Degradation of Diphenyl Ether inSphingobium phenoxybenzoativoransSC_3 Is Initiated by a Novel RingCleavage Dioxygenase

Shu Cai,a,c Li-Wei Chen,b Yu-Chun Ai,c Ji-Guo Qiu,a Cheng-Hong Wang,d

Chao Shi,a Jian He,a Tian-Ming Caib

Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences,Nanjing Agricultural University, Nanjing, Jiangsu, Chinaa; The College of Resources and EnvironmentalSciences, Nanjing Agricultural University, Nanjing, Jiangsu, Chinab; The Institute of Agricultural Resources andEnvironment, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu, Chinac; College of Basic MedicalScience, Jiujiang University, Jiujiang, Jiangxi, Chinad

ABSTRACT Sphingobium phenoxybenzoativorans SC_3 degrades and utilizes diphenylether (DE) or 2-carboxy-DE as its sole carbon and energy source. In this study, we reportthe degradation of DE and 2-carboxy-DE initiated by a novel ring cleavage angulardioxygenase (diphenyl ether dioxygenase [Dpe]) in the strain. Dpe functions at theangular carbon and its adjacent carbon (C-1a, C-2) of a benzene ring in DE (or the2-carboxybenzene ring in 2-carboxy-DE) and cleaves the C-1aOC-2 bond (decarboxyl-ation occurs simultaneously for 2-carboxy-DE), yielding 2,4-hexadienal phenyl ester,which is subsequently hydrolyzed to muconic acid semialdehyde and phenol. Dpe is atype IV Rieske non-heme iron oxygenase (RHO) and consists of three components: ahetero-oligomer oxygenase, a [2Fe-2S]-type ferredoxin, and a glutathione reductase (GR)-type reductase. Genetic analyses revealed that dpeA1A2 plays an essential role in thedegradation and utilization of DE and 2-carboxy-DE in S. phenoxybenzoativorans SC_3.Enzymatic study showed that transformation of 1 molecule of DE needs two moleculesof oxygen and two molecules of NADH, supporting the assumption that the cleavage ofDE catalyzed by Dpe is a continuous two-step dioxygenation process: DE is dioxygen-ated at C-1a and C-2 to form a hemiacetal-like intermediate, which is further deoxygen-ated, resulting in the cleavage of the C-1aOC-2 bond to form one molecule of 2,4-hexadienal phenyl ester and two molecules of H2O. This study extends our knowledgeof the mode and mechanism of ring cleavage of aromatic compounds.

IMPORTANCE Benzene ring cleavage, catalyzed by dioxygenase, is the key andspeed-limiting step in the aerobic degradation of aromatic compounds. As previ-ously reported, in the ring cleavage of DEs, the benzene ring needs to be first dihy-droxylated at a lateral position and subsequently dehydrogenated and openedthrough extradiol cleavage. This process requires three enzymes (two dioxygen-ases and one dehydrogenase). In this study, we identified a novel angular dioxy-genase (Dpe) in S. phenoxybenzoativorans SC_3. Under Dpe-mediated catalysis,the benzene ring of DE is dioxygenated at the angular position (C-1a, C-2), re-sulting in the cleavage of the C-1aOC-2 bond to generate a novel product, 2,4-hexadienal phenyl ester. This process needs only one angular dioxygenase, Dpe.Thus, the ring cleavage catalyzed by Dpe represents a novel mechanism of ben-zene ring cleavage.

KEYWORDS 2-carboxydiphenyl ether, Dpe, Sphingobium, angular dioxygenase,diphenyl ether

Received 12 January 2017 Accepted 19February 2017

Accepted manuscript posted online 10March 2017

Citation Cai S, Chen L-W, Ai Y-C, Qiu J-G, WangC-H, Shi C, He J, Cai T-M. 2017. Degradation ofdiphenyl ether in Sphingobiumphenoxybenzoativorans SC_3 is initiated by anovel ring cleavage dioxygenase. Appl EnvironMicrobiol 83:e00104-17. https://doi.org/10.1128/AEM.00104-17.

Editor Shuang-Jiang Liu, Chinese Academy ofSciences

Copyright © 2017 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Jian He,hejian@njau.edu.cn, or Tian-Ming Cai,ctm@njau.edu.cn.

ENVIRONMENTAL MICROBIOLOGY

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Microbial metabolism of aromatic compounds is a key link in the global carboncycle and also plays an important role in removing a variety of xenobiotics from

the environment. Delineating the degradation mechanisms is critical to facilitate thedevelopment of effective biological solutions to treat aromatic compound-containingwaste streams (1–3). Ring cleavage is the key and speed-limiting step in aromaticcompound degradation (4, 5). Typical aerobic ring cleavage of aromatic compoundsinvolves the following steps: first, the benzene ring is dihydroxylated, resulting in theformation of a structurally stable cis-dihydrodiol intermediate; subsequently, the cis-dihydrodiol intermediate is opened through ortho or meta oxidation (6–9). Certainaromatic compounds, such as benzoate, 3-hydroxybenzoate, m- or p-methylphenol,and 5-chlorosalicylic acid, are also degraded through the gentisate pathway, in whichthe ring cleavage is catalyzed by gentisate 1,2-dioxygenase (10). Furthermore, someangular aromatic compounds, such as diphenyl ether (DE), dibenzo-p-dioxin, dibenzo-furan, fluorine, dibenzothiophene sulfone, and carbazole, are degraded via angulardioxygenation (11). These compounds are dioxygenated at the angular carbon (Ca) andits neighboring carbon to form chemically unstable hemiacetal-like intermediates thatspontaneously split at the CaOO (or CaON, CaOS, or CaOC) bond, resulting in thecleavage of the three-ring structure or DE structure. Subsequent degradation pathwaysare homologous to the pathways that degrade biphenyl or phenol (11, 12). Thedioxygenases catalyzing the angular dioxygenation are termed angular dioxygenases.

DE and its derivatives (DEs) are widely used in the agricultural, pharmaceutical, andchemical industries. DEs are highly persistent due to the presence of a diaryl etherlinkage (13, 14), and they are toxic and carcinogenic to animals and human beings (15).For this reason, the release of DEs into the environment has received considerableattention. Microbial processes are one of the most important paths for the degradationof DEs in soil and water. Several bacterial strains capable of degrading DEs have beenreported, including the DE-degrading strains Sphingomonas sp. strain PH-07 (16),Pseudomonas cepacia Et4 (17), Rhodococcus jostii RHA1 (18), and Sphingomonas sp.strain Ss3 (19); the DE- and 2-carboxy-DE-degrading strain Sphingobium phenoxyben-zoativorans SC_3 (20); the 3- and 4-carboxy-DE-degrading strains Pseudomonas pseu-doalcaligenes POB310 (21), Pseudomonas sp. strain NSS2 (22), Micrococcus sp. strainCPN1 (23), and Sphingobium wenxiniae JZ-1 (24, 25); the dibenzo-p-dioxin-degradingstrain Sphingomonas sp. strain RW1 (26, 27); and the dibenzofuran-degrading strainTerrabacter sp. strain DBF63 (28). Two DE microbial degradation pathways have beenidentified to date. In Sphingomonas sp. PH-07, Pseudomonas cepacia Et4, and Rhodo-coccus jostii RHA1, DE is degraded through lateral dioxygenation (Fig. 1A) (16–18). InPseudomonas pseudoalcaligenes POB310, Pseudomonas sp. NSS2, Sphingobium wenx-iniae JZ-1, Sphingomonas sp. RW1, and Terrabacter sp. DBF63, DEs such as 3- and4-carboxy-DE, dibenzo-p-dioxin, and dibenzofuran are initially degraded via angulardioxygenation (Fig. 1B) (21, 22, 25, 26, 28). Several angular dioxygenases responsible forDE degradation have been reported, including 3-carboxy-DE 1a,6-dioxygenase Pob(21), 3-carboxy-DE 1=a,2=-dioxygenase Pba (25), dibenzo-p-dioxin 1,10a-dioxygenaseDxn (26), and dibenzofuran 4,4a-dioxygenase Dfd (28).

