Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6529-x

ENVIRONMENTAL BIOTECHNOLOGY

Genome-wide investigation of the genes involved in nicotine metabolism in Pseudomonas putida J5 by Tn5 transposon mutagenesis Zhenyuan Xia 1 & Wei Zhang 2 & Liping Lei 1 & Xingzhong Liu 3 & Hai-Lei Wei 3

Received: 6 January 2015 / Revised: 14 February 2015 / Accepted: 7 March 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Pseudomonas putida J5 is an efficient nicotinedegrading bacterial strain isolated from the tobacco rhizosphere. We successfully performed a comprehensive wholegenome analysis of nicotine metabolism-associated genes by Tn5 transposon mutagenesis in P. putida J5. A total of 18 mutants with unique insertions screened from 16,324 Tn5transformants failed to use nicotine as the sole carbon source. Flanking sequences of the Tn5 transposon were cloned with a shotgun method from all of the nicotine-growth-deficient mutants. The potentially essential products of mutated gene were classified as follows: oxidoreductases, protein and metal transport systems, proteases and peptidases, transcriptional and translational regulators, and unknown proteins. Bioinformatic analysis of the Tn5 insertion sites indicated that the nicotine metabolic genes were separated and widely distributed in the genome. One of the mutants, M2022, was a Tn5

insert into a gene encoding a homolog of 6-hydroxy-L-nicotine oxidase, the second enzyme of nicotine metabolism in Arthrobacter nicotinovorans. Genetic and biochemical analysis confirmed that three open reading frames (ORFs) from an approximately 13-kb fragment recovered from the mutant M2022 were responsible for the transformation of nicotine to 3-succinoyl-pyridine via pseudooxynicotine and 3succinoyl semialdehyde-pyridine, the first three steps of nicotine degradation. Further research on these mutants and the Tn5-inserted genes will help us characterize nicotine metabolic processes in P. putida J5.

Keywords Nicotine . Pseudomonas putida . Biodegradation . Tn5

Introduction Zhenyuan Xia and Wei Zhang contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6529-x) contains supplementary material, which is available to authorized users. * Hai-Lei Wei [email protected] 1

Yunnan Academy of Tobacco Agricultural Science, Kunming 650021, Yunnan, China

2

Key Laboratory of Agricultural Environment, Ministry of Agriculture, Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences, 100081 Beijing, China

3

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, 3A Datun Rd, Chaoyang District, 100101 Beijing, China

Nicotine [1-methyl-2-(3-pyridyl-pyrrolidine), C10H14N2], a primary alkaloid component of cigarettes and non-recyclable waste powder from the tobacco manufacturing process, represents up to 3 % (w/w) of the dry mass of tobacco leaves (Armstrong et al. 1998). There is an urgent need in cigarette production and environmental remediation to reduce the nicotine content of cigarettes and tobacco and eliminate tobacco waste (Novotny and Zhao 1999; Sloan and Gelband 2007). Biological treatment with microorganisms is an economical and efficient approach for the manipulation of nicotine content in cigarette production and the detoxification of tobacco wastes containing high concentrations of nicotine (Civilini et al. 1997). Many new nicotine-degrading bacterial genera have been reported, and the related degrading mechanisms also have been investig ated, s uch a s spec ie s of Agrobacterium (Wang et al. 2012; Li et al. 2014), Nocardioides (Ganas et al. 2008), Ochrobactrum (Yu et al.

Appl Microbiol Biotechnol

2014), and Shinella (Qiu et al. 2014). Pseudomonas spp. and Arthrobacter spp. however remain the two most investigated groups for nicotine biodegradation. In recent years, significant advances have been made in the study of the metabolic mechanisms of nicotine biotransformation. In the genus Arthrobacter, the pyridine pathway of nicotine degradation has been thoroughly described, and the related enzymes have been well characterized (Baitsch et al. 2001; Brandsch 2006; Chiribau et al. 2006; Gherna et al. 1965; Igloi and Brandsch 2003; Mihasan et al. 2007; Schenk et al. 1998; Sachelaru et al. 2005). The strains S16 (Yu et al. 2011) and HZN6 (Qiu et al. 2010) are the two best-studied nicotine-degrading Pseudomonas strains. In P. putida S16, a gene cluster designated nic1, which contains the genes encoding nicotine oxidoreductase and 6-hydroxy-3-succinoyl-pyridine (HSP) hydroxylase, was reportedly involved in the catabolism of nicotine to 2,5-dihydroxy-pyridine (2,5-DHP) (Tang et al. 2009). In a more recent report, another gene cluster, nic2, encoding five factors and mediating the conversion of HSP to fumarate, was identified in strain S16 (Tang et al. 2012). The corresponding late steps of the nicotine degradation pathway in P. putida S16 were then shown to pass from 2,5-DHP through the intermediates N-formylmaleamic acid, maleamic acid, maleic acid, and fumaric acid (Tang et al. 2012, 2013). In P. putida S16 and Pseudomonas sp. HZN6, three consecutive genes, nox, pao, and sap, are employed to catalyze the initial three steps of the nicotine degradation processes from nicotine to 3-succinoyl-pyridine (SP), pseudooxynicotine (PN), and 3succinoyl semialdehyde-pyridine (SAP) routinely (Tang et al. 2013; Qiu et al. 2010, 2013). In addition to these two Pseudomonas strains, a novel strain capable of degrading nicotine, Pseudomonas geniculata N1, was isolated recently and sequenced using Illumina High-Seq 2000 genome sequencing (Liu et al. 2014). Compared with other Pseudomonas and Arthrobacter species, strain N1 exhibited a yellowish green color during growth with nicotine as the sole source of carbon and nitrogen (Liu et al. 2014). Identification of metabolic intermediates suggested that strain N1 decomposed nicotine via a unique pathway that was different from those reported for Pseudomonas strains (Liu et al. 2014). Thus, it could be seen that different strains even in the same genus could have distinct nicotine catalyzing mechanisms, although they share some of the same intermediate metabolites. This uncertainty compels the continuation of studies on nicotine-degrading mechanisms in the various Pseudomonas strains. P. putida J5 is an efficient nicotine-degrading strain isolated from the tobacco rhizosphere (Wei et al. 2008). In this study, the nicotine metabolism-associated genes of P. putida J5 were identified using a Tn5-based transposon mutagenesis technique that has been widely used to create mutant libraries in a wide range of genera. In total, 18 nicotine utilizationdeficient mutants were screened from a genome-wide Tn5

insertion library containing 16,324 transformants that, to our knowledge, represents the largest random mutagenesis panel created for Pseudomonas spp. capable of degrading nicotine. Individual mutations were confirmed by Southern blot hybridization and mapped following shotgun cloning and sequencing. The insertion site genes of the 18 mutants were divided into five groups according to their putative functions identified in GenBank (http://www.ncbi.nlm.nih.gov). In this analysis, a gene cluster identified in the nicotinedegradation-deficient mutant M2022 was shown to be responsible for the first three steps of nicotine degradation.

