Molecular characterization of the enzymes ... - Wiley Online Library

Molecular Microbiology (2013) 89(6), 1121–1139 ■

doi:10.1111/mmi.12332 First published online 31 July 2013

Molecular characterization of the enzymes involved in the degradation of a brominated aromatic herbicide Kai Chen,1 Linglong Huang,1 Changfeng Xu,1 Xiaomei Liu,1 Jian He,1 Stephen H. Zinder,2 Shunpeng Li1 and Jiandong Jiang1* 1 Department of Microbiology, Key Lab of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, 210095, Nanjing, China. 2 Department of Microbiology, Cornell University, 272 Wing Hall, Ithaca, NY 14853-5701, USA.

Summary Dehalogenation is the key step in the degradation of halogenated aromatics, while reductive dehalogenation is originally thought to rarely occur in aerobes. In this study, an aerobic strain of Comamonas sp. 7D-2 was shown to degrade the brominated aromatic herbicide bromoxynil completely and release two equivalents of bromides under aerobic conditions. The enzymes involved in the degradation of bromoxynil to 4-carboxy-2-hydroxymuconate-6-semialdehyde, including nitrilase, reductive dehalogenase (BhbA), 4-hydroxybenzoate 3-monooxygenase and protocatechuate 4,5-dioxygenase, were molecularly characterized. The novel dehalogenase BhbA was shown to be a complex of a respiration-linked reductive dehalogenase (RdhA) domain and a NAD(P)H-dependent oxidoreductase domain and to have key features of anaerobic respiratory RdhAs, including two predicted binding motifs for [4Fe-4S] clusters and a close association with a hydrophobic membrane protein (BhbB). BhbB was confirmed to anchor BhbA to the membrane. BhbA was partially purified and found to use NAD(P)H as electron donors. Full-length bhbA homologues were found almost exclusively in marine aerobic proteobacteria, suggesting that reductive dehalogenation occurs extensively in aerobes and that bhbA is horizontally transferred from marine microorganisms. The discovery of a functional reductive dehalogenase and ring-cleavage oxygenases in an aerobe opens up possibilities for basic research as well as the potential application for bioremediation. Accepted 13 July, 2013. *For correspondence. E-mail jiang_jjd@ njau.edu.cn; Tel. (+86) 2584396348; Fax (+86) 2584396314.

© 2013 John Wiley & Sons Ltd

Introduction A large variety of anthropogenic halogenated aromatics have been widely used in industry, agriculture and private households as solvents, degreasing agents and pesticides. Moreover, it has been estimated that hundreds of halogenated aromatics are produced by living organisms or are formed during natural abiogenic processes (Gribble, 2003; 2010). The anthropogenic release and natural production of halogenated aromatics are likely the driving force for the evolution of the microbial capacity to dehalogenate diverse halogenated aromatics, promoting the complex biogeochemical halogen cycle (Adriaens et al., 2003). Moreover, the microbial dehalogenation of halogenated aromatics, by reducing both the recalcitrance to aerobic biodegradation and the risk of forming toxic intermediates during subsequent metabolic steps, has great potential for use in the bioremediation of halogenated aromatic-contaminated sites (Janssen et al., 2001). The investigation of the microbial degradation of halogenated aromatics has led to the detection and elucidation of various dehalogenation mechanisms: reductive, thiolytic, oxidative and hydrolytic (Wang et al., 2010). Reductive dehalogenation is defined as the removal of a halogen substituent from a molecule with the concomitant addition of two electrons, a thermodynamically favourable reaction (Smidt and de Vos, 2004). In anaerobes, halogenated aromatics are reductively dehalogenated as the terminal electron acceptors, which are coupled to energy conservation via the electron transport phosphorylation in a process termed organohalide respiration. It has to be mentioned that the dehalogenated products of respirationlinked reductive dehalogenases (RdhAs) are usually not further converted by anaerobes (Cupples et al., 2005). In contrast to anaerobes, aerobes rarely support reductive dehalogenation, with only a few notable exceptions (de Jong and Dijkstra, 2003; McTamney and Rokita, 2009). In the aerobic strain of Flavobacterium sp. ATCC 39723 (now reclassified as Sphingobium chlorophenolicum), a glutathione S-transferase that reductively dehalogenates tetrachloro-p-hydroquinone (TeCH) to 2,6-dichloro-phydroquinone in the presence of reduced glutathione (GSH) under anaerobic conditions, has been purified (Xun et al., 1992). Another GSH-dependent glutathione S-transferase, LinD, which reductively dehalogenates