In the present study, we report a novel metabolic pathway in S. phenoxybenzoativ-orans SC_3 for DE and 2-carboxy-DE degradation that is different from the previouslyreported lateral and angular dioxygenation mechanisms. A novel Rieske non-heme ironoxygenase (RHO)-type angular dioxygenase, Dpe (diphenyl ether dioxygenase), wasidentified. Under Dpe-mediated catalysis, DE is dioxygenated at the angular carbon andits neighboring carbon (C-1a, C-2), resulting in the cleavage of the C-1aOC-2 bond (butnot the C-1aOO ether bond), generating 2,4-hexadienal phenyl ester, which wasfurther hydrolyzed and cleaved through meta oxidation (Fig. 1C).

RESULTSSubstrate spectrum of S. phenoxybenzoativorans SC_3. In our previous study,

strain SC_3 was isolated from pesticide-manufacturing wastewater-contaminated soil,and polyphasic taxonomic studies revealed that the strain represents a novel species

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Sphingobium phenoxybenzoativorans (20). The substrate spectrum of S. phenoxybenzo-ativorans SC_3 was determined by adding various aromatic compounds to minimal saltmedium (MSM) and monitoring cell growth and substrate disappearance. Figure 2shows that S. phenoxybenzoativorans SC_3 degraded and utilized DE, 2-carboxy-DE,phenol, catechol, and benzoic acid but not 3-carboxy-DE, 4-carboxy-DE, 4-fluorodiphenylether, 4-bromodiphenyl ether, dibenzofuran, dibenzo-p-dioxin, biphenyl, or 2,3-dihydro-xybiphenyl.

Metabolite identification of DE and 2-carboxy-DE degradation by S. phenoxy-benzoativorans SC_3. During the degradation of DE by S. phenoxybenzoativorans SC_3,two metabolites were detected by high-performance liquid chromatography (HPLC) insamples taken after 36 h of incubation (Fig. 3A). Metabolite 1 had a retention time of7.21 min, which was equal to that of the authentic phenol standard. Tandem massspectrometry analysis showed a prominent protonated molecular ion at m/z 93.1 (M �

H)� and a fragment ion peak at m/z 65.1 (loss of a CAO) (Fig. 3B). This tandem massspectrometry feature is consistent with that of the phenol standard. Metabolite 2 had

1 kb

D

C

B

ADioxygenase

Pba

Dioxygenase

Spontaneous

Dehydrogenase

DE 2,3-Dihydroxy diphenyl ether 2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate

3-Carboxy DE Catechol 3-Hydrobenzoate

DE

Dpe

2-Carboxy DE

Dpe2,4-Hexadienal phenyl ester Phenol

dpeB1

dpeA1A2 locus

dpeC locus

orf1 dpeA2 dpeA1 orf2 orf3 orf4 orf5 orf6

dpeB1 dpeB2

dpeC

Hydrolyse

(contig 0020, 61.1 kb)

(contig 0041, 32.5 kb)

NADH2O2 NAD+ NAD+ NADH2 NADH2O2 NAD+

NADH2O2 NAD+

NADH2O2 NAD+

NADH2O2 NAD+

NADH2 NAD+2H2O

O2

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O2

NAD+

+2H2O

+CO2

(contig 0053, 27.5 kb)

(contig 0007, 141.3 kb)

Dpe

Dpe

dpeB2 loci and

+

FIG 1 Different microbial degradation pathways for DEs. (A) Degradation of DE through lateral dioxygenation in Sphingomonas sp. PH-07,Pseudomonas cepacia Et4, and Rhodococcus jostii RHA1; (B) degradation of 3-carboxy-DE through angular dioxygenation in S. wenxiniaeJZ-1; (C) degradation of DE and 2-carboxy-DE catalyzed by Dpe in S. phenoxybenzoativorans SC_3; (D) genetic organization of genes codingfor the components of Dpe. The arrows indicate the size and direction of transcription of the genes; dpeA1 and dpeA2, genes encodingthe � and � subunits, respectively, of the oxygenase component of Dpe; dpeB1 and dpeB2, genes encoding the ferredoxin componentsof Dpe; dpeC1, gene encoding the reductase component of Dpe.

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a retention time of 8.74 min. Tandem mass spectrometry analysis showed prominentprotonated molecular ions at m/z 203.1 (M � H)� and m/z 225.2 (M � Na)� andcharacteristic fragment ion peaks at m/z 80.8 (HCACHOCHACHOCHO) and m/z 109.7(OHCOCHACHOCHACHOCHO) (Fig. 3C). The m/z 203.1 (M � H) peak was equal tothe theoretical molecular weight of the dioxygenation product of DE (DE molecularweight of 170 plus O2 molecular weight of 32), suggesting that the initial step of DEdegradation in S. phenoxybenzoativorans SC_3 is dioxygenation with metabolite 2 asthe product. Theoretically, there are four possible dioxygenation products: 1a,2-dihydroxy-DE, 2,3-dihydroxy-DE, 3,4-dihydroxy-DE, and 4,5-dihydroxy-DE. However, thelast three products do not produce the characteristic fragment ion peaks of m/z 109.7and m/z 80.8. Only 2,4-hexadienal phenyl ester, the cleavage products of 1a,2-dihydroxy-DE,match these characteristic fragment ion peaks. It is interesting that phenol and 2,4-hexadienal phenyl ester were also captured as the intermediates during 2-carboxy-DEdegradation by the strain (see Fig. S1 in the supplemental material).

Based on the identified metabolites, we propose a novel DE catabolic pathway in S.phenoxybenzoativorans SC_3 (Fig. 1C). In this pathway, DE or 2-carboxy-DE is firstdioxygenated at the angular carbon (C-1a) and the adjacent carbon (C-2) to form achemically hemiacetal-like intermediate, which further dioxygenated and opened atthe C-1aOC-2 bond, producing 2,4-hexadienal phenyl ester (2-carboxy-DE is apparentlydecarboxylated simultaneously). 2,4-Hexadienal phenyl ester is further hydrolyzed tophenol and muconic acid semialdehyde. The initial step of DE in this pathway isdifferent from previously reported lateral and angular dioxygenation (Fig. 1A and B). Wedesignated the dioxygenase responsible for the benzene ring cleavage of DE and2-carboxy-DE as Dpe (diphenyl ether dioxygenase) in this study.

Identification of the genes responsible for dioxygenation of DE and 2-carboxy-DE. The draft genome of S. phenoxybenzoativorans SC_3 was initially sequenced usingan Illumina HiSeq 2000 sequencing system. The acquired genome was resolved into127 contigs consisting of 5,044,557 bp, with 4,906 genes predicted. To clone the dpegene, PbaA1 (the oxygenase � subunit of 3-carboxy-DE 1a,2-dioxygenase from

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FIG 2 Degradation and utilization of various aromatic compounds by S. phenoxybenzoativorans SC_3.The cultures were incubated at 30°C on a rotary shaker for 48 h. The data were derived from threeindependent measurements, and the error bars indicate standard deviations.

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S. wenxiniae JZ-1) (25), DxnA1 (the oxygenase � subunit of dibenzo-p-dioxin 1,10a-dioxygenase from Sphingomonas wittichii RW1) (26), and BphA1 (the oxygenase �

subunit of biphenyl 2,3-dioxygenase from Pseudomonas pseudoalcaligenes KF707) (29)were used for BLASTP searches of the genome. The screen resulted in the identificationof three putative dioxygenase gene clusters located on contigs 0027, 0053, and 0084,

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Counts vs. Mass-to-Charge (m/z)0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

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Counts vs. Mass-to-Charge (m/z)70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240

O

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80.80000

A

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Second-order mass spectrometry

Second-order mass spectrometry

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First-order mass spectrometry

FIG 3 HPLC and tandem mass spectrometry analysis of metabolites generated during DE degradation byS. phenoxybenzoativorans SC_3. (A) HPLC spectrum of metabolites generated during DE degradation by S.phenoxybenzoativorans SC_3; (B) tandem mass spectrometry of metabolite 1; (C) tandem mass spectrom-etry of metabolite 2.

