicotine is hydroxylated at the position 6 of pyridine moiety and then cleaves

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the pyrrolidine ring and finally yields nicotine blue and 9.18,26 This catabolic pathway consists of

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six intermediates including 6-hydroxynicotine, 6-hydroxy-N-methylmyosmine,

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6-hydroxy-pseudooxynicotine, 2,6-dihydroxy-pseudooxynicotine, 2,6-dihydroxypyridine and

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2,3,6-trihydroxypyridine.18,26-29 The genes encoding enzymes responsible for nicotine

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degradation via the pyridine pathway have been well characterized from the plasmid pAO1 of A.

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nicotinovorans.26-28 Such catabolism is also observed in Nocardioides sp. JS614.30 Finally, in the

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VPP pathway, nicotine is first converted to 6-hydroxypseudooxynicotine via 6-hydroxynicotine

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and 6-hydroxy-N-methylmyosmine through the pyridine pathway.19

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6-Hydroxy-pseudooxynicotine is then oxidized to yield metabolite 7 that is further degraded to 9

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via the pyrrolidine pathway.19-20 The VPP pathway is elucidated in Shinella sp. HZN7, and two

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mutant genes (nctB, tnp2) are cloned and expressed and shown to be essential for catabolism of

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the first intermediate 6-hydroxynicotine.20,31 Similar catabolic pathways are also found in A.

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tumefaciens, Pusillimonas sp. T2, and Ochrobactrum sp. SJY1.19,32-34 5



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Cotinine is a catabolic intermediate being accumulative during nicotine catabolism in some

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Pseudomonas spp., but no information concerning its further catabolism were found.4,7 Although

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four catabolic pathways of nicotine in various microbial species have been reported, some

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catabolic steps are still not clear and new pathway variations may still occur. In this work, we

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report degradation characteristics of a nicotine-degrading bacterial strain, Pseudomonas sp. S-1,

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and its novel variant of pyrrolidine pathway of nicotine catabolism.

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MATERIALS AND METHODS

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Chemicals and culture media

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(S)-Nicotine (CAS No.: 54-11-5, 99% purity) was provided from Chinese Tobacco Research

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Institute (Qingdao, China). Chromatographic grade solvent including methanol, acetonitrile, and

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formic acid were obtained from Merck & Co., Inc. (Darmstadt, Germany). The other reagents

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were of analytical grade. The mineral salts medium (MSM) was comprised of 0.2 g of K2HPO4,

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0.8 g of KH2PO4, 0.2 g of MgSO4, 0.1 g of CaSO4·H2O, 0.0033 g of NaMoO4 and 0.005 g of

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FeSO4·7H2O in 1000 mL deionized water at pH 7.5. Lysogeny broth (LB) medium was consisted

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of 10.0 g of tryptone, 5.0 g of yeast extract, and 10.0 g of NaCl in 1000 mL deionized water at

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pH 7.5. Solid media of MSM and LB contained 1.8% agar (w/v).

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Enrichment culture and isolation of nicotine-degrading bacterial strains

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Tobacco powdery wastes were collected from Qingzhou Cigarette & Tobacco Industrial Corporation (Weifang, China). Two grams of tobacco sample were weighed in a flask containing 6


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100 mL of sterilized MSM with the addition of 400 mg/L of nicotine. The flask was cultivated on

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an orbital shaker at 30 °C and 150 rpm for 7 d. The culture (1 mL) was then inoculated to 100

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mL of MSM containing 400 mg/L of nicotine followed by cultivation for 7 d. The same process

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was cycled six times for enrichment of nicotine-degrading bacteria. The liquid culture was

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spread on sterilized MSM agar plates that contained 400 mg/L of nicotine. After 7 d of

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cultivation at 30 °C, single pure colonies were picked and then tested their capability to degrade

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nicotine. The isolate S-1 exhibited a high capability of degrading nicotine.