1122 K. Chen et al. ■

A Bxn2 H2O

NH4+

Bromoxynil

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NADPH, H+, O2

NAD(P)H NAD(P)+, Br-

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B bhbE2 bhbD2 bhbF2

tra1

bxn2

orf29 tetR pnbR orf26 orf25

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bhbC tra3 tra4

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Fig. 1. (A) The predicted bromoxynil degradation pathway by Comamonas sp. strain 7D-2; (B) organization of the bromoxynil-degrading gene clusters (2430 bp and 18 374 bp) on the plasmid pBHB. The arrows indicate the size and direction of transcription of each gene or orf. bhbD2E2 and bhbDE, PCA-4,5-dioxygenase alpha and beta subunit genes; bhbF2 and bhbF, HB-3-monooxygenase gene; tra, transposase gene; bxn2, bromoxynil-specific nitrilase gene; orf29, putative transphosphatidylase gene; pnbR, putative regulator gene; orf26, hypothetical MnbA gene; orf25, putative dihydrolipoamide dehydrogenase gene; bhbA, reductive dehalogenase gene; bhbB, extracytoplasmic binding receptor gene; bhbC, putative OmpC family outer membrane porin gene. Abbreviations: DBHB, 3,5-dibromo-4-hydroxybenzoate; BHB, 3-bromo-4-hydroxybenzoate; HB, 4-hydroxybenzoate; PCA, protocatechuate; CHMS, 4-carboxy-2-hydroxymuconate-6-semialdehyde and TCA, tricarboxylic acid.

2,5-dichlorohydroquinone (2,5-DCHQ) to chlorohydroquinone and hydroquinone, has also been characterized in Sphingomonas paucimobilis UT26 (Miyauchi et al., 1998). In Corynebacterium sepedonicum KZ-4 and coryneform bacterium strain NTB-1, a NADPH-dependent reductive ortho dehalogenation of 2,4-dichlorobenzoyl-coenzyme A to 4-chlorobenzoyl-coenzyme A has been reported (Romanov and Hausinger, 1996). In these two strains, Mg·ATP and coenzyme A (CoA) are required to stimulate the reductive dehalogenation reaction. In addition, in the facultative aerobe Rhodopseudomonas palustris RCB100, 3-chlorobenzoyl-CoA has been reported to be reductively dehalogenated to benzoyl-CoA under anaerobic conditions (Egland et al., 2001). In this type of reaction, CoA is also required to initiate the reductive dehalogenation, and 3-chlorobenzoate is used as the carbon source rather than as the terminal electron acceptor (Egland et al., 2001; Kuntze et al., 2011). Recently, Burkholderia sp. strain SJ98 (DSM 23195) has been shown to reductively dehalogenate 2-chloro-4-nitrophenol to p-nitrophenol under aerobic conditions (Pandey et al., 2011). To date, glutathione S-transferase family dehalogenase is the only reported reductive dehalogenase (RDase) in aerobes. In general, very little is known regarding dehalogenases involved in reductive dehalogenation in aerobes. Bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), a halogenated aromatic herbicide, is widely used for the postemergence control of annual broad-leaved weeds in cereals, maize, sorghum, flax, allium species, mint and grass seed crops (Roberts et al., 1998). The extensive use of bromoxynil has also caused environmental pollution and ecosystem damage (Miller et al., 1995; Semchuk et al., 2003). Bromoxynil is classified as a group C pos-

sible human carcinogen and is considered developmentally toxic by the US Environmental Protection Agency (Cupples et al., 2005); therefore, the dissipation of bromoxynil in the environment requires further study. Although the isolation of microorganisms capable of degrading bromoxynil (McBride et al., 1986; Vesela et al., 2010; Cai et al., 2011), the identification of bromoxynil metabolites (Topp et al., 1992; Rosenbrock et al., 2004), the cloning of the bromoxynil-specific nitrilase (Stalker and McBride, 1987; Stalker et al., 1988) and the reductive debromination of bromoxynil to 4-hydroxybenzonitrile by the anaerobe Desulfitobacterium chlororespirans (Cupples et al., 2005) have been reported, the complete degradation pathway of bromoxynil has not yet been determined, and the enzymes involved in the degradation, particularly in the dehalogenation, have not been characterized. In this study, a strictly aerobic strain of Comamonas sp. 7D-2, which was capable of utilizing bromoxynil as the sole source of carbon and nitrogen for growth, was isolated, and the complete degradation pathway of bromoxynil, including the successively reductive debromination steps (Fig. 1A), was elucidated based on the identification of metabolites and the molecular characterization of the degradation gene clusters (Fig. 1B). The presence and activity of enzymes involved in bromoxynil degradation including nitrilase, RDase, 4-hydroxybenzoate 3-monooxygenase (HB-3-monooxygenase) and protocatechuate 4,5dioxygenase (PCA-4,5-dioxygenase) were demonstrated. The major objectives of this work were to characterize the novel RDase used for the debromination of bromoxynil derivatives in the examined aerobic strain and to investigate the origin of this RDase.

© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 89, 1121–1139

Aerobic dehalogenation of brominated aromatic herbicide 1123 0.16

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Fig. 2. Degradation of bromoxynil (◆) and the cell growth of strain 7D-2 (•) with the release of bromide ions (▲) when bromoxynil was used as the sole source of carbon and nitrogen (in MM without NH4NO3 and NaCl) under aerobic conditions. The data are represented as the mean ± standard deviation for triplicate incubations. When the error bar is not visible it is within the data point.