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respectively. All of these gene clusters consist of at least one oxygenase � subunit geneand one oxygenase � subunit gene. The oxygenase � subunits in the three clustersshowed 29% to 37%, 28% to 32%, and 19% to 25% identities with BphA1, PbaA1, andDxnA1, respectively.

To determine which dioxygenase gene cluster is responsible for the ring cleavage ofDE, DNA fragments (approximately 4 kb) containing each of the three putative geneclusters and 1-kb upstream sequences were amplified and ligated into the broad-host-range vector pBBR1MCS-5, and the resulting plasmids were introduced into Escherichiacoli DH5� and S. wenxiniae JZ-1 for whole-cell transformation experiments. Only strainJZ-1 containing the fragment from contig 0053 acquired the ability to degrade DE (Fig.4A) and 2-carboxy-DE (data not shown); thus, the dioxygenase gene cluster on contig0053 encodes Dpe. Furthermore, the HPLC spectrum showed that the metabolites2,4-hexadienal phenyl ester and phenol were accumulated in cells of strain JZ-1(pBBR0053) with incubation for 36 h but disappeared after incubation for 72 h (Fig. 4B,C, and D), indicating that strain JZ-1 could further degrade the two metabolites. E. coliDH5� containing the contig 0053 fragment did not degrade DE or 2-carboxy-DE, whichcould be caused by the absence of a suitable electron transport component (ETC) tosupply reducing power to the oxygenase component of Dpe in E. coli.

To further investigate the physiological roles of the dpe gene cluster in catabolismof DE or 2-carboxy-DE in vivo, a mutant of S. phenoxybenzoativorans SC_3 withdisruption of dpeA1 was constructed by a single-crossover homologous recombinationtechnique. The SC_3ΔdpeA1 mutant (with dpeA1 disruption) completely lost the abilityto degrade and utilize DE and 2-carboxy-DE as carbon sources, while the dpeA1-complemented strain SC_3ΔdpeA1[pBBR-dpeA1] regained the ability to degrade andutilize DE (Fig. 5) and 2-carboxy-DE (data not shown). These results demonstrated that

0 12 24 36 48 60 720

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)

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JZ-1(pBBR) JZ-1(pBBR0027) JZ-1(pBBR0053) JZ-1(pBBR0084)

A DE

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Phenol

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FIG 4 Degradation of DE by recombinant S. wenxiniae JZ-1 expressing the dioxygenase gene cluster. (A) Time course of DE conversion by S. wenxiniaeJZ-1(pBBR) (containing the pBBR1MCS-5 vector), JZ-1(pBBR0027) (containing the dioxygenase gene cluster from contig 0027), JZ-1(pBBR0053) (containing thedioxygenase gene cluster from contig 0053), and JZ-1(pBBR0084) (containing the dioxygenase gene cluster from contig 0084). The data were derived from threeindependent measurements, and the error bars indicate standard deviations. (B to D) HPLC analysis of DE degradation in MSM containing 0.5 mM DE inoculatedwith JZ-1(pBBR0053) with incubation for 0 (B), 36 (C), and 72 (D) h, respectively.

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the dpeA1 is essential for S. phenoxybenzoativorans SC_3 to degrade and utilize DE and2-carboxy-DE as carbon and energy sources.

ORF analysis of the dpe gene cluster and screening of the genes encoding theETCs for Dpe. A search for open reading frames (ORFs) revealed that there were twoadjacent genes (Fig. 1D), designated dpeA1 and dpeA2, showing similarity to the � and� subunits, respectively, of the oxygenase components of some reported dioxygenases(Table 1). dpeA1 is 1,383 bp in length and encodes a protein of 481 amino acids,whereas dpeA2 is 561 bp in length and encodes a protein of 187 amino acids. BLASTanalysis indicated that DpeA1 shares identities with HcaA1 (3-phenylpropionate 2,3-dioxygenase from Bordetella bronchiseptica E010, 38% identity) (30), BphA1 (biphenyl2,3-dioxygenase from Pseudomonas pseudoalcaligenes KF707, 37% identity) (29), PbaA1

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FIG 5 Deletion and complementation of dpeA1. (A) Degradation efficiency of DE by strains SC_3, SC_3ΔdpeA1, andSC_3ΔdpeA1[pBBR5-dpeA1] and the growth curves for these strains in MSM supplemented with 0.5 mM DE. A solid lineindicates the concentration of residual DE; a dotted line indicates the cell concentration. Symbols: e, no inoculation; �, �,and Œ, inoculation with SC_3, SC_3ΔdpeA1, and SC_3ΔdpeA1[pBBR5-dpeA1], respectively. (B) Growth of strains on MSM platesupplemented with 1.0 mM DE; quadrants 1, 2, and 3 were streaked with strains SC_3, SC_3ΔdpeA1[pBBR5-dpeA1], andSC_3ΔdpeA1, respectively.

TABLE 1 ORF analysis of the gene loci involved in DE and 2-carboxy-DE degradation in S. phenoxybenzoativorans SC_3

LocusGenename Proposed product

Position inlocus (nt)

Productsize (bp) Homologus protein (GenBank accession no.), source % Identity

dpeA1A2 orf1 Integrase 8770–9789 340 Integrase (WP_040702686.1), Novosphingobiumnitrogenifgens DSM 19370

97

dpeA2 Diphenyl ether dioxygenase� subunit

10417–10978 187 Biphenyl 2,3-dioxygenase � subunit (KAK52445.1),Bordetella bronchiseptica OSU054

40

dpeA1 Diphenyl ether dioxygenase� subunit

10982–12447 488 3-Phenylpropionate/cinnamic acid dioxygenase �subunit, Bordetella bronchiseptica E010

38

orf2 LysR transcriptional regulator 12591–13385 265 LysR family transcriptional regulator (WP_045059569.1),Pseudomonas sp. ES3-33

30

orf3 2,3-Dihydroxybiphenyl1,2-dioxygenase

13610–14491 294 BphC (2,3-dihydroxybiphenyl 1, 2-dioxygenase)(AEA36111.1), Cupriavidus sp. Ch34

53

orf4 TonB-dependent receptor 14582–16801 740 TonB-dependent receptor (KGB52036), Sphingopyxis sp.LC363

42

orf5 Dehydrogenase 16936–17733 266 Short-chain dehydrogenase (WP_043975662.1),Novosphingobium sp. P6W

61

orf6 Hydrolase 17754–18548 265 BphD (KEH15370.1), Medicago truncatula A17 49

dpeB1 dpeB1 [2Fe-2S] ferredoxin 301–622 107 [2Fe-2S] ferredoxin (KCZ94256.1), Hyphomonas johnsoniiMHS-2

71

dpeB2 dpeB2 [2Fe-2S] ferredoxin 301–616 105 [2Fe-2S] ferredoxin (WP_021690373.1), Novosphingobiumtardaugens ARI-1

79

dpeC1 dpeC1 GR-type reductase 401–1625 408 GR-type reductase (WP_007012794.1), Novosphingobiumpentaromativorans US6-1