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Identification and characterization of the isolate S-1

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Isolate S-1 was characterized by morphological, physiological and biochemical tests. Fresh

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cells of strain S-1 were fixed by glutaraldehyde, lyophilized, and then morphologically examined

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under a JEM-1200EX transmission electron microscope (TEM) (Jeol Ltd., Tokyo, Japan). The

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physiological and biochemical tests of S-1 were performed according to Bergey’s Manual.35

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Genomic DNA from isolate S-1 was obtained with the bacterial DNA kit (Tiangen Biotech,

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Beijing, China). The PCR amplification of 16S rRNA sequence was performed as described

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previously.10 Amplification products were purified on a SanPrep PCR purification column

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(Shanghai Sangon biotech, Shanghai, China) and sequenced by General Biosystems Co. Ltd

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(Hefei, China). A neighbor-joining tree was performed with MEGA 6.0. The nicotine-degreding

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strain, designated as S-1, was deposited in China General Microbiological Culture Collection

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Center (Beijing, China) (accession number 15003). 7



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Biodegradation of nicotine by isolate S-1 Isolate S-1 was pre-cultured in a flask that contained100 mL of LB media at 30 °C and 150

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rpm. The cells at the logarithmic phase (12 h) were harvested by centrifugation at 8000g for 5

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min, and then immediately washed thrice with 20 mL of 0.9% aseptic NaCl solution and

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resuspended with MSM. For nicotine degradation assays, the final OD600 of cells was adjusted to

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0.6 (5.3×108 cfu/mL) as determined with a UV-1800 spectrophotometer (Shimadzu Corp., Kyoto,

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Japan) at 600 nm.

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To achieve optimal conditions for nicotine degradation by isolate S-1, cultivation

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temperature and pH of medium were determined. A 100-mLflask that contained 20 mL of MSM

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with the addition of 400 mg/L of nicotine was inoculated with 0.6 mL of fresh cell suspensions

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(5.3×108 cfu/mL) of isolate S-1. The flask was then cultivated at 30 °C on a shaker set at 150

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rpm. Nicotine residues were determined after cultivations for 2, 4, 6, 8, 10 and 12 h. The whole

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culture was extracted with acetonitrile (20 mL). The mixture was sonicated for 1 min. The

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mixture solution was then collected in a 50-mL volumetric flask, followed by the volume

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adjustment with aqueous acetonitrile (1:1, v/v). The sample was filtered through a 0.45-µm nylon

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filter. Each treatment included three repetitions. The control was inoculated without cell

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suspensions of isolate S-1 under the same conditions as the treatment. Nicotine was analyzed on

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a 1260 series high performance liquid chromatograph (HPLC) (Agilent Technologies, Santa

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Clara, CA). The chromatographic separation was obtained on a 250 mm × 4.6 mm i.d., 5 µm, 8


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SB-C18 column (Agilent Technologies, Santa Clara, CA) at 30 °C. The column flow rate was 1.0

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mL/min with a mixture of 0.02 mol/L potassium phosphate buffers and methanol (90:10, v/v),

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pH 3.0, as the elution solution. The determination wavelength was 254 nm. Average recoveries

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of nicotine ranged from 88.4% to 97.5% with relative standard deviations of 1.8-8.9% at

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concentrations of 0.1, 100 and 800 mg/L. The limit of detection and limit of quantitation were

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0.012 and 0.041 mg/kg, respectively.

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Effects of cultivation temperatures on nicotine catabolism by strain S-1 in MSM were tested

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at 25, 28, 30, 37 and 42 °C. Effects of pH on nicotine catabolism were evaluated at pH 5, 6, 7, 8,

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and 9 which was adjusted with the phosphate buffer (0.2 mol/L NaH2PO4 and Na2HPO4). Effects

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of initial concentrations on nicotine catabolism were performed at 100, 400, and 800 mg/L.

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Identification of nicotine metabolites

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To determine nicotine catabolic intermediates, cell suspensions (5.3×108 cfu/mL) of strain

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S-1 was added to an Erlenmeyer flask, which contained 400 mg/L of nicotine in 20 mL of MSM.