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Results Growth of Comamonas sp. strain 7D-2 on bromoxynil Strain 7D-2 was capable of utilizing bromoxynil as the sole source of carbon and nitrogen for growth under aerobic conditions (Fig. 2). Strain 7D-2 completely degraded 0.3 mM bromoxynil within 28 h with the cellular OD600 increasing from 0.05 to 0.135. It was found that 0.56 mM (near 0.6 mM) bromide was released when 0.3 mM bromoxynil was degraded, showing that strain 7D-2 could remove both bromines from bromoxynil. Supplementation with vitamin B12, CoA or GST in minimal salts medium (MM) did not increase the dehalogenation rate (data not shown). Strain 7D-2 was shown to be an obligate aerobe that cannot grow with bromoxynil or 3,5-dibromo-4hydroxybenzoate (DBHB) as an electron acceptor under anaerobic conditions even when acetate, formate, pyruvate, succinate, glucose or Luria–Bertani (LB) broth was used as the electron donor. Although strain 7D-2 cells did not grow under anaerobic conditions, approximately 7.8% of 0.3 mM DBHB was dehalogenated within 72 h under such conditions (< 10% of that observed under aerobic conditions).

quencing. BLASTp analysis of the predicted open reading frames (orfs) against the NCBI database revealed a potential bromoxynil-degrading gene cluster (denoted bhb) (Fig. 1B). Gene bxn2 encodes a nitrilase The nitrilase encoded by bxn2, when cloned and expressed in Escherichia coli DH5α (pUC-bxn2), efficiently hydrolysed bromoxynil to an equivalent amount of DBHB (see Supplementary Fig. S1), as confirmed by comparison with the standard DBHB compound and by high-performance liquid chromatography (HPLC)-mass spectrometry (MS) identification (Fig. 3A, C and D). Due to its utilization by E. coli cells, only a small fraction (< 14 μM) of the ammonia released by nitrile hydrolysis was detected in the growth medium (Fig. S1). The amino acid sequence of Bxn2 is 99% identical to the nitrilase of K. ozaenae (Stalker and McBride, 1987; Stalker et al., 1988) (Accession No. P10045) and contains a Glu–Lys– Cys catalytic triad. Bxn2 is also bromoxynil-specific, preferring aromatic nitrile containing two meta-positioned halogen atoms, and cannot hydrolyse the nitrile group in chlorothalonil or 4-hydroxybenzonitrile (data not shown). BhbA is a novel iron-sulphur cluster-containing RDase

Cloning of the bromoxynil-degrading gene clusters Strain 7D-2 was found to harbour a large plasmid, which was designated pBHB. A plasmid-cured mutant, Comamonas sp. strain 2B, was obtained through subculturing in medium lacking bromoxynil. Strain 2B lost the bromoxynildegrading ability and the bromoxynil nitrilase gene, which could be amplified from strain 7D-2 with the primers designed according to the sequence of the bromoxynilspecific nitrilase gene (bxn) from Klebsiella ozaenae (Stalker and McBride, 1987; Stalker et al., 1988), indicating that bromoxynil-degrading gene(s) are located on pBHB. The 119 kb pBHB was sequenced by 454 pyrose-

No dehalogenase activity was detected in the plasmid-cured strain 2B carrying the empty vector. However, resting cells of strain 2B carrying pMCS2-7k completely dehalogenated 0.2 mM DBHB to 4-hydroxybenzoate (HB) via 3-bromo-4-hydroxybenzoate (BHB) within 16 h in phosphate-buffered saline (PBS) containing 10 mM sodium succinate. The metabolites BHB and HB were also identified by comparisons with the standard compounds and by HPLC-MS (Fig. 3B, E and F). Because hydrogen, not hydroxyl, was added to the aromatic ring during dehalogenation, the dehalogenation reaction was defined as a reductive dehalogenation.



A DBHB Bromoxynil

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Br

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D

CN

COOH

Br

Br

Br

OH

OH

Bromoxynil

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COOH

COOH

Br OH

OH

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HB

Fig. 3. Identification of the upper pathway metabolites during bromoxynil degradation by HPLC-MS. A. HPLC profile of DBHB [Rt (min) = 6.3] produced from bromoxynil [Rt (min) = 12.3] hydrolysis by E. coli DH5α (pUC-bxn2). B. The HPLC profiles of BHB [Rt (min) = 4.1] and HB [Rt (min) = 2.7] produced from the reductive dehalogenation of DBHB [Rt (min) = 6.3] by Comamonas sp. strain 2B carrying pMCS2-7k. C–F. The mass spectra showed the prominent protonated molecular ions at m/z = 275.7 [M-H]− (C), m/z = 294.8 [M-H]− (D), m/z = 215.7 [M-H]− (average) (E) and m/z = 136.9 [M-H]− (F), enabling the assignment of the molecular ions [M−] at m/z = 277, m/z = 296, m/z = 217 and m/z = 138; therefore, the compounds were identified as bromoxynil, DBHB, BHB and HB respectively.