79

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(3-carboxy-DE 1a,2-dioxygenase from S. wenxiniae JZ-1, 32% identity) (25), CarAa (carbazole1,9a-dioxygenase from Sphingomonas sp. XLDN-2-5, 30% identity) (31), and DxnA1(dibenzo-p-dioxin 1,10a-dioxygenase from Sphingomonas wittichii RW1, 25% identity) (26).Sequence alignment analysis revealed that the presence of conserved sequences for aRieske [2Fe-2S] domain (CXHX18CX2H) and a nonheme Fe(II) domain (EX4DX2HX4H) (32) inDpeA1 (Fig. S2), suggesting that Dpe is an RHO. In the phylogenetic tree constructed byneighbor-joining (NJ), maximum likelihood (ML), and minimum evolution (ME) algorithmsbased on the oxygenase � submit of many RHOs, DpeA1 forms a subcluster with theoxygenase � subunit of the dioxygenase (function unknown) on contig 0084 but isseparated from other oxygenase � subunits of reported dioxygenases (Fig. 6). DpeA2 sharesidentities with BphA2 (biphenyl 2,3-dioxygenase from Burkholderia xenovorans LB400, 41%identity) (33, 34) and HcaA2 (3-phenylpropionate 2,3-dioxygenase from E. coli, 38% identity)(30). These analyses suggested that dpeA1 and dpeA2 are likely to encode the oxygenase �

and � subunits of Dpe, respectively.All reported RHO dioxygenases require ETCs to transfer reducing power to the

oxygenase component, but we found no evidence of genes encoding the ETCs (ferre-doxin and ferredoxin reductase) in the vicinity of dpeA1A2. Potentially, the ferredoxinand ferredoxin reductase genes do not cluster with dpeA1A2. Similar phenomena havealso been observed for certain RHO genes responsible for xenobiotic metabolism (1, 26,32, 35, 36). According to Kweon et al. (3), RHO dioxygenases possess two types offerredoxin, namely, the [2Fe-2S] type and the [3Fe-4S] type, and three types ofreductase, namely, the glutathione reductase (GR) type, the FNRN (ferredoxin-NADP�

reductase with the [2Fe-2S] ferredoxin domain connected to the N terminus of theflavin binding domain) type, and the FNRC (ferredoxin-NADP� reductase with the[2Fe-2S] ferredoxin domain connected to the C terminus of the NAD domain) type. ORFanalysis of the SC_3 genome predicted three putative [2Fe-2S]-type ferredoxin genes(dpeB1, dpeB2, and dpeB3) and a putative [3Fe-4S]-type ferredoxin gene (dpeB4), threeputative GR-type reductase genes (dpeC1, dpeC2, and dpeC3), two FNRC-type reductasegenes (dpeC4 and dpeC5), and an FNRN-type reductase gene (dpeC6).

Expression of the genes encoding the oxygenase and putative ETCs andreconstruction of Dpe. dpeA1, dpeA2, and the putative ferredoxin and reductase geneswere individually overexpressed in E. coli BL21(DE3), and the C-terminally His-taggedfusion proteins were purified to apparent homogeneity by Ni2�-nitrilotriacetic acid(NTA) affinity chromatography (Fig. S3). The purified ferredoxins and reductases wereassembled with DpeA1 and DpeA2 in various combinations in vitro. Dpe activitiestoward DE and 2-carboxy-DE were detected only when the reaction mixture containedDpeA1, DpeA2, the [2Fe-2S]-type ferredoxin DpeB1 or DpeB2, and the GR-type reduc-tase DpeC1. Thus, Dpe is a type IV RHO and consists of three components: a hetero-oligomer oxygenase, a [2Fe-2S]-type ferredoxin, and a GR-type reductase. The otherferredoxins and reductases did not act as the ETC for Dpe. HPLC and tandem massspectrometry analysis demonstrated the conversion of DE to 2,4-hexadienal phenylester (Fig. S4). The activities of DpeA1A2B1C1 toward DE and 2-carboxy-DE were 3.04and 5.13 �mol/min/mg, respectively, whereas the activities of DpeA1A2B2C1 toward2-carboxy-DE were 4.16 and 7.09 �mol/min/mg, respectively. 2-Carboxy-DE appears tobe more easily degraded than DE. Other DEs, such as 3- and 4-carboxy-DE,4-fluorodiphenyl ether, and 4-bromodiphenyl ether, could not be degraded, indicatingthat their biodegradability is significantly affected by group substitutions and positions.Biphenyl, 2,3-dihydroxybiphenyl, dibenzofuran, and carbazole were also not the sub-strate of Dpe.

Amounts of oxygen and NADH required for transformation of DE. As shown inFig. 7A, when the concentration of the added NADH in the enzyme mixture was lessthan 2-fold of that of DE, the amount of transformed DE was approximately half (0.44-to 0.49-fold) of the amount of the added NADH; when the concentration of the addedNADH was more than 2-fold of that of DE, the added DE was completely transformeddue to the excess of the added NADH. These results indicated that transformation of

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FIG 6 Phylogenetic tree constructed based on the alignment of DpeA1 with the � subunits of many RHOs. The multiple-alignmentanalysis was performed with ClustalX v2.0, the phylogenetic tree was constructed by the neighbor-joining (NJ), maximum-likelihood(ML), and minimum evolution (ME) methods using MEGA 5.0, and bootstrap values (based on 1,200 replications) are indicated atbranch nodes. Filled circles indicate that the corresponding branches were also recovered using the ML and ME algorithms.Boldface indicates the oxygenase � subunits of the three putative dioxygenases in this study. Underlining indicates � subunits ofpreviously reported angular dioxygenases. Bar, 0.02 substitution per nucleotide position. Each item was arranged in the followingorder: protein name, GI number, and strain name.

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one molecule of DE needs two molecules of NADH. As for the determination of theoxygen requirement, the predetermined dissolved oxygen in the mixture was 0.29 mM(9.2 mg/liter). When the concentration of DE was 0.15 mM or higher, the transformedDE was 0.13 to 0.14 mM, which was approximately half (0.45- to 0.49-fold) of thedissolved oxygen; when the concentration of DE in the enzyme mixture was less than0.15 mM, almost all of the added DE was transformed due to the excess oxygen (Fig.7B). These results indicated that transformation of one molecule of DE needs twomolecules of oxygen.

1H NMR analysis of the metabolite of DE and 2-carboxy-DE catalyzed by Dpe.1H nuclear magnetic resonance (NMR) analysis was carried out to further confirm themolecular structure of the product of DE or 2-carboxy-DE catalyzed by Dpe. Theproduct was purified using silica gel column chromatography. The 1H NMR spectrum ofthe product of DE is shown in Fig. 8, and the detailed chemical shifts are listed in TableS2. Seven target peaks were detected. The chemical shifts of the seven peaks were asfollows: (i) a split peak of 5.183 ppm corresponding to CH of the C�C double bond; (ii)a peak of 7.427 ppm corresponding to CH of the C�C double bond; (iii) a peak of 7.605ppm corresponding to CH of the C�C double bond; (iv) a peak of 5.517 ppmcorresponding to CH of the C�C double bond; (v) a peak of 8.035 ppm correspondingto the C�O of the aldehyde group; (vi) a peak of 7.325 ppm corresponding to the twohydrogens of the benzene ring; and (vii) a split peak of 7.513 to 7.432 ppm corre-sponding to the other three hydrogens of the benzene ring. The correspondinghydrogens are all marked in the chemical structural formula. This finding furtherconfirms the identification of the product as 2,4-hexadienal phenyl ester. The 1H NMRdata for the product of 2-carboxy-DE (Fig. S5) are similar to those of the product of DE,indicating that 2-carboxy-DE was also converted to 2,4-hexadienal phenyl ester by Dpe.

Verification of gene transcription. To assess the relative transcriptional levels ofgenes related or putatively related to DE and 2-carboxy-DE degradation in S. phenoxy-benzoativorans SC_3, we performed real-time quantitative PCR (RT-qPCR) to comparethe mRNA levels of the genes dpeA1, dpeA2, dpeB1, dpeB2, and dpeC1 in cells with orwithout DE or 2-carboxy-DE induction. dpeA1, dpeA2, and dpeC1 were significantlyupregulated in DE- and 2-carboxy-DE-induced cells (Fig. 9). These three genes wereexpressed 26.3- to 63.2-fold and 48.4- to 224.4-fold higher in DE and 2-carboxy-DEmedium, respectively, than in glucose medium. 2-Carboxy-DE was a better inducer thanDE. The results further demonstrated the involvement of the enzymes encoded bydpeA1, dpeA2, and dpeC1 in both DE and 2-carboxy-DE catabolism in S. phenoxyben-

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FIG 7 Amounts of transformed DE in the enzyme mixture added with different concentrations of NADH (A) and different concentrations ofDE (B). (A) The concentrations of added DE were 0.25 and 0.50 mM, and the concentrations of NADH were 0.10, 0.15, 0.20, 0.25, 0.50, 1.00,and 2.00 mM. The reaction mixture was incubated at 30°C for 120 min. (B) The concentration of added NADH in the mixture was 1.00 mM,and the concentrations of DE were 0.05, 0.10, 0.15, 0.20, 0.25, and 0.30 mM. The dissolved oxygen is predetermined to be 0.29 mM (9.2mg/liter). The mixture in a closed tube was covered with paraffin oil to isolate air and incubated at 30°C for 120 min.