Materials and methods Chemicals (S)-Nicotine (99 %) was purchased from Sigma-Aldrich (Seelze, Germany) for standard measurements, and PN (98 %) and SP (98 %) were obtained from Toronto Research Chemicals (Toronto, Canada). All analytical- and highperformance liquid chromatography (HPLC)-grade reagents were from Merck China Co., Ltd (Shanghai, China). The intermediate metabolites of nicotine were identified by liquid chromatography-mass spectrometry (LC-MS) in accordance with previous reports (Tang et al. 2013; Qiu et al. 2010, 2013). Bacterial strains and growth conditions Strains and plasmids are listed in Table 1. P. putida J5 (collection number CGMCC AS1828) and its derivatives were grown in Luria-Bertani (LB) medium at 28 °C, whereas Escherichia coli DH5α and S17-1 (λ-pir) were grown at 37 °C in LB medium. M9 minimal medium was used for biparental mating (Wei et al. 2009). Nicotine inorganic medium (NIM) was used for the screening of nicotine-degradationdeficient mutants (Wei et al. 2008). For plasmid propagation and selection of mutants and transformants, media were supplemented with antibiotics as follows: ampicillin (Amp, 50 μg/ml), kanamycin (Km, 50 μg/ml), tetracycline (Tet, 20 μg/ml), and chloramphenicol (Cm, 20 μg/ml). Creation of a P. putida J5 transposon mutant library Tn5 mutants were created by biparental mating of the recipient cell J5 with the donor cell E. coli S17-1 (λ-pir) containing plasmid pUTKm (Herrero et al. 1990). Overnight cultures of donor and recipient cells were mixed in a 1.5-ml microfuge tube at a ratio of 1:1 (v/v). Cells were pelleted by brief centrifugation and washed once with autoclaved distilled water. The bacterial pellet was then suspended in 100 μl autoclaved, distilled water and spotted onto LB plates. After a 12-h incubation at 30 °C, bacteria were harvested by washing with 1 ml

HB101

Sambrook et al. (1989)

Apr; ColE1 origin

pUC19

Heeb et al. (2002)

Tetr, pVS1-derived lacIq-Ptac shuttle vector

pK18mobsacB containing about 2-kb fragment for ndaA mutation

pME6032

pK18ΔndaA

This study

TaKaRa, Dalian, China Thomas et al. (2009)

Cm ; ColE1 origin Kmr, pMB1 mob sacB; SucS

pHSG399 pK18mobsacB

r

Stratagene, Santa Clara, USA Thomas et al. (2009)

Herrero et al. (1990)

Ap ; ColE1 origin Cmr; ColE1 replicon with RK2 transfer region, helper plasmid

r

Apr; Kmr; delivery plasmid for Tn5; R6K replicon

Sambrook et al. (1989)

Δ(gpt-proA)62 gln V44 recA13

pBluescript II SK+ pRK600

Plasmids pUTKm

Sambrook et al. (1989) Sambrook et al. (1989)

This study This study This study This study This study Thomas et al. (2009)

thi pro hsdRhsdM+recARP4-2-Tc::Mu-Km::Tn7 λpir

Escherichia coli DH5α

DH5α(λ-pir)

M4525 M5731 M16336 M9648 M2748 P. fluorescens Pf0-1

This study This study This study This study This study This study This study This study This study This study This study This study This study

Wei et al. (2008)

Source

F- recA1 endA1 hsdR17 supE44 thi-1 gyrA96 relA1Δ (argF-lacZYA) I169Φ80lacZ ΔM15

Nicotine-degrading-deficient mutant inserted with Tn5 inserted into a sec-independent periplasmic gene Nicotine-degrading-deficient mutant inserted with Tn5 inserted into a molybdate ABC transporter gene Nicotine-degrading-deficient mutant inserted with Tn5 inserted into a molybdate ABC transporter gene Nicotine-degrading-deficient mutant inserted with Tn5 inserted into a molybdate ABC transporter gene Nicotine-degrading-deficient mutant inserted with Tn5 inserted into an ABC transporter gene Nicotine-degrading-deficient mutant inserted with Tn5 inserted into a TldD/PmbA peptidase-encoding gene Nicotine-degrading-deficient mutant inserted with Tn5 inserted into a M23/M37 peptidase-encoding gene Nicotine-degrading-deficient mutant inserted with Tn5 inserted into a peptidase S16-encoding gene Nicotine-degrading-deficient mutant inserted with Tn5 inserted into a glycine cleavage system transcriptional repressor gene Nicotine-degrading-deficient mutant inserted with Tn5 inserted into a heavy metal two component sensor gene Nicotine-degrading-deficient mutant inserted with Tn5 inserted into a nicotine oxidase-encoding gene Nicotine-degrading-deficient mutant inserted with Tn5 inserted into a quinone oxidoreductase-encoding gene Nicotine-degrading-deficient mutant inserted with Tn5 inserted into a glutaryl-CoA dehydrogenaseencoding gene Nicotine-degrading-deficient mutant inserted with Tn5 inserted into a 2OG-Fe(II) oxygenase-encoding gene Nicotine-degrading-deficient mutant inserted with Tn5 inserted into a glycerate dehydrogenase Nicotine-degrading-deficient mutant inserted with Tn5 inserted into a β-hydroxydecanoyl ACP dehydratase-encoding gene Nicotine-degrading-deficient mutant inserted with Tn5 inserted into a gene with unknown function Nicotine-degrading-deficient mutant inserted with Tn5 inserted into a gene with unknown function Wild type

Apr; wild type

Pseudomonas putida J5

M15738 M728 M9502 M430 M9505 M2735 M11614 M2744 M12440 M10225 M2022 M406 M506

Characteristics

Strains and plasmids used in this study

Strains or plasmids

Table 1

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This study This study

This study This study This study This study This study This study This study This study This study This study This study This study This study