To clarify the specific gene(s) performing the reductive dehalogenation, the dehalogenation activities of strain 2B carrying different genes of the bhbABC cluster were analysed (Fig. 4A). No dehalogenation activity was detected when only bhbB or bhbC was expressed (data not shown), while the expression of bhbA yielded dehalogenation activity (Fig. 4A). From these results, we concluded that bhbA is the RDase gene. The deduced BhbA protein sequence contains 1071 amino acids and has a calculated molecular mass of

117.9 kDa, which is approximately twice as large as previously reported respiratory RdhAs (usually 40–65 kDa). Based on the domain search results, BhbA is a complex of an N-terminal respiratory RdhA domain and a C-terminal NAD(P)H-dependent oxidoreductase domain. The N-terminal of BhbA showed considerable homology to respiratory RdhAs (32–46%) rather than other Fe–S cluster-containing modules. Interestingly, BhbA possesses some typical features of the respiratory RdhAs that are primarily found in strict anaerobes (Smidt and de


Aerobic dehalogenation of brominated aromatic herbicide 1125

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Fig. 4. A. The identification of the key regions involved in reductive dehalogenation. A schematic of DNA regions fused with the same promoter sequence is shown to the left. The relative dehalogenation activities of the resting cells of strain 2B harbouring each complementary plasmid with DBHB as the substrate are shown to the right. B. The dehalogenase activities in cytoplasmic and membrane fractions of strain 2B-bhbAB and strain 2B-bhbA respectively. White column, membrane fraction; black column, cytoplasmic fraction. B, BamHI; X, XbaI.

Vos, 2004), such as the C1–C5 conserved regions, two conserved binding motifs for Fe–S clusters and a close association with a hydrophobic membrane protein (Figs 5 and 6). The consensus sequences of the binding motifs (CXXCXXCXXXCP) for two 4Fe-4S clusters or for one 4Fe-4S cluster and one 3Fe-4S cluster were identified at amino acids 558–568 and 604–615 (Fig. 5). However, the complete twin arginine (TAT) signal peptide (RRXFXK) and the predicted vitamin B12-binding motif DXHXXG . . . SXL . . . GG (Ludwig and Matthews, 1997; Hölscher et al., 2004) were not identified. The entire BhbA amino acid sequence showed 49–91% similarities with putative RDases and oxidoreductases (NAD-binding/iron-sulphur cluster-binding protein) from Hydrogenophaga and a variety of marine proteobacteria (Fig. 7). However, these annotated RDases and oxidoreductases were all deduced from genome sequencing (Gordon and Betty Moore Foundation Marine Microbial Genome Sequencing Project (http://camera.calit2.net/ microgenome/); Lee et al., 2007; Moran et al., 2007; Kang et al., 2011), and none of these proteins have been functionally confirmed. To the best of our knowledge, our current study is the first to confirm the reductive dehalogenation function of this type of RDase. BhbB anchors BhbA to the membrane The gene bhbB is located 40 bp upstream of bhbA and in the same orientation as bhbA. Each of the start codons of bhbA and bhbB is preceded by a putative ribosome binding site. In addition, no termination sites were identified in the region between bhbA and bhbB, suggesting they are co-transcribed. BhbB (337 aa) has a calculated molecular mass of 35.7 kDa. BhbB shows 41–82% similarities to certain hypothetical extracytoplasmic binding receptors and contains a conserved tripartite tricarboxylate trans-

porter family receptor (TctC) domain. TMpred and TMHMM analysis showed that BhbB encodes a highly hydrophobic protein with two strong transmembrane helices (Fig. 6B). Although the complementary expression of bhbA alone resulted in dehalogenation activity in resting cells, its activity was only approximately 10% that of bhbAB (Fig. 4A). Because the same promoter sequence (in the Q2 fragment) was used in all recombinant plasmids, we concluded that BhbB significantly increased the dehalogenation activity. In a 12% polyacrylamide sodium dodecyl sulphate (SDS)-gel electrophoresis analysis of the membrane fraction from strain 2B-bhbAB (expressing bhbAB), an extra 35 kDa band besides the 117 kDa BhbA band was observed compared with that of strain 2B (Supplementary Fig. S2). The extra protein was identified as BhbB using matrix-assisted laser desorption ionization-time of flight/ time of flight (MALDI-TOF/TOF) MS (with 66% coverage), showing that BhbB was membrane located. To investigate the interaction between BhbB and BhbA, the dehalogenase activities in the cytoplasmic and membrane fractions from strain 2B-bhbAB and strain 2B-bhbA (only expressing bhbA) were analysed respectively (Fig. 4B). It was found that 275.4 U of dehalogenase activity (91.1% of total activity) was solubilized by the detergent Triton X-100 from the membrane fraction of strain 2B-bhbAB. While in strain 2B-bhbA, only 37.9 U of dehalogenase activity was found in the membrane fraction, which is only approximately 13.8% of that in strain 2B-bhbAB (Fig. 4B). These results confirmed that the function of BhbB is to anchor BhbA to the membrane. BhbC contains 211 aa and displays similarities (34– 80%) to certain OmpC family outer membrane porins, which typically allow certain solutes to cross the outer membrane. However, BhbC expression did not exhibit any significant effect on BhbA activity in our resting cell and extract assays (Fig. 4A).