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zoativorans SC_3. It is interesting that there were no obvious differences between thetranscriptional levels of the two ferredoxin genes dpeB1 and dpeB2 in cells grown withglucose, DE, and 2-carboxy-DE. A possible reason is perhaps that dpeB1 and dpeB2 aremultifunctional and constitutively expressed in S. phenoxybenzoativorans SC_3.

DISCUSSION

In previous reports of the ring cleavage of aromatic compounds, the ring needs tobe pre-dihydroxylated by monooxygenase or dioxygenase and subsequently opened

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FIG 8 1H NMR spectra of the DE metabolite catalyzed by the recombinant enzyme DpeA1A2B2C1. The metabolite was purifiedby silica gel column chromatography. Chemical shifts of hydrogens are displayed on the chemical structure.

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FIG 9 Transcriptional levels of genes dpeA1, dpeA2, dpeB1, dpeB2, and dpeC1 (from left to right) with orwithout DE or 2-carboxy-DE induction. mRNA expression levels of the seven target genes were estimatedusing RT-qPCR and the 2�ΔΔCT method. The 16S rRNA gene was used as the reference gene. Results arethe mean values for three independent experiments, and error bars indicate standard deviations.

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by intradiol dioxygenase (ortho pathway), extradiol dioxygenase (meta pathway), orgentisate 1,2-dioxygenase (gentisate pathway) (6–10). Multiple enzymes are involved inthe process. For example, the cleavage of DE through lateral dioxygenation requiresthree enzymes: a dioxygenase catalyzing the ring dihydroxylation, a dehydrogenase,and a dioxygenase catalyzing the ring cleavage. In the present study, we found a novelring cleavage mechanism of DEs in S. phenoxybenzoativorans SC_3. The ring cleavageoccurs at the angular position (C-1a, C-2), not at the lateral position, and requires onlyone dioxygenase.

Through genome sequencing and comparison and enzymatic studies, we identifiedan angular dioxygenase, Dpe, responsible for the ring cleavage of DE or 2-carboxy-DE.Dpe is a type IV RHO consisting of a hetero-oligomer oxygenase (DpeA1A2), a [2Fe-2S]-type ferredoxin (DpeB1 or DpeB2), and a GR-type reductase (DpeC1). DpeA1, the �

subunit of the oxygenase component of Dpe, shares very low sequence identities (nomore than 38%) with other reported enzymes and forms a separate branch with otheroxygenase � subunits of reported dioxygenases in NJ, ML, and ME trees constructedbased on the oxygenase � subunit of RHOs, indicating that Dpe is a novel dioxygenase.Although DpeA1 shares some sequence identities with some angular dioxygenases andalso functions at the angular position, the difference is that the angular dioxygenationcatalyzed by Dpe results in the benzene ring cleavage of DE, generating a newmetabolite, 2,4-hexadienal phenyl ester, whereas previously reported angular dioxy-genations led to the cleavage of the CaOO (or -N, -S, or -C) bond, forming two benzenerings.

Previous studies suggested that dioxygenases can catalyze only a one-step reaction,such as dihydroxylation (3, 18, 29, 31), dealkylation (32, 35, 36), or ring cleavage (5, 9,17), and transformation of one molecule of substrate needs one molecule of oxygenand one molecule of NADH. It is interesting that in this study, we found that transfor-mation of one molecule of DE catalyzed by Dpe needs two molecules of oxygen andtwo molecules of NADH. Based on this result and the structures of DE and 2,4-hexadienal phenyl ester, we propose an assumption that the cleavage of DE catalyzedby Dpe is a continuous two-step dioxygenation process: DE is first dioxygenated at C-1aand C-2 to form a hemiacetal-like intermediate with the consumption of one moleculeof oxygen and one molecule of NADH, and then the hemiacetal-like intermediate isfurther dioxygenated, resulting in the cleavage of the C-1aOC-2 bond to form onemolecule of 2,4-hexadienal phenyl ester and two molecules of H2O. In this process, onemolecule of oxygen and one molecule of NADH are needed (Fig. 1C).

MATERIALS AND METHODSStrain and growth conditions. The bacterial strains and plasmids used in this study are listed in

Table 2. S. phenoxybenzoativorans SC_3 was cultured aerobically at 30°C in R2A medium (BD Difco)or minimal salt medium (MSM) (25) containing 0.5 mM DE or 2-carboxy-DE as a carbon source. S.wenxiniae JZ-1 was cultured aerobically in R2A medium at 30°C. The E. coli strains were aerobicallycultured in LB medium (BD Difco) at 37°C. Kanamycin (50 �g/ml), gentamicin (50 �g/ml), ampicillin(100 �g/ml), streptomycin (50 �g/ml), or chloramphenicol (34 �g/ml) was added to the medium asnecessary.

Biotransformation and intermediate identification. Cells of strain SC_3 grown in 100 ml of R2Amedium until the mid-log phase were collected by centrifugation (5,000 � g, 10 min), washed twice withfresh MSM, and resuspended in 10 ml of MSM as the stock culture. Stock culture (1%, vol/vol) was theninoculated into a 100-ml Erlenmeyer flask containing 20 ml of MSM supplemented with eachsubstrate (DE, 2-carboxy-DE, 3-carboxy-DE, 4-carboxy-DE, 4-fluorodiphenyl ether, 4-bromodiphenylether, biphenyl, 2,3-dihydroxybiphenyl, dibenzofuran, dibenzo-p-dioxin, phenol, catechol, and ben-zoic acid) at a final concentration of 0.5 mM. The flask was incubated aerobically at 150 rpm and 30°Con a rotary shaker for 48 h. Experiments were performed in three parallel flasks, and experimentswith negative controls inoculated with sterilized cells were carried out under the same conditions.Bacterial growth was monitored by serial dilution and counting colonies on the plates, and theconcentration of each substrate was determined by HPLC as described in “Analytical method” below.To study the metabolites of DE and 2-carboxy-DE, 2-ml samples were drawn from the MSM cultureat regular intervals, and the metabolites were identified by HPLC-tandem mass spectrometry analysisas described below.

Genome sequencing and ORF analysis. Total DNA was extracted by the phenol chloroformextraction procedure as described by Sambrook and Russell (37). Draft genome sequencing wascompleted by the Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China), 300-bp shotgun

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libraries were constructed, and paired-end sequencing was performed on an Illumina HiSeq 2000. Theraw reads were assembled using the SOAP de novo assembler (version 1.05; http://soap.genomics.org.cn/soapdenovo.html). Gene prediction was performed with the Glimmer 3.02 system. For phylogeneticanalysis of DpeA1, protein sequences were aligned with CLUSTAL_X (38). The phylogenetic tree wasconstructed using MEGA 5.0 software (39). Evolutionary distances were calculated by the Kimuratwo-parameter model (40), and clustering was performed with neighbor-joining (NJ), maximum-likelihood (ML) and minimum evolution (ME) algorithms (41). Confidence values for the branches of thephylogenetic trees were determined by performing bootstrap analyses (based on 1,200 resamplings).

Functional verification of the putative genes responsible for DE degradation. The DNA frag-ments containing each of the three putative genes and their native promoters were amplified from thegenomic DNA of strain SC_3 using the respective primers listed in Table S1 in the supplemental material,digested, and ligated into the corresponding sites of the broad-host-range vector pBBR1MCS-5. Therecombinant plasmids were then transformed into E. coli DH5� competent cells. After verification bysequencing, the recombinant plasmids were introduced into strain JZ-1, which does not degrade DE and2-carboxy-DE, via triparental mating using pRK600 as a helper. The recombinants were studied for theirabilities to degrade DE and 2-carboxy-DE by performing whole-cell biotransformation in MSM asdescribed by Liu et al. (42).

Expression of the putative genes encoding the Dpe components and purification of theproducts. The dpeA1, dpeA2, ferredoxin, and reductase genes were amplified from the genomic DNA ofstrain SC_3 using PrimeSTAR HS DNA polymerase with the primers listed in Table S1. These genes werecloned into pET-29a(�). The recombinant plasmids were then transferred into E. coli BL21(DE3). Geneexpression and purification of the C-terminally His-tagged proteins were performed as described by Fangand Zhou (43). The expression levels of the proteins were examined by SDS-PAGE.