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The flask was cultivated at 30 °C and 150 rpm. Ultrapure water (80 mL) was then added to the

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flask after cultivations for 4, 6, 8, 10 and 12 h. After centrifugation at 12000g for 20 min, the

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supernatants were lyophilized with an FD5-4 freeze-dryer at -80 °C, followed by redissolution

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with 30 mL of methanol. The dissolved samples were filtered through a 0.45-µm nylon filter and

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identified with an Accela liquid chromatograph coupled with LTQ Orbitrap XL high resolution

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mass spectrometer (LC-HRMS) (Thermo Fisher Scientific, Waltham, MA). A 150 mm × 2.1 mm 9



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i.d., 3 µm, Hypersil Gold C18 analytical column (Thermo Fisher Scientific, San Jose, CA) was

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used to separate metabolites, which was eluted with the mixture of 0.1% formic acid and

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acetonitrile (50:50, v/v) at a flow rate of 0.2 mL/min. Mass spectrometry was operated with an

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electrospray ionization (ESI) source with voltage of 4.0 kV in a positive mode. Voltage of tube

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lens was 110 V. The vaporizer and capillary temperatures was 280 °C and 275 °C, respectively.

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Nitrogen (purity 99.999%) was the sheath, sweep and auxiliary gases at a flow rates of 20, 0.45

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and 5 arbitrary units, respectively. The retention time of metabolites was between 1.43 min and

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2.04 min.

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Metabolites 6 and 7 were isolated and purified with a Waters 2695 preparative HPLC

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(pre-HPLC) (Waters Corp., Milford, MA) as follows. The samples were concentrated to dryness

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at 40 °C on a rotary concentrator under vacuum for further purification by a pre-HPLC. The

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pre-HPLC was coupled with a 150 mm × 30 mm i.d., 5 µm, kromasil C18 column (AkzoNobel,

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Bohus, Sweden) and a UV detector set at 254 nm. The samples were initially eluted with 5%

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aqueous acetonitrile (containing 0.1% trifluoroacetic acid), increased linearly to 25% aqueous

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acetonitrile in 12 min, followed by a sharp increase to 95% in 2 min and decreased to 5%

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acetonitrile in 2 min. The column flow rate was 35 mL/min at 25 °C. Compounds 6 and 7 were

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individually collected and then freeze-dried under vacuum. The chemical structures of 6 and 7

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were identified on the basis of 1D and 2D nuclear magnetic resonance (NMR) spectroscopic and

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high-resolution mass spectrometric analyses. NMR spectra were recorded on an Agilent 600MHz 10


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NMR spectrometer (Agilent Technologies, Palo Alto, CA) and a Bruker Avance III 400 NMR

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spectrometer (Bruker Biospin Corp., Fällanden, Switzerland). Chemical shifts were reported in

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ppm (δ) relative to tetramethylsilane (TMS) using dimethyl-d6 sulfoxide (DMSO-d6) as solvent.

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Data analysis

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Degradation percentage (%) and degradation rate (mg/L/h) were obtained according to the

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previous method described by Wu et al.36 Data presented by the average values and standard

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deviations were calculated with Microsoft Excel 2016 (Microsoft Corp., Redmond, WA).

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One-way analysis of variance was done with SPSS 17.0 package (SPSS, Inc., Chicago, IL).

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Differences between treatments were compared with Duncan's test at P value < 0.05.

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RESULTS AND DISCUSSION

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Isolation and identification of nicotine-degrading bacteria

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Strain S-1, isolated from a tobacco powdery waste, can utilize nicotine as a sole source of

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carbon and energy. Strain S-1 is a Gram-negative, rod-shaped bacterium. Its dimensions are 1.32

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to 1.99 by 0.68 to 0.73 µm under TEM. Morphology of S-1 colonies on LB plate is light yellow,

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smooth, round and opaque. Strain S-1 can utilize glucose as carbon source, but not maltose,

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sucrose, lactose and starch. The Methyl Red test was positive, but negative in Voges-Proskauer

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test, indicating that strain S-1 belongs to the genus Pseudomonas. The 16S rRNA gene sequence

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of isolate S-1 (1440 bp) was obtained and deposited in the NCBI GenBank database (accession

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No. MG557558). 16S rRNA gene sequence of S-1 had 99% similarity with that of Pseudomonas 11



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sp. (Figure 1). Therefore, strain S-1 was denoted as Pseudomonas sp. S-1.