Fig. 5. Primary sequence alignment of the N-terminal region of BhbA and representative respiratory RdhAs. The conserved sequence motifs (C1–C5, two Fe-S and RR) are indicated. The conserved cysteine residues for the binding of two Fe-S clusters are marked by five-pointed stars. The first cysteine in the second binding motif is replaced by glycine or located 12 amino acids upstream. 7D_BhbA, Comamonas sp. 7D-2 N-terminal of BhbA (AFV28964); DKBC1_PrdA, Desulfitobacterium sp. KBC1 PCE-RDase (BAE45338); DY51_PceA, Desulfitobacterium Y51 PCE-RDase; Dr_PceA, Dehalobacter restrictus PCE-RDase (CAD28790); Dm_TceA, Dehalococcoides mccartyi TCE-RDase (AAN85588); Dhaf_CprA5, Desulfitobacterium hafniense strain PCP-1 3,5-DCP-RDase (AAQ54585); Ddehal_CprA, Desulfitobacterium dehalogenans o-CP-RDase (AAD44542); Dchloro_CprA, Desulfitobacterium chlororespirans o-CP-RDase (AAL84925). RDase, reductive dehalogenase; PCE, tetrachloroethene; TCE, trichloroethene; DCP, dichlorophenol and CP, chlorophenol.

The NAD(P)H-dependent oxidoreductase domain is necessary for reductive dehalogenation The NAD(P)H-dependent oxidoreductase domain of BhbA also contains a ferredoxin reductase-like NAD(H)binding domain and an iron-binding domain. Deletion of any of the region led to the complete loss of the RDase activity (Fig. 4A). From the results, we concluded that the iron-binding motif, NAD-binding motif and flavin-binding motif in the NAD(P)H-dependent oxidoreductase domain were also essential for reductive dehalogenation, and they were likely to play key roles in electron transfer from NAD(P)H to the halogenated aromatics. Partial purification of BhbA The dehalogenase from strain 2B-bhbAB was enriched from the membrane fraction using Mono Q chromatography (elution at 200 mM NaCl, pH 8.0). The purification

results are shown in Table 1. A 35.8-fold enrichment with a yield of 12% was achieved. From 6 g of wet cell mass, 0.42 mg of protein with a specific activity of near 1640 U mg−1 was obtained. An 8% polyacrylamide SDSgel electrophoresis analysis revealed the enrichment of an approximately 117 kDa band that was also observed in strain 2B-bhbAB but not in strain 2B (Fig. 8A). The size of the band was in agreement with the molecular mass deduced from the amino acid sequence of BhbA. The enriched band was excised and analysed by MALDI-TOF/TOF MS. MASCOT V2.1 MS/MS data searches identified 19 peptides that matched the predicted BhbA sequence (with 22% coverage). The results confirmed that the purified band was BhbA. The peptide DRPVHLGSFPSER (maximal ion score 48), which is located at residues 8–20 of BhbA sequence, was identified, showing that BhbA translation began from the first ATG codon and verified that the two arginines at



A

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DKBC1_PrdAB (463 aa, 103 aa)

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SCH4B_RDH (1070 aa) HPBC _RDBA (339 aa, 1071 aa) 7D_BhbBA (337 aa, 1071 aa)

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Fig. 6. Arrangements of the gene clusters for RDases and their corresponding anchoring proteins from strain 7D-2 and representative organohalide respiring microorganisms. A. The conserved RDase features, including the RR signal peptide RRXFXK (blue) and two 4Fe-4S clusters (black) and the association with a hydrophobic membrane protein, are indicated. The amino acid numbers of each RDase and anchoring protein are shown in the parentheses. The flavin-binding motif (green), NAD-binding motif (light blue) and the iron-binding motif (purple) in the NAD(P)H-dependent oxidoreductase domain of BhbA are also highlighted. DKBC1_PrdAB, Desulfitobacterium sp. KBC1 PCE-RDase and putative membrane-bound protein (BAE45338 and BAE45339); DY51_PceAB, Desulfitobacterium Y51 PCE-RDase and putative anchor protein; Dr_PceAB, Dehalobacter restrictus PCE-RDase and putative anchor protein (CAD28790 and CAD28791); Dm_TceAB, Dehalococcoides mccartyi TCE-RDase and putative anchor protein (AAN85588 and AAN85589); Dhaf_CprA5B5, Desulfitobacterium hafniense strain PCP-1 3,5-DCP-RDase and putative membrane anchor protein (AAQ54585 and AAQ54586); Ddehal_CprBA, Desulfitobacterium dehalogenans putative membrane-bound protein and o-CP-RDase (AAD44541 and AAD44542); Dchloro_CprBA, Desulfitobacterium chlororespirans putative membrane docking protein and o-CP-RDase (AAL84924 and AAL84925); HPBC _RDBA, Hydrogenophaga sp. PBC putative extracytoplasmic binding receptor and putative RDase (ZP_10155045 and ZP_10155044); SCH4B_RDase, Silicibacter sp. TrichCH4B putative RDase (ZP_05739592); 7D_BhbBA, Comamonas sp. 7D-2 membrane anchor protein and RDase (AFV2896 and AFV28964). B. The prediction of the transmembrane helices of the amino acid sequence of BhbB by the software TMpred. Two helices (20–38 aa and 105–121 aa) with scores greater than 500 were detected.