Measurement of Dpe activity. Dpe activity was tested in a 1-ml mixture containing 50 mMphosphate buffer (pH 7.0), the components of Dpe (0.30 �g of DpeA1, 0.10 �g of DpeA2, 0.30 �g of eachferredoxin, and 0.60 �g of each reductase), 1 mM NADH, 0.5 mM Fe2�, and 1 mM Mg2�. In controlsamples, individual Dpe components were added separately. The assay mixtures were preincubated for5 min at 30°C, and reactions were initiated by adding each substrate (DE, 2-carboxy-DE, 3-carboxy-DE,4-carboxy-DE, biphenyl, 2,3-dihydroxybiphenyl, 4-fluorodiphenyl ether, 4-bromodiphenyl ether, diben-zofuran, or carbazole) at a final concentration of 0.5 mM. The mixtures were incubated at 30°C for 60 minand terminated by boiling at 100°C for 3 min. Disappearance of the above-mentioned substrates wasmonitored by HPLC, and the products were identified by HPLC-tandem mass spectrometry and 1H NMRas described below. One unit of enzyme activity was defined as the amount of enzyme required toconvert 1 �mol of substrate per minute.

Stoichiometry determination of NADH and oxygen required for DE cleavage. For stoichiometrydetermination of NADH, the enzyme mixture was added with 0.25 or 0.50 mM DE and 0.10, 0.15, 0.20,

TABLE 2 Strains and plasmids used in this study

Strain or plasmid Characteristic(s) Source

StrainsS. phenoxybenzoativorans SC_3 Degrades DE and 2-carboxy-DE; Smr 20S. phenoxybenzoativorans

SC_3ΔdpeA1SC_3 mutant with dpeA1 gene disrupted This study

S. wenxiniae JZ-1 Unable to degrade 2-carboxy-DE; Smr 46E. coli DH5� Host strain for cloning vector TaKaRaE. coli BL21(DE3) Host strain for expression vector TaKaRaE. coli HB101(pRK600) Conjugation helper strain; Cmr 47

PlasmidspJQ200SK Suicide vector; Gmr 48pJQ-dpeA1 pJQ200SK carrying partial dpeA1 gene This studypBBR1MCS-5 Broad-host-range cloning vector; Gmr 49pBBR0027 pBBR1MCS-5 derivative carrying the dioxygenase gene cluster from contig 0027 This studypBBR0053 pBBR1MCS-5 derivative carrying the dioxygenase gene cluster from contig 0053 This studypBBR0084 pBBR1MCS-5 derivative carrying the dioxygenase gene cluster from contig 0084 This studypBBRdpeA1 pBBR1MCS-5 derivative carrying dpeA1 This studypET-29a(�) Expression vector; Kmr TaKaRapET-dpeA1 pET-29a(�) derivative carrying dpeA1 This studypET-dpeA2 pET-29a(�) derivative carrying dpeA2 This studypET-dpeB1 pET-29a(�) derivative carrying dpeB1 This studypET-dpeB2 pET-29a(�) derivative carrying dpeB2 This studypET-dpeB3 pET-29a(�) derivative carrying dpeB3 This studypET-dpeB4 pET-29a(�) derivative carrying dpeB4 This studypET-dpeC1 pET-29a(�) derivative carrying dpeC1 This studypET-dpeC2 pET-29a(�) derivative carrying dpeC2 This studypET-dpeC3 pET-29a(�) derivative carrying dpeC3 This studypET-dpeC4 pET-29a(�) derivative carrying dpeC4 This studypET-dpeC5 pET-29a(�) derivative carrying dpeC5 This studypET-dpeC6 pET-29a(�) derivative carrying dpeC6 This study

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0.25, 0.50, 1.00, or 2.00 mM NADH. After incubation at 30°C for 120 min, the consumption of DE in themixture was monitored by HPLC. As for oxygen, the concentration of the dissolved oxygen in the mixturewas predetermined by using a dissolved oxygen meter (HACH HQ30D53303000) and then the mixturewas added with enzyme, 1.0 mM NADH, and 0.05, 0.10, 0.15, 0.20, or 0.30 mM DE. The mixture was placedin a closed tube and covered with paraffin oil to isolate air. After incubation at 30°C for 120 min, theconsumption of DE was monitored by HPLC.

Gene disruption and complementation. The gene dpeA1 was disrupted by single-crossover ho-mologous recombination (44). Briefly, the fragments used for homologous recombination were amplifiedfrom the genomic DNA of strain SC_3 using the primers listed in Table S1. These fragments were ligatedinto the suicide plasmid pJQ200SK, resulting in the recombinant plasmid pJQ-dpeA1. Then, pJQ-dpeA1was transferred into strain SC_3 via triparental mating. A mutant with a single recombination event wasscreened based on resistance to streptomycin and gentamicin on R2A agar. The single recombinationevent of the mutant was examined by PCR. The target gene in the mutant was divided into two separateparts by pJQ200SK. To perform gene complementation, pBBR5-dpeA1 was constructed by fusing the PCRproduct of dpeA1, including its native promoter, into HindIII- and XbaI-digested pBBR1MCS-5. Theplasmid was then transformed into the SC_3ΔdpeA1 mutant via triparental mating. The abilities ofSC_3ΔdpeA1 and SC_3ΔdpeA1[pBBR-dpeA1] to utilize DE and 2-carboxy-DE were determined by moni-toring cell growth together with substrate consumption.

RNA preparation and transcription analysis. RT-qPCR was carried out to demonstrate whether thegenes with predicted involvement in DE degradation were induced by DE and 2-carboxy-DE. Strain SC_3was cultured aerobically in 100 ml of MSM supplemented with 0.5 mM glucose, DE, or 2-carboxy-DE. Thecells were harvested at mid-log phase by centrifugation (5,000 � g, 10 min). RNA extraction wasperformed using Tiangen RNAprep Pure RNA extraction kits according to the manufacturer’s instructions.RT-qPCR was performed on a CFX Connect real-time PCR detection system (Bio-Rad, Hercules, CA) in a25-�l reaction volume using iQ SYBR green supermix (Bio-Rad) and the primers described in Table S1.The relative change in gene expression was calculated using the 2�ΔΔCT method (45). The 16S rRNA genewas used as an internal control, and all samples were analyzed in triplicate.

Analytical method. Each sample from culture and enzymatic reaction was mixed with an equalvolume of methanol and vortexed vigorously for 10 min. Each sample was then centrifuged at 15,000 �g at 4°C for 20 min before the supernatant was collected. For HPLC analysis, the mobile phase wasmethanol and ultrapure water with 0.3% phosphoric acid (75:25, vol/vol). The flow rate was 1 ml/min, andthe detection wavelength was 230 nm. The separation column (internal diameter, 4.6 mm; length, 25 cm)was filled with Kromasil 100-5C18. Authentic DE, 2-carboxy-DE, and phenol standards had retention timesof 12.3, 11.4, and 7.2 min, respectively; 3-carboxy-DE, 4-carboxy-DE, benzoic acid, dibenzofuran, dibenzo-p-dioxin, biphenyl, 4-fluorodiphenyl ether, 4-bromodiphenyl ether, and 2,3-diphenyl ether had retentiontimes of 12.8, 13.9, 6.8, 19.6, 20.2, 15.4, 16.2, 17.3, and 13.2 min, respectively. The metabolites producedduring DE and 2-carboxy-DE degradation were identified by electron spray ionization (ESI) tandem massspectrometry (Agilent G6410B triple quadrupole mass spectrometer). The mass spectrometer was operated inthe ESI mode and in the negative-ion mode. The ESI conditions were as follows: a gas temperature of 350°C,a capillary voltage of 4.0 kV, a nebulization pressure of 30.0 lb/in2, and a gas flow rate of 10.0 V/min. Thesecond-order mass spectrometry conditions were as follows: a fragmentor voltage of 90 V and a collisionenergy of 10 to 25 eV. For 1H NMR analysis, the DE and 2-carboxy-DE metabolites were purified using silicagel column chromatography; the mobile phase was ligroin-chloroform-methyl cyanide at a ratio of 20:6:5.Dimethyl sulfoxide (DMSO) was used to dissolve the purified product. 1H spectra were recorded (BrukerAvance III; 500 MHz). The results were analyzed using Bruker Topspin 3.1 software.