Source

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autoclaved distilled water, diluted, and spread on M9 plates containing Km. Resulting colonies were manually inoculated into individual wells of 96-microwell plates containing LB broth supplemented with Km. Following overnight growth, the wells were supplemented with glycerol at a 20 %w/v final concentration, and the plates were stored at −80 °C. Screening of nicotine-degradation-deficient mutants

pK18mobsacB containing about 2-kb fragment for ndaB mutation pK18mobsacB containing about 2-kb fragment for ndaC mutation pK18mobsacB containing about 2-kb fragment for ndaD mutation pK18mobsacB containing about 2-kb fragment for ndaE mutation pK18mobsacB containing about 2-kb fragment for ndaF mutation pK18mobsacB containing about 2-kb fragment for ndaG mutation pK18mobsacB containing about 2-kb fragment for ndaH mutation pME6032 containing the full length of ndaA gene pME6032 containing the full length of ndaB gene pME6032 containing the full length of ndaC gene pME6032 containing the full length of ndaD gene pME6032 containing the full length of ndaE gene pME6032 containing the full length of ndaF gene

pME6032 containing the full length of ndaG gene pME6032 containing the full length of ndaH gene

pK18ΔndaB pK18ΔndaC pK18ΔndaD pK18ΔndaE pK18ΔndaF pK18ΔndaG pK18ΔndaH pME-ndaA pME-ndaB pME-ndaC pME-ndaD pME-ndaE pME-ndaF

pME-ndaG pME-ndaH

Strains or plasmids

Table 1 (continued)

Characteristics

To screen nicotine-degradation-deficient mutants, Tn5 transformants were individually picked with sterilized toothpicks to stab-inoculate LB and NIM plates. After plates were cultured for 48 h at 28 °C, the mutant candidates that failed to grow on NIM were picked from the LB plates and subjected to selection for further confirmation. Purified, single colonies of individual nicotine-growth-deficient mutants were inoculated in LB liquid medium and cultured to saturation at 30 °C. Fifty microliters of culture was transferred into 5 ml of LB and NIM liquid with 1 g/l of nicotine and cultured for 12 h at 30 °C. Then, all of the samples were centrifuged for 5 min at 12, 000 rpm. Nicotine concentrations in the supernatants were determined using Agilent 1100 series high-pressure liquid chromatography (HPLC) (Agilent, Santa Clara, USA) and an Agilent C-18 column (5 μm, 4.6×150 mm) as described previously (Wei et al. 2009). All experiments were independently performed at least twice with three replicates each time. Southern blot hybridization analysis and determination of transposon insertion sites DNA from P. putida J5-derived transposon insertion mutants was isolated according to previously described procedures. Genomic DNA was isolated (Wei et al. 2009), and Southern blot transfer of PstI-digested chromosomal DNA was performed according to Sambrook et al. (1989). A PCR product containing a partial Km resistance gene was used as the hybridization probe. Primer pairs Km421 and Km1016 (Table S1 in the Supplementary Material) were designed according to the sequence of plasmid pHSG299 (GenBank no. M19415) to amplify a 0.6-kb Km resistance gene fragment from pHSG299. Hybridization and detection were performed according to the protocol of the digoxigenin DNA labeling and detection kit (Roche Molecular Biochemicals, Lewes, UK). Shotgun cloning was performed to determine the transposon insertion sites in selected mutants. Chromosomal DNA samples were restricted with PstI, BamHI, or EcoRI and ligated into pUC19, pHSG399, or pBluescript II SK. E. coli DH5α transformants were selected on LB medium containing Km. Positive clones were sequenced with Tn5-39 and Tn5-1571 (Table S1 in the Supplementary Material) to determine the precise location of transposon insertions. Sequences were then compared to the protein sequence database (GenBank) using

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the BlastX algorithm (Altschul et al. 1990). For each mutant, the junction between the transposon sequence (the Tn5 inverted repeat sequence ending with GTG TAT AAG AGA CAG) and the genomic DNA sequence and the 9-bp target duplication (a characteristic of Tn5 insertions) were identified. Construction of individual gene-disrupted mutants Eight unmarked deletion mutants of nda genes were created by double recombination of each gene-flanking region. Briefly, two fragments carrying the left and right flanking regions of each individual gene were amplified with HighFidelity PCR MM (BioLabs, Ipswich, USA) using primer pairs (Table S1 in the Supplementary Material). For one gene, each fragment was purified and ligated for 1 h. The forward primer of the left fragment and the reverse primer of the right fragment were used to amplify the ligation product. After purification, the big fragment harboring the left and right fragments was inserted into the SmaI site of the plasmid pK18mobsacB (Schäfer et al. 1994). The resulting pK18mobsacB derivative vectors were transformed into strain J5 by triparental mating with the helper plasmid pRK600 to introduce the shortened target genes into the chromosome of strain J5 (Keen et al. 1988; Wei and Zhang 2006). The Kmresistant transformants were selected and cultured in 5-ml liquid LB medium without any antibiotics for overnight. And, 0.1-ml culture was transferred to 5-ml fresh liquid LB medium for another 12 h. On LB plates, 0.1 ml culture was diluted and poured. The colonies that have lost Km resistance were selected to be confirmed a double crossover event with colony PCR using the forward and reverse primers. Heterologous expression of nicotine degradation genes in Pseudomonas fluorescens Pf0-1 Gene fragments were amplified with High-Fidelity PCR MM (BioLabs, Ipswich, USA) using the custom primer pairs (Table S1 in the Supplementary Material). Each fragment was digested with appropriate restriction enzymes and then cloned into the broad-range vector pME6032 (Heeb et al. 2002). Recombinant plasmids were transformed into P. fluorescens Pf0-1 (Thomas et al. 2009) by triparental mating with the helper plasmid pRK600 as described above. P. fluorescens Pf0-1 and their derivative strains were cultured in LB medium for 12 h, harvested by centrifugation, washed twice, and resuspended in sterile H2O, and the optical density at 600 nm (OD600) was adjusted to 1. An aliquot of the cells (5 %) was inoculated in NIM supplemented with 100 mg/l substrate (nicotine, PN, SAP, or SP) and incubated for 24 h at 30 °C and 200 rpm. Concentrations of the substrates were determined by HPLC analysis, as described previously (Wei et al. 2008).

Nucleotide sequence accession number The nucleotide sequence rescued from M2022 in the present study was deposited in GenBank under the accession number KJ462515.