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Growth of strain S-1 and nicotine degradation in MSM

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Cell biomass of S-1 (OD600) increased rapidly during 6-20 h of cultivation and reached a

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maximum at 20 h (Figure 2). Greater than 97% nicotine (400 mg/L) was degraded by S-1 after

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cultivation for 10 h and completely degraded by 12 h. The cell biomass of S-1 continued increase

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after complete degradation of nicotine, suggesting that nicotine catabolic intermediates could

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undergo further catabolism and provide carbon source to support strain S-1 growth. Statistical

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analysis indicated that the decrement of nicotine concentration had a significant positive

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correlation with the increment of growth during degradation process (P < 0.05, r2 = 0.988),

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suggesting that the growth of S-1 mainly depended on nicotine utilization. Previous studies

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indicated a positive correlation between nicotine degradation and HF-1 biomass.37 Wang et al.

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observed that overall simultaneous cell growth of the Pseudomonas sp. strain CS3 and nicotine

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catabolism occurred in liquid culture4.

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Effect of temperature on nicotine biodegradation

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Figure 3A shows nicotine biodegradation (400 mg/L) at 25-42 °C. The percentage of

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nicotine degradation was less than 2% in the cell free controls. The extent of nicotine

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degradation accounted for 43.9%, 62.9%, and 92.6% at 25, 28, and 30 °C, respectively, after 8 h

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of cultivation, and the corresponding degradation rates were 20.4, 30.3, and 44.6 mg/L/h. The

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nicotine degradation efficiencies by strain S-1 were statistically different (P < 0.05) among the 12


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three temperatures and increased with the rise in temperature. However, only 10.5% and 10.1%

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of nicotine was degraded by strain S-1 at 37 °C and 42 °C. These results indicated the optimal

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temperature of nicotine degradation was 30 °C (P < 0.05).

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Nicotine biodegradation was remarkably affected by temperature mainly because

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temperature has significant influence on microbial proliferation. Our results were in good

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agreement with those reported by wang et al.4 The suitable temperature for degrading nicotine by

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Pseudomonas sp. CS3 was 30 °C and the nicotine degradation ability was sharply decreased

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when temperature was over 37 °C or lower than 25 °C.4 Similarly, nicotine-degrading activity of

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Pseudomonas sp. Nic22 was stable under the cultivation temperature of 30-34 °C, and the

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activity decreased at temperatures below 28 °C and above 34 °C.7 Pseudomonas stutzeri ZCJ

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degraded nicotine at an optimal temperature of 37 °C.38 Shinella sp. HZN7 degraded nicotine

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efficiently within 30-35 °C.39 Wang et al. also found that P. putida S16 had a high nicotine

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degradation efficiency at 30 °C.14

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Effect of pH on nicotine biodegradation

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A negligible amount of nicotine was degraded in the controls during the entire experimental

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period (Figure 3B). The degradation rates were 37.0, 37.3, 43.3, 21.0, and 0.8 mg/L/h at pH 5.0,

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6.0, 7.0, 8.0, and 9.0, respectively, after 8 h of cultivation (Figure 3B). This indicates that

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degrade efficiency of nicotine increased as the pH increased from 5.0 to 7.0 but decreased

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significantly (P < 0.05) with the rise in pH from 7.0 to 9.0. The result indicated that neutral pH 13



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condition is suitable for nicotine degradation by isolate S-1. Growth of isolate S-1 was studied in

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sterilized MSM supplemented with 400 mg/L of nicotine under different pH (5-9) conditions.

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The biomass of the isolate S-1 reached maximum at pH 7.0 (data not shown). Nicotine

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degradation was related to the biomass of the isolate S-1 at pH 5.0-9.0, which agreed with the

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previous report that pH affects the growth of microorganisms and thus influences the degradation

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of xenobiotic compounds.40 In the present study, the maximum degradation rate of nicotine was

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obtained at pH 7.0, which may be attributed to biosynthesis and expression of some degradation

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enzymes of strain S-1.41 Additionally, over 98.6% of nicotine at 400 mg/L was degraded within a

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pH rage of 5-8 after 10 h of cultivation (Figure 3B), suggesting the degradation of nicotine by

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S-1 favored conditions that varied from weakly acidic to weakly alkaline, whereas stronger