residues 22–23 were not part of a twin arginine transport signal. To unambiguously confirm that the enriched 117 kDa band is indeed the enzyme catalysing the reductive dehalogenation reaction, an approach that involves native PAGE followed by RDase activity assays with gel slices and subsequent identification of proteins using MALDITOF/TOF MS was performed. Strong RDase activity towards DBHB was found to be mostly constrained in a gel segment corresponding to the position of slice ID 4 in a parallel Coomassie-stained lane (Fig. 8B). MALDI-TOF/ TOF MS analysis of the slice showed that a total of six peptides matched that of BhbA, resulting in a sequence coverage of 7% for the protein (Supplementary Table S1). Although a non-RDase protein (RNA polymerase) was also detected in the gel slice, BhbA showed the highest number of peptide hits (six hits). These data confirmed that BhbA is indeed the RDase catalysing the dehalogenation reaction. Weak RDase activities were detected in the segments

corresponding to positions of slice ID 2 and 3 (Fig. 8B), although BhbA peptides were not detected. Notably, BhbB peptides were also detected in gel slices with strong or weak RDase activities (Fig. 8B, slice ID 2, 3 and 4; Table S1), indicating the interaction between BhbA and BhbB. BhbA characteristics No reductive dehalogenation activity was detected when purified BhbA was used in the absence of any cofactors (Table 2). However, activity was detected in the membrane fraction, indicating that an unknown component in the preparation was serving as an electron donor. The cofactors NADPH and NADH (0.2 mM) greatly increased the activity of the purified BhbA. NAD(P)H has been proven to enhance reductive dehalogenations (Tsuchiya and Yamaha, 1984; Vollmer et al., 1993; Romanov and Hausinger, 1996). Thus, we concluded that NAD(P)H


1128 K. Chen et al. ■ 100 100

*Phaeobacter gallaeciensis DSM 17395 (AFO92526) *Phaeobacter gallaeciensis 2.10 (AFO88639)

60

*Nautella italica R11 (EEB70068)

95

*Phaeobacter arcticus MED193 (EAQ46899)

100

*Leisingera aquimarina SK209-2-6 (EBA18313) *Ruegeria sp. TM1040 (ABF62547)

92

100

*Ruegeria mobilis TrichCH4B (EEW60663) *Roseobacter denitrificans OCh 114 (ABG33540) 100

*Roseobacter litoralis Och 149 (AEI92404) *Planktotalea frisia HTCC2083 (EDZ42005) *Ruegeria sp. TW15 (ZP_08861284)

100

*Ruegeria sp. KLH11 (EEE36585)

54

100

*Silicibacter lacuscaerulensis ITI-1157 (EEX08396) 53

*Ruegeria pomeroyi DSS-3 (AAV93904) *Roseibium sp. TrichSKD4 (EFO34197) *Jannaschia sp. CCS1 (ABD56891)

63

*Ahrensia sp. R2A130 (EFL88602) *Thalassiobium sp. R2A62 (EET46325) *Sulfitobacter litoralis GAI101 (EEB83384)

93 100

*Oceanibulbus indolifex HEL-45 (EDQ05638) *Rhodobacterales bacterium HTCC2255 (EAU52318)

99

*Sulfitobacter sp. EE-36 (EAP83333) 100

*Sulfitobacter sp. NAS-14.1 (EAP79417)

*Phaeobacter daeponensis Y4I (EDZ48635)

100

*Rhodobacteraceae bacterium HTCC2150 (EBA05478)

100 95

*Loktanella maricola CCS2 (EBA12491) *Nitratireductor pacificus pht-3B (EKF20174) *Pseudovibrio sp. FO-BEG1 (AEV39478)

99 100 100 100

*Pseudovibrio sp. JE062 (EEA92265) Hydrogenophaga sp. PBC (EIK89721) BhbA (Comamonas sp. 7D-2) (AFV28965) *Nitratireductor pacificus pht-3B (EKF20868)

63

*Congregibacter litoralis NOR5-3 (EED32850) *Nitratireductor pacificus pht-3B (EKF18105)

0.1

Fig. 7. The phylogenetic tree of BhbA and homologous putative RDases from different hosts constructed according to the neighbour-joining method. The significant bootstrap values are shown at the nodes and expressed as a percentage of 1000 replicates. The related strains marked with asterisks are marine microorganisms. The underlined strains are facultative anaerobes, while the others are aerobes. The sequence accession numbers of the RDases are in parentheses.



Table 1. BhbA purification from Comamonas sp. strain 2B-bhbAB.