Results Construction of a P. putida J5 transposon library In this study, we constructed a transposon mutant library with 16,324 mutants of P. putida J5 by conjugative transfer of a mini-Tn5 from E. coli S17-1 (λ-pir). Combined conjugation and transposition in J5 occurred at a frequency of 3.5×10−6 per donor colony-forming units (cfu) and 2.8×10−7 per recipient cfu. Assuming that the transposon was randomly inserted and that all the genes had the same probability of being targeted, the probability of finding a mutation within a particular gene was defined as P=1−(1−X / G)n, a simplified model where P is the probability of finding a mutation within a particular gene, X is the average length of the genes, G is the genome length, and n is the number of clones within the library (Reznikoff and Winterberg 2008; Laia et al. 2009). As the genome of strain J5 has not yet been sequenced, our analysis is based on P. putida S16 genome information (Yu et al. 2011). Therefore, the probability of finding a mutation within a given gene according to the P. putida S16 genome was 93.4–96.1 % (Yu et al. 2011), which was adequate for the discovery of nicotine-degradationassociated genes. Screening for defective growth in nicotine and identification of transposon insertion sites To select mutants from the mutagenized library of P. putida J5 that were growth deficient in minimal medium with nicotine (1 g/l) (NIM) as the sole carbon resource, Tn5-inserted transformants of the library were spotted on NIM plates with a positive growth control on LB plates. After 72 h of incubation at 28 °C, 18 mutants unable to use nicotine as the only carbon source (NIC−) were isolated from a total of 16,324 transconjugants (Fig. 1a). This inability was also confirmed in NIM broth and LB broth with 1 g/l of nicotine; there was no increase of the OD600 in 72 h following inoculation at an OD600 of 0.05 for these strains in NIM and no nicotine degradation in LB medium. We first assessed the Tn5 insertion number in NIC− mutants using Southern blot analysis. As is shown in Fig. 1b, all mutants carried a single Tn5 insertion. Hybridization patterns also showed that insertions were in different locations, suggesting that mutants bore independent transposon insertions. Transposon integration sites were then identified by shotgun

Appl Microbiol Biotechnol Fig. 1 Confirmation of growth and transposon insertion events of the nicotine-degrading-deficient mutants. a Eighteen mutants lost the ability to utilize nicotine (1 g/ l) as the only carbon source. b The Tn5 insertion number in the nicotine-degrading-deficient mutants was determined by Southern blot hybridization using a kanamycin resistance gene fragment as a probe. All mutants carried a single copy of Tn5 insertion

cloning, a commonly used technique to clone the flanking DNA sequences of transposon insertion sites. Analysis of the DNA sequences flanking the transposons at either end revealed that each insertion was flanked by the 9-bp duplication that is characteristic of Tn5 insertions (Berg and Berg 1983). Most flanking sequences of the insertion sites could be mapped in the genome sequence of P. putida strain S16 with the exception of mutant M16336 (Table 2). Comparison of the resulting sequences with those found in GenBank revealed that six of the mutants had insertions into genes similar to those encoding known oxidoreductases, five mutants had insertions into genes with similarity to protein secretion- and metal transport-related genes, three had insertions into genes with similarity to those encoding peptidases, two had insertions into regulator-encoding genes, and two into genes had no similarity to any other protein sequence in the GenBank database. In the six oxidoreductases, five could be mapped in the P. putida S16 genome sequence, with the exception of mutant M16336 (Table 2).

Identification of a gene cluster responsible for the first three steps of nicotine degradation In the mutant M2022, the Tn5 transposon was inserted in a gene encoding nicotine oxidase, which has been reported to be a crucial nicotine degradation enzyme in Pseudomonas sp. HZN6 (Qiu et al. 2010, 2013) and P. putida S16 (Tang et al. 2013). We only cloned an approximately 3.4-kb fragment containing the transposon with the shotgun method using PstI, but this fragment did not cover the full length of the nicotine oxidase gene. BamHI was then used to clone a longer fragment, and an approximately 13-kb fragment was recovered from mutant M2022. That clone was fully sequenced, with the removal of the transposon sequence, and assembled into an 11,022-bp fragment containing eight intact open reading frames (ORFs) (Table 3, Fig. 2). The gene interrupted by Tn5 transposon, designated nicotine-degrading-associated A gene (ndaA), encoded a 482-residue protein with a predicted molecular mass of 52.4 kDa. The predicted pI, 6.4, indicated

c

b

a

478 (482) 200 (201) 270 (393) 25 (207) 305 (321) 110 (156) 90 (262) 71 (229) 10 (363) 65 (229) 85 (310) 415 (480) 310 (473) 80 (806) 160 (186) 145 (450) 45 (162) 90 (384)

AEJ14620 (99) AEJ11872 (95) AEJ10714 (96) AEJ15535 (79) AEJ11398 (89) None AEJ15390 (96) AEJ13858 (93) AEJ13859 (88) AEJ13858 (93) AEJ12102 (97) AEJ15322 (95) AEJ11016 (97) AEJ14744 (96) AEJ14522 (93) AEJ11433 (97) AEJ12316 (96) AEJ14095 (93)

The GenBank accession numbers of closest relatives in P. putida S16 and its identity (inside the bracket) are shown

I, oxidoreductases; II, protein secretion- and metal transport-related proteins; III, peptidases, IV, gene expression regulators; V, unknown proteins

V

IV

III

II

I

Insertion site (aa)a Accession no./identity (%)b Typec

The numbers inside the brackets are the amino acid sequence length of the protein being disrupted. The numbers outside the brackets are the codon numbers of insertion sites

Putative nicotine oxidase, low identical to 6-HLNO wrbA, flavodoxin/nitric oxide synthase, quinone oxidoreductase gcdH, glutaryl-CoA dehydrogenase Oxidoreductase, 2OG-Fe(II) oxygenase Glycerate dehydrogenase, 2-hydroxyacid dehydrogenase fabA, β-hydroxydecanoyl ACP dehydratases (FabA) Sec-independent periplasmic protein translocator TatC modB, molybdate ABC transporter, permease protein modC, molybdenum import ATP-binding protein modB, molybdate ABC transporter, permease protein ABC transporter, ATP-binding protein TldD/PmbA family protein, peptidase U62 Peptidase, M23/M37 family ATP-dependent protease La, peptidase S16 Glycine cleavage system transcriptional repressor Heavy metal two component sensor protein CopS Unknown protein Unknown protein