Accession number(s). The GenBank accession number of the draft genome sequence of strain SC_3is MINO00000000, the accession number of the DNA fragment containing the dpeA1A2 cluster isKX823577, the accession numbers of fragments containing dpeB1, dpeB2, dpeB3, and dpeB4 areKX823576, KT319225, KX823578, and KX823579, respectively, and the accession numbers of fragmentscontaining dpeC1, dpeC2, dpeC3, dpeC4, dpeC5, and dpeC6 are KT319226, KX823581, KX823582,KX823583, KX823585, and KX823584, respectively.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.00104-17.

SUPPLEMENTAL FILE 1, PDF file, 0.9 MB.

ACKNOWLEDGMENTSThis work was supported by the National Natural Science Foundation of China

(31560033 and 31600087) and the Program for New Century Excellent Talents inUniversity (NCET-13-0861).

REFERENCES1. Yan X, Gu T, Yi ZQ, Huang JW, Liu XM, Zhang J, Xu XH, Xin ZH, Hong Q,

He J, Spain JC, Li SP, Jiang JD. 2016. Comparative genomic analysis ofisoproturon-mineralizing sphingomonads reveals the isoproturon cata-

bolic mechanism. Environ Microbiol 18:4888 – 4906. https://doi.org/10.1111/1462-2920.13413.

2. Chen K, Huang LL, Xu CF, Liu XM, He J, Zinder SH, Li SP, Jiang JD. 2013.

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Molecular characterisation of the enzymes involved in the degradationof a brominated aromatic herbicide. Mol Microbial 89:1121–1139.https://doi.org/10.1111/mmi.12332.

3. Kweon O, Kim SJ, Baek S, Chae JC, Adjei MD, Baek DH, Kim YC, CernigliaCE. 2008. A new classification system for bacterial Rieske non-heme ironaromatic ring-hydroxylating oxygenases. BMC Biochem 9:11. https://doi.org/10.1186/1471-2091-9-11.

4. Liu SH, Su TT, Zhang C, Zhang WM, Zhu DY, Su J, Wei TD, Wang T, HuangY, Guo LM, Xu SJ, Zhou NY, Gu LC. 2015. Crystal structure of PnpCD, atwo-subunit hydroquinone 1,2-dioxygenase, reveals a novel structuralclass of Fe2�-dependent dioxygenases. J Biol Chem 290:24547–24560.https://doi.org/10.1074/jbc.M115.673558.

5. Brennerova MV, Josefiova J, Brenner V, Pieper DH, Junca H. 2009. Met-agenomics reveals diversity and abundance of meta-cleavage pathwaysin microbial communities from soil highly contaminated with jet fuelunder air-sparging bioremediation. Environ Microbiol 11:2216 –2227.https://doi.org/10.1111/j.1462-2920.2009.01943.x.

6. Zhang WM, Zhang JJ, Jiang X, Chao HJ, Zhou NY. 2015. Transcriptionalactivation of multiple operons involved in para-nitrophenol degradationby Pseudomonas sp. strain WBC-3. Appl Environ Microbiol 81:220 –230.https://doi.org/10.1128/AEM.02720-14.

7. Mahiudddin M, Fakhruddin AN, Abdullah AM. 2012. Degradation ofphenol via meta cleavage pathway by Pseudomonas fluorescens PU1.ISRN Microbiol 2012:741820. https://doi.org/10.5402/2012/741820.

8. Vilchez-Vargas R, Junca H, Pieper DH. 2010. Metabolic networks, micro-bial ecology and ‘omics’ technologies: towards understanding in situbiodegradation processes. Environ Microbiol 12:3089 –3104. https://doi.org/10.1111/j.1462-2920.2010.02340.x.

9. Vaillancourt FH, Bolin JT, Eltis LD. 2006. The ins and outs of ring-cleavingdioxygenases. Crit Rev Biochem Mol 41:241–267. https://doi.org/10.1080/10409230600817422.

10. Liu K, Liu TT, Zhou NY. 2013. HbzF catalyzes direct hydrolysis of maleyl-pyruvate in the gentisate pathway of Pseudomonas alcaligenes NCIMB.9867. Appl Environ Microbiol 79:1044 –1047. https://doi.org/10.1128/AEM.02931-12.

11. Nojiri H, Habe H, Omori T. 2001. Bacterial degradation of aromaticcompounds via angular dioxygenation. J Gen Appl Microbiol 47:279 –305. https://doi.org/10.2323/jgam.47.279.

12. Sato SI, Nam JW, Kasuga K, Nojiri H, Yamane H, Omori T. 1997. Identifi-cation and characterization of genes encoding carbazole 1,9a-dioxygenase in Pseudomonas sp. strain CA10. J Bacteriol 179:4850 – 4858.https://doi.org/10.1128/jb.179.15.4850-4858.1997.

13. Masse R, Messier F, Peloquin L, Ayotte C, Sylvestre M. 1984. Microbialbiodegradation of 4-chlorobiphenyl, a model compound of chlorinatedbiphenyls. Appl Environ Microbiol 47:947–951.

14. Bedard DL, Haberl ML, May RJ, Brennan MJ. 1987. Evidence for novelmechanisms of polychlorinated biphenyl metabolism in Alcaligenes eu-trophus H850. Appl Environ Microbiol 53:1103–1112.

15. Rahman F, Langford KH, Scrimshaw MD, Lester JN. 2001. Polybrominateddiphenyl ether (PBDE) flame retardants. Sci Total Environ 275:1–17.https://doi.org/10.1016/S0048-9697(01)00852-X.

16. Kim YM, Nam IH, Murugesan K, Schmidt S, Crowley DE, Chang YS. 2007.Biodegradation of diphenyl ether and transformation of selected bro-minated congeners by Sphingomonas sp. PH-07. Appl Microbiol Biotech-nol 77:187–194. https://doi.org/10.1007/s00253-007-1129-z.

17. Pfeifer F, Truper HG, Klein J, Schacht S. 1993. Degradation of diphenyle-ther by Pseudomonas cepacia Et4: enzymatic release of phenol from2,3-dihydroxydiphenylether. Arch Microbiol 159:323–329. https://doi.org/10.1007/BF00290914.

18. Robrock KR, Mohn WW, Eltis LD, Cohen LA. 2011. Biphenyl and ethyl-benzene dioxygenases of Rhodococcus jostii RHA1 transform PBDEs.Biotechnol Bioeng 108:313–321. https://doi.org/10.1002/bit.22952.

19. Schmidt S, Wittich RM, Erdmann D, Wilkes H, Francke W, Fortnagel P.1992. Biodegradation of diphenyl ether and its monohalogenated de-rivatives by Sphingomonas sp. strain Ss3. Appl Environ Microbiol 58:2744 –2750.

20. Cai S, Shi C, Zhao JD, Cao Q, He J, Chen LW. 2015. Sphingobiumphenoxybenzoativorans sp. nov., a 2-phenoxybenzoic-acid-degradingbacterium. Int J Syst Evol Microbiol 65:1986 –1991. https://doi.org/10.1099/ijs.0.000209.

21. Dehmel U, Engesser KH, Timmis KN, Dwyer DF. 1995. Cloning, nucleotidesequence, and expression of the gene encoding a novel dioxygenaseinvolved in metabolism of carboxydiphenyl ethers in Pseudomonas pseu-

doalcaligenes POB310. Arch Microbiol 163:35– 41. https://doi.org/10.1007/BF00262201.

22. Wittich RM, Schmidt S, Fortnagel P. 1990. Bacterial degradation of 3- and4-carboxybiphenyl ether by Psuedomonas sp. NSS2. FEMS Microbiol Lett67:157–160. https://doi.org/10.1111/j.1574-6968.1990.tb13854.x.