Purification step

Protein (mg)

Activitya (U)

Yield (%)

Specific activity (U mg−1)

Purification (fold)

Crude extract Cytoplasmic fraction Membrane fraction Solubilized fraction Mono Q, pH 8.0

122.2 77.5 48.9 21.5 0.42

5611 458 3922 3852 691

100 8 70 69 12

45.9 5.9 80 180 1640

1.0 – 1.7 3.9 35.8

a. One unit of activity is defined as the reduction of 1 nmol of DBHB per minute.

extracts prepared from cultures grown on MM with sodium succinate still contained dehalogenase activity. The optimum temperature range for BhbA dehalogenation activity was 25–30°C, and the optimum pH was 7.5. BhbA was active over a temperature range of 10–45°C and a pH range of 5.5–9.5. BhbA was completely inactivated above 50°C or at pH > 10. BhbA was completely inhibited by 0.1 mM Hg2+, Zn2+, Cd2+ or Cu2+ and partially inhibited by Al3+, Co2+ and Mn2+. The addition of 0.1 mM Ca2+, Fe3+, Fe2+, Mg2+ or Li+ had no significant effect on activity. Incubation of BhbA with 5 mM EDTA for 1 h did not inhibit activity. BhbA activity was completely inhibited by 5 mM sodium dithionite or 10 mM sodium thiosulphate. However, sodium sulphite (50 mM) had no effect on BhbA activity. The addition of 0.5 mM 1-iodopropane and 2 mM dithiothreitol did not inhibit the debrominating activity when incubated in the dark, suggesting that cob(III)alamin was not involved in the catalytic cycle of the BhbA dehalogenation reaction.

served as the electron donors in our reductive dehalogenation process. When NADPH was used as the cofactor, the relative activity was 2.82-fold higher than that of NADH (Table 2). Based on the dissociation constant (Km) and catalytic efficiency (Vmax/Km) values of NADPH (97.6 ± 7.6 μM and 489.0 ± 20.9 min−1·mg−1 respectively) and NADH (127.5 ± 8.2 μM and 98.1 ± 5.9 min−1·mg−1 respectively), NADPH was predicted to be the best cofactor of BhbA in vivo. The addition of FAD and FMN slightly enhanced BhbA activity when NADH was used as a cofactor (Table 2). However, FMN, FAD, NAD+, Fe2+, reduced MV and GST + ATP alone did not enhance the activity. There were no significant differences between the BhbA activities when the dehalogenation reactions were performed under anaerobic or aerobic conditions, although the dehalogenation activity of resting cells was approximately 10-fold higher in presence of oxygen than in the absence. BhbA was not oxygen sensitive. Upon exposure to air at 4°C, approximately 100% of the BhbA activity was retained after 24 h, and approximately 60% activity remained after 48 h. These BhbA properties contrast with the extremely oxygen-labile respiratory RdhAs (usually the oxygen sensitivity half-life (t1/2) is less than 330 min) (Holliger et al., 1999; Maillard et al., 2003), for which reductive dehalogenation reaction requires strictly anaerobic conditions. BhbA was constitutively expressed and cell

A

B M

1

2

3

kDa 200.0

116.0

BhbA substrate specificity BhbA had a Km of 37.6 ± 5.2 μM and Vmax/Km of 1504.5 ± 209.6 min−1·mg−1 for DBHB and Km of 148.9 ± 23.8 μM and Vmax/Km of 371.0 ± 47.9 min−1·mg−1 for BHB, showing that BhbA preferred the substrate DBHB to BHB.

Slice ID

RDase activity

1 2 3 4

w w

5

−

BhbA

97.2

66.4

44.3


− +

Fig. 8. A. SDS-PAGE analysis of the partially purified BhbA from strain 2B-bhbAB. The resolving gel was 8% and was stained with Coomassie Brilliant Blue G250 (Amresco, USA). Lane M: protein molecular weight marker (high); lane 1: membrane fraction from strain 2B; lane 2: membrane fraction from strain 2B-bhbAB; and lane 3: pooled fractions after Mono Q-chromatography elution at 200 mM NaCl. The arrow indicates the enrichment of the 117 kDa BhbA protein. B. Native PAGE analysis of the membrane fraction from strain 2B-bhbAB, indicating the positions of gel slices. RDase activities in unstained gel slices corresponding to the Coomassie-stained gel slices are indicated. –, no RDase activity; w, weak activity; +, strong activity.


Table 2. The effect of potential cofactors on BhbA activity. Cofactor(s)a


BhbA BhbA + FMN BhbA + FAD BhbA + NAD+ BhbA + NADH BhbA + NADPH BhbA + NADH + FAD BhbA + NADH + FAD + Fe2+ BhbA + NADH + FMN + Fe2+ BhbA + NADPH + FAD BhbA + NADPH + FAD + Fe2+ BhbA + NADPH + FMN + Fe2+ BhbA + GST + ATP BhbA + MV

ND ND ND ND 35.4 ± 3.39 100 ± 4.51 44.0 ± 5.82 44.8 ± 4.18 47.5 ± 3.47 101.3 ± 5.13 101.3 ± 4.66 102.6 ± 5.95 ND ND

a. Reaction mixtures (3 ml) containing partially purified BhbA (5 μg ml−1) and 0.1 mM DBHB were supplemented with cofactors. The reactions were initiated by substrate addition and terminated after 60 min by the addition of 15 μl sodium dithionite (1 M). The relative activity of BhbA with 0.2 mM NADPH as the cofactor was set at 100%. GST, reduced glutathione; MV, reduced methyl viologen; ND, not detected.