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alkaline conditions inhibited the degradation. Similarly, wang et al. reported the most favorable

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pH for degradation of nicotine by Pseudomonas sp. CS3 was 7.0.4 The suitable pH ranges for

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nicotine catabolism were between 7.0 and 8.0 for Acinetobacter sp. TW and between 6.0 and 7.0

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for Sphingomonas sp. TY.42 A stable degradation rate of nicotine by Pseudomonas geniculata N1

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occurred at pH 6.0-7.5.11

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Effect of initial concentration on nicotine degradation

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Figure 3C shows the degradation efficiency of nicotine at 100, 400, and 800 mg/L. Nicotine

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concentration remained unchanged without strain S-1 during the whole experiment period (data

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not shown). After 12 h of cultivation, the degradation percentages of nicotine were 100%, 100%, 14


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and 93.9% at 100, 400, and 800 mg/L, respectively, and the corresponding biodegradation rates

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of nicotine were 7.6, 30.6, and 59.5 mg/L/h. This indicated that complete degradation of nicotine

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at 100, 400, and 800 mg/L occurred in 6, 10, and 12 h, respectively, suggesting that

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Pseudomonas sp. S-1 has a high efficiency in nicotine degradation.

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Identification of nicotine metabolites

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Seven major intermediates (2, 3, 4, 5, 6, 7 and 8) of nicotine were detected during the

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degradation process by Pseudomonas sp. S-1, including two metabolites 6 and 7 that were

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characterized by HRMS and NMR after purification (Figure 5). After cultivation for 6 h, the

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positive-ion mass spectrum of metabolite 2 derived from nicotine showed a peak of [M + H]+ ion

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at m/z 161.1076 (Figure 4A), which might be the protonated form of nicotine after

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dehydrogenation. Thus, 2 was proposed to be

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1-methyl-2-(pyridin-3-yl)-3,4-dihydro-2H-pyrrolium, which was different from the first-step

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metabolite 10 in the general pyrrolidine pathway in the Gram-negative Pseudomonas genus.

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The mass spectrum of metabolite 3 showed a molecular ion at m/z 177.1020 [M + H]+

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(Figure 4B) and was proposed to be cotinine (3). Similarly, cotinine and its analogue,

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5-(pyridin-3-yl)-1H-pyrrol-2(3H)-one (14), were observed during nicotine degradation by

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Shinella sp. HZN14,43-44 and P. plecoglossicida TND35.5 The harmful cotinine (3) has been

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found in human and microorganisms.7 Understanding mechanism of nicotine catabolism in

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Pseudomonas sp. S-1 might provide insights into nicotine catabolism in human. 15



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Metabolites 4 and 5 were identified on the basis of molecular ions m/z 179.1177 (ESI+, M +

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1) and 193.0968 (ESI+, M + 1) (Figures 4C and 4D). Compared to the exact mass of the

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metabolites, 4 and 5 were proposed to be N-methyl-4-(pyridin-3-yl)butanamide and

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N-methyl-4-oxo-4-(pyridin-3-yl)butanamide. Interestingly, 4 and 5 were observed for the first

288

time during bacterial catabolism of nicotine.

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Metabolites 6 and 7 were identified as 3-succinyl pyridine (6) and

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6-hydroxy-3-succinylpyridine (7), respectively, based on HRMS and NMR analysis after

291

purification. The molecular ion peaks of 6 and 7 were at m/z 180.0651 (ESI+, M + 1) and

292

196.0601 (ESI+, M + 1) (Figures 4E and 4F), respectively, which were in accordance with

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calculated mass from molecular formula C9H9O3N and C9H9O4N. 1H NMR and 13C NMR spectra

294

of 6 and 7 (Table 1) were consistent with previously reported data.45-46 Combined with HRMS

295

and NMR data, the structures of metabolites 6 and 7 were unambiguously assigned to be those as

296

shown in Figures 4E and 4F. Metabolite 8 was presumed to be 2,5-dihydroxypyridine (8), based

297

on the ion at m/z 161.1077 (Figure 4G).