BamHI PstI EcoRI PstI PstI PstI PstI PstI PstI PstI PstI PstI PstI PstI PstI PstI PstI PstI

M2022 M406 M506 M4525 M5731 M16336 M15738 M728 M9502 M430 M9505 M2735 M11614 M2744 M12440 M10225 M9648 M2748

pUC19 pBluescript pBluescript pHSG399 pHSG399 pBluescript pBluescript pBluescript pBluescript pBluescript pHSG399 pBluescript pBluescript pBluescript pBluescript pHSG399 pHSG399 pHSG399

Endonuclease for cloning Fragment length (kb) Cloning vector Predicted function of interrupted gene

Mutant 13.0 4.8 4.3 4.8 2.8 4.0 3.0 6.3 6.3 6.3 2.8 7.0 4.5 4.0 6.6 6.0 2.8 3.5

Mapping transposon insertion sites in the 18 nicotine-degrading-deficient mutants

Table 2

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Appl Microbiol Biotechnol Table 3

Predicted gene products on the nda gene cluster

Gene product Length Position

Gene product description

Closest relative in P. putida S16 GenBank accession no. Identity (%)

NdaH NdaG NdaF NdaE

115 390 512 170

931-1278 1387-2559 2579-4117 4749-5261

Cupin_2, superfamily Putative aromatic amino acid transporter, tryptophan/tyrosine permease Transcription regulator, PucR family, purine catabolism PurC-like protein Endoribonuclease L-PSP, YjgF-like protein

AEJ14623 AEJ14622 AEJ14621 AEJ14586

99 100 93 73

NdaA NdaB NdaC NdaD

482 132 496 474

5322-6770 6802-7200 7233-8723 8716-10140

Flavin-containing amine oxidase Cytochrome c-like protein Flavin-containing amine oxidase 3-Succinoylsemialdehyde-pyridine dehydrogenase

AEJ14620 None AEJ14619 AEJ14618

99 None 100 99

that the protein was a neutral enzyme. A homology search of NCBI databases revealed that the amino acid sequence of NdaA shared 99, 84, and 28 % identity, respectively, with the flavin-containing nicotine oxidase NicA2 from P. putida S16 (Tang et al. 2013), NOX from Pseudomonas sp. HZN6 (Qiu et al. 2013), and 6-hydroxy-L-nicotine oxidase (6HLNO) from A. nicotinovorans (Kachalova et al. 2010). Three more ORFs were found downstream of the ndaA gene. Of these ORFs, ndaB, located directly downstream of ndaA, was the most homologous to the genes encoding cytochrome C-like

proteins (accession no. COG3474) that was not present in P. putida S16 and Pseudomonas sp. HZN6. The proteins encoded by ndaC and ndaD shared significant identities with nicotine oxidase (accession no. AEJ14619) and SAP dehydrogenase (accession no. AEJ14618) in P. putida S16, respectively. Notably, both NdaA and NdaC are monoamine oxidases (accession no. COG1231) according to homology blast. Also, there is 38 % identity between these two nicotine oxidases. Another ORF, named ndaE, lies immediately upstream of ndaA. The deduced amino acid sequence of

Fig. 2 Schematic representation of the genes responsible for the initial steps of nicotine degradation from P. putida J5. a Organization of the cloned genes from M2022 and different gene constructs. b Nicotine and intermediate-degrading abilities of P. fluorescens Pf0-1 harboring different gene constructs. Ability (plus sign); no ability (minus sign). P. fluorescens Pf0-1 and the mutants were cultured in LB medium

overnight. An aliquot of the cells (5 %) was inoculated in NIM supplemented with 100 mg/l substrates, respectively, and incubated at 30 °C and 200 rpm. Culture samples were removed after 24 h to be measured the residual substrates with HPLC. c Proposed the first three steps of pyrrolidine pathway of nicotine catabolism in P. putida J5

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Fig. 3 Growth profiles and resting cell reactions of individual gene deletion mutants of the nda cluster. a Growth curves of strain J5 and the eight gene deletion mutants with nicotine as sole carbon source. b HPLC analysis of nicotine degradation by resting cells of strain J5 and the gene deletion mutants. P. putida J5 and the mutants were cultured in LB

medium overnight. An aliquot of the cells (5 %) was inoculated in NIM supplemented with 1 g/l nicotine and incubated at 30 °C and 200 rpm. Culture samples were removed periodically to be measured the absorbance at 600 nm and the residual nicotine with HPLC. Presented data are average of duplicate independent growth experiments

ndaE shared 73 % identity with anendoribonuclease (accession no. AEJ14586) of P. putida S16, but the gene encoding the endoribonuclease in P. putida S16 is not clustered with the nicotine degradation gene nicA2 (Tang et al. 2013). The other three intact ORFs (ndaF, ndaG, and ndaH) are detailed in Table 3. Based on sequence similarity and the putative nicotine degradation pathway, we surmised that this gene cluster was responsible for the early steps of nicotine degradation. To confirm this hypothesis, we made eight independent unmarked deletion mutants of the eight gene candidates so that expression of the downstream genes was not affected by gene disruption. We then measured cell growth in nicotine and nicotine conversion by resting cells. Mutants of ndaA, ndaB, ndaC, ndaD, and ndaE lost the ability to use nicotine as the sole carbon and nitrogen source, while disruptions of ndaF, ndaG, and ndaH had no effect on growth with nicotine as the sole carbon source (Fig. 3a). Of the eight single-gene mutants, only resting cells of J5ΔndaA failed to metabolize nicotine (Fig. 3b). These results indicated that ndaA played a crucial role in the first step of nicotine metabolism. To further characterize the function of ndaA and its neighboring gene sets, constructs harboring different gene set were made and heterologously expressed in the nicotine-degradation-deficient bacterium P. fluorescens Pf0-1 (Thomas et al. 2009). The degradative ability of the transformants for nicotine, PN, and SAP was evaluated, and results are shown in Fig. 2b. Expression of the ndaABCD large fragment allowed strain Pf0-1 to degrade nicotine, PN, and SAP completely. SP was detected in all of the cell extracts containing the three substrates. In the following assays, we found that ndaA, ndaC, and ndaD were required for the conversion of nicotine to PN, PN to SAP, and SAP to SP, respectively, while ndaB, ndaE, and ndaFGH were not involved in the first three steps of nicotine degradation. Therefore, the first three steps of nicotine degradation in P. putida J5 were proposed to pass through the pyrrolidine

pathway, which is similar to the degradative pathways of P. putida S16 (Tang et al. 2013) and Pseudomonas sp. HZN6 (Qiu et al. 2010, 2013) (Fig. 2c).