23. Tallur PN, Megadi VB, Ninnekar HZ. 2008. Biodegradation of cyperme-thrin by Micrococcus sp. strain CPN1. Biodegradation 19:77– 82. https://doi.org/10.1007/s10532-007-9116-8.

24. Wang BZ, Guo P, Hang BJ, Li L, He J, Li SP. 2009. Cloning of a novelpyrethroid-hydrolyzing carboylesterase gene from Sphingobium sp. JZ-1and characterization of the gene product. Appl Environ Microbiol 75:5496 –5500. https://doi.org/10.1128/AEM.01298-09.

25. Wang CH, Chen Q, Wang R, Shi C, Yan X, He J, Hong Q, Li SP. 2014. Anovel angular dioxygenase gene cluster encoding 3-phenoxybenzoate1=,2=-dioxygenase in Sphingobium wenxiniae JZ-1. Appl Environ Micro-biol 80:3811–3818. https://doi.org/10.1128/AEM.00208-14.

26. Armengaud J, Happe B, Timmis K. 1998. Genetic analysis of dioxindioxygenase of Sphingomonas sp. strain RW1: catabolic genes dispersedon the genome. J Bacteriol 180:3954 –3966.

27. Hartmann EM, Armengaud J. 2014. Shotgun proteomics suggests in-volvement of additional enzymes in dioxin degradation by Sphingomo-nas wittichii RW1. Environ Microbiol 16:162–176. https://doi.org/10.1111/1462-2920.12264.

28. Kasuga K, Nitta A, Kobayashi M, Habe H, Nojiri H, Yamane H, Omori T,Kojima I. 2013. Cloning of dfdA genes from Terrabacter sp. strain DBF63encoding dibenzofuran 4,4a-dioxygenase and heterologous expressionin Streptomyces lividans. Appl Microbiol Biotechnol 97:4485– 4498.https://doi.org/10.1007/s00253-012-4565-3.

29. Taira K, Hirose J, Hayashida S, Furukawa K. 1992. Analysis of bph operonfrom the polychlorinated biphenyl-degrading strain of Pseudomonaspseudoalcaligenes KF707. J Biol Chem 267:4844 – 4853.

30. Diaz E, Ferrandez A, Garcia JL. 1998. Characterization of the hca clusterencoding the dioxygenolytic pathway for initial catabolism of3-phenylpropionic acid in Escherichia coli K-12. J Bacteriol 180:2915–2923.

31. Gai ZH, Wang XY, Liu XR, Tai C, Tang HZ, He XF, Wu G, Deng ZX, Xu P.2010. The genes coding for the conversion of carbazole to catechol areflanked by IS6100 elements in Sphingomonas sp. strain XLDN-2-5. PLoSOne 5:e10018. https://doi.org/10.1371/journal.pone.0010018.

32. Chen Q, Wang CH, Deng SK, Wu YD, Li Y, Yao L, Jiang JD, Yan X, He J, LiSP. 2014. A novel three-component Rieske non-heme iron oxygenase(RHO) system catalyzing the N-dealkylation of chloroacetanilide herbi-cides in sphingomonads DC-6 and DC-2. Appl Environ Microbiol 80:5078 –5085. https://doi.org/10.1128/AEM.00659-14.

33. Mondello FJ. 1989. Cloning and expression in Escherichia coli of Pseu-domonas strain LB400 genes encoding polychlorinated biphenyl degra-dation. J Bacteriol 171:1725–1732. https://doi.org/10.1128/jb.171.3.1725-1732.1989.

34. Erickson BD, Mondello FJ. 1992. Nucleotide sequencing and transcrip-tional mapping of the genes encoding biphenyl dioxygenase, a multi-component polychlorinated-biphenyl-degrading enzyme in Pseudomo-nas strain LB400. J Bacteriol 174:2903–2912. https://doi.org/10.1128/jb.174.9.2903-2912.1992.

35. Herman PL, Behrens M, Chakraborty S, Chrastil BM, Barycki J, Weeks DP.2005. A three-component dicamba O-demethylase from Pseudomonasmaltophilia, strain DI-6: gene isolation, characterization, and heterolo-gous expression. J Biol Chem 280:24759 –24767. https://doi.org/10.1074/jbc.M500597200.

36. Gu T, Zhou CY, Sørensen SR, Zhang J, He J, Yu PW, Yan X, Li SP. 2013. Thenovel bacterial N-demethylase PdmAB is responsible for the initial stepof N,N-dimethyl-substituted phenylurea herbicides degradation. ApplEnviron Microbiol 79:7846 –7856. https://doi.org/10.1128/AEM.02478-13.

37. Sambrook J, Russell D. 2001. Molecular cloning: a laboratory manual, 3rded. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

38. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997.The clustal-x window interface: flexible strategies for multiple sequencealignment aided by quality analysis tools. Nucleic Acids Res 25:4876 – 4882. https://doi.org/10.1093/nar/25.24.4876.

39. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011.MEGA5: molecular evolutionary genetics analysis using maximum like-lihood, evolutionary distance, and maximum parsimony methods. MolBiol Evol 28:2731–2739. https://doi.org/10.1093/molbev/msr121.

40. Kimura M. 1980. A simple method for estimating evolutionary rate of

Novel Dioxygenase Catalyzing DE Ring Cleavage Applied and Environmental Microbiology

May 2017 Volume 83 Issue 10 e00104-17 aem.asm.org 15

on April 22, 2021 by guest

http://aem.asm

.org/D

ownloaded from

base substitutions through comparative studies of nucleotide se-quences. J Mol Evol 16:111–120. https://doi.org/10.1007/BF01731581.

41. Saitou N, Nei M. 1987. The neighbor-joining method: a new method forreconstructing phylogenetic trees. Mol Biol Evol 4:406 – 425.

42. Liu H, Wang SJ, Zhang JJ, Dai H, Tang H, Zhou NY. 2011. Patchworkassembly of nag-like nitroarene dioxygenase genes and the3-chlorocatechol degradation cluster for evolution of the2-chloronitrobenzene catabolism pathway in Pseudomonas stutzeriZWLR2-1. Appl Environ Microbiol 77:4547– 4552. https://doi.org/10.1128/AEM.02543-10.

43. Fang T, Zhou NY. 2014. Purification and characterization of salicylatehydroxylase, a three-component monooxygenase from Ralstonia sp.strain U2. Appl Microbiol Biotechnol 98:671– 679. https://doi.org/10.1007/s00253-013-4914-x.

44. Leloup L, Ehrlich SD, Zagorec M, Deville FM. 1997. Single-crossoverintegration in the Lactobacillus sake chromosome and insertionalinactivation of the ptsI and lacL genes. Appl Environ Microbiol 63:2117–2123.

45. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression datausing real-time quantitative PCR and the 2�ΔΔCT method. Methods25:402– 408. https://doi.org/10.1006/meth.2001.1262.

46. Wang BZ, Ma Y, Zhou WY, Zheng JW, Zhu JC, He J, Li SP. 2011.Biodegradation of synthetic pyrethroids by Ochrobactrum tritici strainpyd-1. World J Microb Biotechnol 10:2315–2324.

47. Bible AN, Stephens BB, Ortega DR, Xie Z, Alexandre G. 2008. Functionof a chemotaxis-like signal transduction pathway in modulating mo-tility, cell clumping, and cell length in the alphaproteobacteriumAzospirillum brasilense. J Bacteriol 190:6365– 6375. https://doi.org/10.1128/JB.00734-08.

48. Quandt J, Hynes MF. 1993. Versatile suicide vectors which allow directselection for gene replacement in Gram-negative bacteria. Gene 127:15–21. https://doi.org/10.1016/0378-1119(93)90611-6.

49. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, Peter-son KM. 1995. Four new derivatives of the broad-host-range cloningvector pBBR1MCS, carrying different antibiotic-resistance cassettes.Gene 166:175–176. https://doi.org/10.1016/0378-1119(95)00584-1.

Cai et al. Applied and Environmental Microbiology

May 2017 Volume 83 Issue 10 e00104-17 aem.asm.org 16

on April 22, 2021 by guest

http://aem.asm

.org/D

ownloaded from