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Catabolic pathway of nicotine by strain S-1

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The catabolic pathway of nicotine by Pseudomonas sp. S-1 is proposed in Figure 5A. The

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nicotine degradation was first postulated to dehydrogenation of the pyrrolidine moiety of

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nicotine to produce a positively charged intermediate by protonation, compound 2, and then this

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metabolite was oxidized at position 5 of the pyrrolidine to yield intermediate 3. Compound 3 was 16


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degraded to a new intermediate 4 by carbon-nitrogen bond cleavage of pyrrole ring between

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positions 1 and 5. Compound 4 was further oxidized to form another new intermediate 5.

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Interestingly, these two new metabolites produced by strain S-1 were observed during nicotine

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catabolism, which differed from metabolites of other nicotine-degrading microorganisms. This

307

may be attributed to the split of carbon-nitrogen bond of the pyrrolidine of nicotine which

308

occurred at positions 1 and 5 not at positions 1 and 2. Subsequently, 5 was hydrolyzed and

309

released methylamine to form intermediate 6 which was hydroxylated at position 6ʹ of the

310

pyridine to form intermediate 7. Compound 7 was further degraded to produce intermediate 8

311

that would be further mineralized.

312

Bacteria, particularly in the genus Pseudomonas, could catabolize numerous hazardous

313

compounds.23 To date, the predominant nicotine-degrading Gram-negative Pseudomonas spp.

314

mainly followed six pyrrolidine pathways and formed various nicotine intermediates (Figure 5).

315

Catabolic pathway of nicotine and its molecular mechanisms in P. putida S16 as described above

316

have been well elucidated. Nicotine was converted into 6 through 10 and pseudooxynicotine (11)

317

by nicotine oxidoreductase (NicA) in P. putida S16 (Figure 5C).21 Similar to this pathway,

318

nicotine catabolism by Pseudomonas sp. HZN6 was also documented (Figure 5B). In contrast to

319

strain S16, Pao and sap genes encoded pseudooxynicotine amine oxidase and

320

3-succinoylsemialdehyde pyridine dehydrogenase, respectively, which are responsible for

321

catalyzing the conversion of 11 to 3-succinoylsemiadehyde pyridine (12) and 6 in strain HZN6 17



322

(Figure 5B).15 P. plecoglossicida TND35 exhibited a novel nicotine catabolic pathway that

323

differed from that in Pseudomonas sp. HZN6 and P. putida S16 (Figure 5D).5 Pseudomonas sp.

324

HF-1, Pseudomonas sp. Nic22, and Pseudomonas sp. CS3 followed other three incomplete

325

pyrrolidine pathways (a, b, and c) such as nicotine to cotinine, nicotyrine (16), and to myosmine

326

(19) (Figure 5E). Pseudomonas sp. CS3 may start with hydroxylation at position 5 on the

327

pyrrolidine ring to produce 2'-hydroxynicotine (17) that is then converted by pathway b to

328

cotinine (Figure 5E).4 In the incomplete degradation pathway by Pseudomonas sp. HF-1, Nic22,

329

and CS3, cotinine and its analogue are potentially harmful to human health and the

330

environment.7,44,47 On the basis of intermediates detected, Pseudomonas sp. S-1 exhibited a

331

novel intact degradation pathway and no cotinine was found to be accumulated (Figure 5A).

332

Pseudomonas sp. S-1 capable of efficiently degrading nicotine was successfully isolated

333

and its degradation characteristics were determined. Pseudomonas sp. S-1 employed a novel

334

variant pyrrolidine pathway. The present study provides an insight to using nicotine-degrading

335

microorganisms to decontaminate sites contaminated by tobacco wastes as well as enhances the

336

understanding of the catabolic diversity of nicotine in Pseudomonas species. Related genes and

337

enzymes in strain S-1 which mediate nicotine catabolism need to be studied further to understand

338

the mechanisms of gene regulation and expression.

339

Supporting Information

340

Electron transmission micrographs of strain S-1 and comparison of four distinct nicotine 18


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341

catabolic pathways are provided in the supporting materials.