Discussion Although large numbers of genomes for microorganisms have been sequenced and published, the function of various genes in any given genome still need to be determined. Pseudomonas is the primary nicotine-degrading bacterial genus with species capable of using nicotine as a sole carbon source (Wada and Yamasaki 1953). The genetic analysis of the nicotine-degrading bacterium P. putida J5 is in its infancy. In this work, a P. putida J5 insertion library consisting of 16,324 mutants was created based on the mini-Tn5 system. Further analysis of the random mutants, selected for the inability to grow on minimal medium with nicotine as the sole carbon, has validated the efficiency of the library. Across the library, 94 % of the sequence reads of nicotine-degradation-deficient mutants, with the exception of mutant M16336 where Tn5 was inserted into a gene encoding β-hydroxydecanoyl ACP dehydratase (FabA), were mapped to the genome of P. putida S16, a well-studied nicotine-degrading bacterium (Tang et al. 2013). Interestingly, three mutants were inserted with Tn5 in genes of the molybdenum transport system, in which two mutants were inserted in the modB gene although the other 15 insertions were relatively evenly distributed around the genome. In the nicotine-degrading-deficient mutants, the Tn5 insertions of M406 and M5731 are very close to the end of target genes (Table 1) which suggests that more careful and detailed analysis should be performed to check whether the downstream genes are involved in nicotine utilization. Metabolism of nicotine by bacteria is a complex biocatalytic process. In pioneer research of nicotine-degrading, gram-

Appl Microbiol Biotechnol Fig. 4 Phylogenetic tree of 16S rDNA from eight nicotinedegrading Pseudomonas strains. The phylogenetic tree is constructed using the molecular evolutionary genetics analysis tool (MEGA 4.1) by neighborjoining (NJ) method (Tamura et al. 2007). The repeated bootstrapping for 1000 times was performed

positive Arthrobacter spp., at least 40 ORF products of the 165 predicted plasmid pAO1 ORFs cooperated in nicotine catabolism (Igloi and Brandsch 2003; Ganas et al. 2009). They were subdivided into three groups: those involved in nicotine breakdown to nicotine blue, those involved in Nmethylaminobutyrate degradation, and those involved in molybdenum cofactor (MoCo) biosynthesis (Ganas et al. 2009). In this study, the proteins of P. putida J5 from genes with Tn5 insertion sites were divided into five groups: oxidoreductases, protein secretion- and metal transport-related proteins, peptidases, gene expression regulators, and unknown proteins, according to putative functions recovered from GenBank. Although the oxidoreductases might be directly involved in early steps of nicotine biodegradation, we also found two potential degradation regulators and five protein secretion- and metal transport-related genes. Three mutants had insertions into genes of the molybdate ABC transporter system. A remarkable feature of the pAO1 genome is a gene cluster for the nearly complete pathway of molybdenum cytosine dinucleotide (MCD) cofactor biosynthesis (Ganas et al. 2009). The large subunits of nicotine dehydrogenase and ketone dehydrogenase perform MoCo biosynthesis (Sachelaru et al 2006). Nicotinic acid dehydrogenases identified from Bacillus niacini, Eubacterium barkeri, and P. fluorescens also belong to the class of molybdo-iron/sulfur flavoproteins (Nagel and Andreesen 1990; Gladyshev et al. 1994; Hurh et al. 1994). In P. putida S16, the key step in the conversion of SP to HSP is catalyzed by a multienzyme reaction consisting of molybdopterin-binding oxidase, molybdopterin dehydrogenase, and (2Fe-2S)-binding ferredoxin with molybdenum molybdopterin cytosine dinucleotide as a cofactor (Tang et al. 2013). We predict from these reports that molybdate could be important for nicotine metabolism, especially for early steps of biodegradation. Further studies of the three mutants (M430, M728, and M9502) and the related genes (modABC) would help determine how molybdo-iron is involved in nicotine degradation. In terms of the peptidases, although we do not know the role of the peptidases involved

in nicotine metabolism, it has been reported that the 2,5-DHP dioxygenase gene (hpo) in the nicotine degradation strain P. putida S16 has amino peptidase activity and is predicted to belong to the leucine M29 peptidase family (Yao et al. 2013). In this study, a 11-kb fragment was cloned from the nicotine-degradation-deficient mutant M2022 which was required for the first three steps of nicotine biodegradation. Eight intact ORFs were found from that large fragment, and of these, ndaA, a gene for a protein homologous to amine oxidase and 6HLNO, was targeted by the Tn5 transposon. Expression of a minimum fragment containing ndaABCD enabled the non-nicotine-degrading bacterial strain Pf0-1 to degrade nicotine to SP. Similar results have been reported with the strain Pseudomonas sp. HZN6 (Qiu et al. 2013). The nox gene, a homolog of ndaA expressed in the non-nicotinedegrading bacterium P. putida KT2440, enabled the bacterium to degrade nicotine and yielded an equimolar amount of PN in nicotine medium (Qiu et al. 2013). Another two genes, designated pao (homologous of ndaC) and sap (homologous of ndaD), were responsible for the transformation of PN to SAP and SAP to SP when expressed in recombinant E. coli BL21 (Qiu et al. 2010). Our present study verified the first three steps of nicotine biodegradation by Pseudomonas sp. HZN6. These results might help to identify the flanking sequences of nox and sap. In a recent study, MS-based spectral counting and quantitative reverse transcription-PCR were used to identify the proteins and genes involving in key processes of nicotine degradation (Tang et al. 2013). nicA2 (PPS_4081, homologous of ndaA), pnao (PPS_4080, homologous of ndaC), and sapd (PPS_4079, homologous of ndaD) were more abundantly transcribed under nicotine conditions than under glycerol conditions (Tang et al. 2013). RT-qPCR analysis revealed that these three genes appeared to be upregulated in nicotineinduced P. putida S16 (Tang et al. 2013). Our research and the previous two studies (Qiu et al. 2013; Tang et al. 2013) clearly indicated that the biodegradation of nicotine through

Appl Microbiol Biotechnol

PN, SAP, and SP is the common pathway for Pseudomonas spp. Although nicA1, a rare oxidoreductase-encoding gene, whose product was also reported in P. putida S16 to convert nicotine to 3-succinoylpyridine through PN, we did not obtain any related mutants in our Tn5 insertion library. In the same way, nicotine-degradation-related genes, including nicA1, hspA, and hspB in P. putida S16, were not found in the genome sequence of strain N1 (Liu et al. 2014). It has been proposed that there may be novel nicotine degradation genes and a novel nicotine catabolic pathway(s) present in strain N1 (Liu et al. 2014). The genome of P. putida J5 has not been sequenced, so we are not sure if there is another nicotinedegrading pathway present. Based on the phylogeny of nicotine-degrading Pseudomonas strains based on 16S rDNA sequences, we can see that the strain J5 is more closely associated with strain S16 than with HZN6 (Fig. 4). The genome sequence of P. putida S16 was preliminarily analyzed due to the high identity of nicotine-degradationassociated genes in P. putida J5. However, we could not find any homologs of the ndaB gene from P. putida S16. In addition, one of the eight ORFs in the 11-kb fragment directly involved in nicotine degradation, named ndaE, had 73 % identity to PPS_4045 of P. putida S16, which was nevertheless not clustered with nicA2, pnao, and sapd. Interestingly, there are two IS4 transposon elements located at the corresponding upstream and downstream sites of the 11-kb fragment in the genome sequence of P. putida S16. ISs of the IS4 family are delimited by short imperfect inverted repeat sequences (IR) which encode transposases being required for transposition and inserting, leading to directly repeated sequences at the insertion site (Chandler and Mahillon 2002). Similar elements were found in P. putida KT2440 (Cánovas et al. 2003) and Pseudomonas aeruginosa (Aubert et al. 2006), where an IS4 family element was involved in mobilization and expression of β-lactam resistance genes. These elements not only in bacteria and archaea but also in eukaryota are involved in a wide variety of biological transformations that cause the genome reshuffling and evolution (de Palmenaer et al. 2008). This study presents the results of an extensive mutagenesis project generating 16,324 transposon insertion mutants. Clearly, additional work is required to fully understand the phenotypes of these randomly constructed mutants. Complementation of the disrupted genes and/or independent generation of further mutants in the same gene is needed to provide confirmation for the observed phenotypes. Furthermore, sequencing and characterization of the upstream and downstream regions of the insertion genes will also be helpful for the comprehensive investigation of the mechanism of nicotine metabolism. Acknowledgments This work was supported by a grant from the International Foundation for Science (F/4583-1) to Hai-Lei Wei and a grant from the National Natural Science Foundation of China (30760011) to Liping Lei.

Conflict of interest The authors have no conflict of interest to declare.

References Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410 Armstrong DW, Wang X, Ercal N (1998) Enantiomeric composition of nicotine in smokeless tobacco, medicinal products, and commercial reagents. Chiriality 10:587–591 Aubert D, Naas T, Héritier C, Poirel L, Nordmann P (2006) Functional characterization of IS1999, an IS4 family element involved in mobilization and expression of β-lactam resistance genes. J Bacteriol 188:6506–6514 Baitsch D, Sandu C, Brandsch R, Igloi GL (2001) Gene cluster on pAO1 of Arthrobacter nicotinovorans involved in degradation of the plant alkaloid nicotine: cloning, purification, and characterization of 2, 6dihydroxypyridine 3-hydroxylase. J Bacteriol 183:5262–5267 Berg DE, Berg CM (1983) The prokaryotic transposable element Tn5. Nat Biotechnol 1:417–435 Brandsch R (2006) Microbiology and biochemistry of nicotine degradation. Appl Microbiol Biotechnol 69:493–498 Cánovas D, Cases I, de Lorenzo V (2003) Heavy metal tolerance and metal homeostasis in Pseudomonas putida as revealed by complete genome analysis. Environ Microbiol 5:1242–1256 Chandler M, Mahillon J (2002) Insertion sequences revisited. In: Craig NL, Craigie R, Geller M, Lambowitz AM (eds) Mobile DNA II. ASM Press, Washington D.C., pp 305–366 Chiribau CB, Mihasan M, Ganas P, Igloi GL, Artenie V, Brandsch R (2006) Final steps in the catabolism of nicotine. FEBS J 273: 1528–1536 Civilini M, Domenis C, Sebastianutto N, de Bertoldi M (1997) Nicotine decontamination of tobacco agro-industrial waste and its degradation by micro-organisms. Waste Manag Res 15:349–358 dePalmenaer D, Siguier P, Mahillon J (2008) IS4 family goes genomic. BMC Evol Biol 8:18 Ganas P, Sachelaru P, Mihasan M, Igloi GL, Brandsch R (2008) Two closely related pathways of nicotine catabolism in Arthrobacter nicotinovorans and Nocardioides sp. strain JS614. Arch Microbiol 189:511–517 Ganas P, Igloi GL, Brandsch R (2009) The megaplasmid pAO1 of Arthrobacter nicotinovorans and nicotine catabolism. In: Steinbüchel A, Schwartz E (eds) Microbial megaplasmids. Springer Berlin Heidelberg, Germany, pp 271–282 Gherna RL, Richardson SH, Rittenberg SC (1965) The bacterial oxidation of nicotine. VI. The metabolism of 2,6dihydroxypseudooxynicotine. J Biol Chem 240:3669–3674 Gladyshev VN, Khangulov SV, Stadtman TC (1994) Nicotinic acid hydroxylase from Clostridium barkeri: electron paramagnetic resonance studies show that selenium is coordinated with molybdenum in the catalytically active selenium-dependent enzyme. Proc Natl Acad Sci U S A 91:232–236 Heeb S, Blumer C, Haas D (2002) Regulatory RNA as mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescens CHA0. J Bacteriol 184:1046–1056 Herrero M, de Lorenzo V, Timmis KN (1990) Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol 172:6557–6567 Hurh B, Yamane T, Nagasawa T (1994) Purification and characterization of nicotinic acid dehydrogenase from Pseudomonas fluorescens. J Ferment Bioeng 78:19–26 Igloi GL, Brandsch R (2003) Sequence of the 165-kilobase catabolic plasmid pAO1 from Arthrobacter nicotinovorans and identification