342

AUTHOR INFORMATION

343

*Corresponding author: Tel/Fax: +86-551-65786296; E-mail: [email protected];

344

[email protected]

345

Author contributions: Dr. D. Pan wrote the paper and was responsible for identification of

346

metabolites. Ms. M. Sun performed the experiments of degrading-bacteria isolation and

347

degradation characteristics. Ms. Y. Wang and Dr. P. Lv performed pre-HPLC and HRMS

348

experiments. Dr. X. Wu conceived the project and revised the paper. Dr. H. Cao conducted

349

characterization of some metabolites. Dr. Q. X. Li interpreted the data and revised the manuscript.

350

Dr. R. Hua revised the manuscript.

351

Funding: This work was supported in part by the National Natural Science Foundation of

352

China (31572033), the National Key Research and Development Program of China

353

(2016YFD0200201-1), the Natural Science Fund for Distinguished Young Scholars of Anhui

354

Province (1808085J16), the Natural Science Research Project of Higher Education of Anhui

355

(KJ2017A162), and the National Training Programs of Innovation and Entrepreneurship for

356

Undergraduates (201610364020).

357

Notes: The authors declare no competing financial interest.

358 359 19



360

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361

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CYP82E4 in Tobacco Leaf. J. Agric. Food Chem. 2012, 60, 11586-11591.

26


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FIGURE CAPTIONS

484

Figure 1. Phylogenetic tree based on 16S rRNA gene sequences of strain S-1 and related species

485

by the neighbor-joining approach. Bootstrap values obtained with 1000 repetitions are indicated

486

at the nodes.

487 488

Figure 2. Cell growth and nicotine degradation by Pseudomonas sp. S-1.

489 490

Figure 3. Effects of: (A) temperature; (B) pH; and (C) initial substrate concentrations on nicotine

491

degradation by Pseudomonas sp. S-1.

492 493

Figure 4. Mass spectra of nicotine metabolites by Pseudomonas sp. S-1.

494 495

Figure 5. Proposed catabolic pathway of nicotine by Pseudomonas sp. S-1 (A) and the reported

496

various pyrrolidine pathways for nicotine degradation followed by (B) Pseudomonas sp. HZN6,

497

(C) Pseudomonas putida S16, (D) Pseudomonas plecoglossicida TND35, and (E) Pseudomonas

498

sp. HF-1, Pseudomonas sp. Nic22 and Pseudomonas sp. CS3. NicA: nicotine oxidoreductase;

499

PNAO: pseudooxynicotine amine oxidase; SAPD: 3-succinoylsemialdehyde pyridine

500

dehydrogenase; SPM: 3-succinoyl pyridine dehydrogenase; HspA and HspB:

501

6-hydroxy-3-succinoyl pyridine hydroxylase. 27



Table 1. 1H- and 13C NMR Chemical Shifts for Two Metabolites of Nicotine Identified by NMR 3-succinoyl pyridine 1

H NMR (600 MHz, DMSO-d6)

13

C NMR (150 MHz, DMSO-d6)

1

H NMR (400 MHz, DMSO-d6)

13

C NMR (100 MHz, DMSO-d6)

δ 9.18 (d, J = 2.0 Hz, 1H), 8.83 (dd, J = 1.6, 5.6 Hz, 1H), 8.37-8.40 (m, 1H), 7.64 (dd, J = 4.8, 8.0 Hz, 1H), 3.1 (t, J = 7.2 Hz, 2H), 2.61 (t, J = 7.2 Hz, 2H). δ 198.1, 174.1, 152.3, 148.2, 137.6, 132.6, 125.0, 33.9, 28.1. 6-hydroxy-3-succinoyl pyridine δ 12.13 (s, 2H), 8.25 (d, J = 2.4 Hz, 1H), 7.86 (dd, J = 2.4, 9.6 Hz, 1H), 6.37 (d, J = 9.6 Hz, 1H), 3.05 (t, J = 6.4 Hz, 2H), 2.51 (t, J = 6.4 Hz, 2H). δ 194.1, 173.8, 162.4, 140.8, 138.2, 119.5, 116.1, 31.9, 27.8

28


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Figure 1

29



Figure 2

30


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Figure 3

31



Figure 4

32


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Figure 5

33



Table of Contents Graphic

ACS Paragon Plus Environment 34

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Characterization of Nicotine Catabolism through a Novel Pyrrolidine

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