Appl Microbiol Biotechnol of a pAO1-dependent nicotine uptake system. J Bacteriol 185:1976– 1989 Kachalova GS, Bourenkov GP, Mengesdorf T, Schenk S, Maun HR, Burghammer M, Riekel C, Decker K, Bartunik HD (2010) Crystal structure analysis of free and substrate-bound 6-hydroxy-l-nicotine oxidase from Arthrobacter nicotinovorans. J Mol Biol 396:785–799 Keen NT, Tamaki S, Kobayashi D, Trollinger D (1988) Improved broadhost-range plasmids for DNA cloning in gram-negative bacteria. Gene 70:191–197 Laia ML, Moreira LM, Dezajacomo J, Briati JB, Ferreria CB, Ferro M, Silva A, Ferro J, Oliveira J (2009) New genes of Xanthomonas citri subsp. citri involved in pathogenesis and adaption revealed by a transposon-based mutant library. BMC Microbiol 9:9–12 Li H, Xie K, Huang H, Wang S (2014) 6-Hydroxy-3-succinoylpyridine hydroxylase catalyzes a central step of nicotine degradation in Agrobacterium tumefaciens S33. PLoS ONE 9(7):e103324 Liu Y, Wang L, Huang K, Wang W, Nie X, Jiang Y, Li P, Liu S, Xu P, Tang H (2014) Physiological and biochemical characterization of a novel nicotine-degrading bacterium Pseudomonas geniculata N1. PLoS ONE 9:e84399 Mihasan M, Chiribau CB, Friedrich T, Artenie V, Brandsch R (2007) An NAD (P) H-nicotine blue oxidoreductase is part of the nicotine regulon and may protect Arthrobacter nicotinovorans from oxidative stress during nicotine catabolism. Appl Environ Microbiol 73(8):2479–2485 Nagel M, Andreesen JR (1990) Purification and characterization of the molybdoenzymes nicotinate dehydrogenase and 6hydroxynicotinate dehydrogenase from Bacillus niacini. Arch Microbiol 154:605–613 Novotny TE, Zhao F (1999) Consumption and production waste: another externality of tobacco use. Tob Control 8:75–80 Qiu J, Ma Y, Wen Y, Chen L, Wu L, Liu W (2010) Functional identification of two novel genes from Pseudomonas sp. strain HZN6 involved in the catabolism of nicotine. Appl Environ Microbiol 78: 2154–2160 Qiu J, Ma Y, Zhang J, Wen Y, Liu W (2013) Cloning of a novel nicotine oxidase gene from Pseudomonas sp. strain HZN6 whose product nonenantioselectively degrades nicotine to pseudooxynicotine. Appl Environ Microbiol 79:2164–2171 Qiu J, Wei Y, Ma Y, Wen R, Wen Y, Liu W (2014) A novel (S)-6hydroxynicotine oxidase gene from Shinella sp. strain HZN7. Appl Environ Microbiol 80(18):5552–5560 Reznikoff WS, Winterberg KM (2008) Transposon-based strategies for the identification of essential bacterial genes. Methods Mol Biol 416:13–26 Sachelaru P, Schiltz E, Igloi GL, Brandsch R (2005) An α/β-fold C-C bond hydrolase is involved in a central step of nicotine catabolism by Arthrobacter nicotinovorans. J Bacteriol 187:8516–8519 Sachelaru P, Schiltz E, Brandsch R (2006) A functional mobA gene for molybdopterin cytosine dinucleotide cofactor biosynthesis is required for activity and holoenzyme assembly of the heterotrimeric nicotine dehydrogenases of Arthrobacter nicotinovorans. Appl Environ Microbiol 72:5126–5131 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor

Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73 Schenk S, Hoelz A, Krauss B, Decker K (1998) Gene structures and properties of enzymes of the plasmid-encoded nicotine catabolism of Arthrobacter nicotinovorans. J Mol Biol 284:1323–1339 Sloan FA, Gelband H (2007) Cancer control opportunities in low- and middle-income countries. Institute of Medicine (U.S.), Committee on Cancer Control in Low- and Middle-Income Countries, National Academies Press, Washington DC Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599 Tang H, Wang L, Meng X, Ma L, Wang S, He X, Wu G, Xu P (2009) Novel nicotine oxidoreductase-encoding gene involved in nicotine degradation by Pseudomonas putida strain S16. Appl Environ Microbiol 75:772–778 Tang H, Yao Y, Wang L, Yu H, Ren Y, Wu G, Xu P (2012) Genomic analysis of Pseudomonas putida: genes in a genome island are crucial for nicotine degradation. Sci Rep 2:377 Tang H, Wang L, Wang W, Yu H, Zhang K, Yao Y, Xu P (2013) Systematic unraveling of the unsolved pathway of nicotine degradation in Pseudomonas. PLoS Genet 9:e1003923 Thomas WJ, Thireault CA, Kimbrel JA, Chang JH (2009) Recombineering and stable integration of the Pseudomonas syringae pv. syringae 61 hrp/hrc cluster into the genome of the soil bacterium Pseudomonas fluorescens Pf0-1. Plant J 60:919–928 Wada E, Yamasaki K (1953) Mechanism of microbial degradation of nicotine. Science 117:152–153 Wang S, Huang H, Xie K, Xu P (2012) Identification of nicotine biotransformation intermediates by Agrobacterium tumefaciens strain S33 suggests a novel nicotine degradation pathway. Appl Microbiol Biotechnol 95(6):1567–1578 Wei HL, Zhang LQ (2006) Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24. Anton Leeuw Int J G 89:267–280 Wei HL, Lei LP, Xia ZY, Liu XZ (2008) Characterization of a novel aerobic nicotine-biodegrading strain of Pseudomonas putida. Ann Microbiol 58:41–45 Wei HL, Lei L, Liu S, Xia Z, Liu X, Liu P (2009) PanB is involved in nicotine metabolism in Pseudomonas putida. Int Biodeterior Biodegrad 63:988–992 Yao Y, Tang H, Ren H, Yu H, Wang L, Zhang W, Behrman EJ, Xu P (2013) Iron (II)-dependent dioxygenase and N-formylamide deformylase catalyze the reactions from 5-hydroxy-2-pyridone to maleamate. Sci Rep 3:3235 Yu H, Tang H, Wang L, Yao Y, Wu G, Xu P (2011) Complete genome sequence of the nicotine-degrading Pseudomonas putida strain S16. J Bacteriol 193:5541–5542 Yu H, Li Y, Tang H, Xu P (2014) Genome sequence of a newly isolated nicotine-degrading bacterium, Ochrobactrum sp. SJY1. Genome Announc 2(4):e00720-14