In addition to DBHB and BHB, the chlorinated hydroxybenzoate counterparts 3,5-dichloro-4-hydroxybenzoate (DCHB) and 3-chloro-4-hydroxybenzoate (CHB) were also dehalogenated, as summarized in Table 3. Dehalogenation occurred exclusively in the ortho position with respect to the hydroxyl group. The compound 3,5dibromobenzoate, which lacks the hydroxyl group, was not dehalogenated. An ortho-hydroxyl group was not sufficient for dehalogenation by BhbA, and the para-carboxyl group relative to the hydroxyl group also affected dehalogenation activity. For example, only one bromide of bromoxynil (in which carboxyl is substituted by the nitrile) and 2,6dibromophenol (which lacks the carboxyl group) was dehalogenated, at an even lower rate (7.6% and 21.4% respectively). Moreover, 3,5-dichloro-4-hydroxylnitrobenzene (in which the carboxyl is substituted by the nitro-group) and 2,3-, 2,4-, 2,5-dichlorophenol (DCP), 4-bromophenol, 2-chlorobenzoate, 4-chlorobenzoate, 3-bromobenzoate and hexachlorobenzene (which lack either a para-carboxyl group relative to the hydroxyl group or an ortho-hydroxyl group) were not dehalogenated. Chlorinated alkanoic acids (dichloroacetic acid and trichloroacetic acid) and chlorinated ethene (tetrachloroethylene) were also not dehalogenated (data not shown).

Functional confirmation of the bhbDE, bhbF, bhbD2E2 and bhbF2 genes The bhbFDE genes are located just upstream of the bhbAB genes and are transcribed in the same direction with bhbAB (Fig. 1B). Gene bhbF, bhbDE and bhbB

overlap with each other and share a single promoter, indicating that these genes constitute an operon. The amino acid sequence of BhbF has 76% identity to the HB-3-monooxygenase from Comamonas testosteroni CNB-2 (Accession No. YP_003278458) (Ma et al., 2009). BhbD and BhbE display 40% and 56% identities to the PCA-4,5-dioxygenase alpha and beta chains, LigA and LigB, from S. paucimobilis (Accession No. P22635.1 and P22636.1) (Noda et al., 1990) and 43% and 55% identities to PmdA and PmdB from Comamonas sp. strain E6 (Accession No. AB462808.1) (Kamimura et al., 2010). However, the activities of HB-3-monooxygenase and PCA-4,5-dioxygenase were not detected in resting cell experiments or crude enzyme activity assays when bhbF and bhbDE were expressed in E. coli DH5α (pUC-bhbF and pUC-bhbDE). The bhbF orf with a predicted GTG start codon is only 801 bp long, which is shorter than previously reported HB-3-monooxygenase genes. The BhbE sequence contains all predicted active sites and Fe(II) binding sites compared with the catalytic beta subunit of PCA-4,5-dioxygenase. However, the reason for our inability to detect PCA-4,5-dioxygenase activity is unknown. It is possible that bhbF and bhbDE lost the HB-3-monooxygenase and PCA-4,5-dioxygenase activities through recombination and/or rearrangement during the evolution of the bhb cluster. However, the possibility of bhbDEF possessing some as-yet unknown functions cannot be excluded. The predicted bhbE2D2F2 cluster was found 31 kb upstream of the bhb cluster on plasmid pBHB (Fig. 1B) and transcribed in the opposite direction. The amino acid sequence of BhbF2 has 79% identity with the HB-3monooxygenase from C. testosteroni CNB-2 (Accession No. YP_003278458). The complete bhbF2 orf is 1182 bp long. BhbF2 was functionally expressed in E. coli DH5α (pUC-bhbF2) and transformed HB to protocatechuate (PCA), as identified by MS/MS (Fig. S3C and D). The possibility of the oxygenic dehalogenation of BHB to PCA by BhbF2 was excluded because HB, which should not appear in an oxygenic dehalogenation process, was detected. These results indicate that two successive reductive dehalogenation reactions occurred instead of a reductive plus oxygenic dehalogenation in the DBHB dehalogenation process (Fig. 1A). BhbD2 and BhbE2 show 40% and 54% amino acid identities to LigA and LigB and 45% and 58% identities to PmdA and PmdB respectively. The complete bhbDE orf is 1252 bp long. BhbD2E2 were also functionally expressed in E. coli DH5α (pUC-bhbD2E2) and transformed PCA to a yellow product, which was further identified as 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) by MS/MS (Fig. S3E and F). The activities of HB-3-monooxygenase and PCA-4,5dioxygenase in the crude extract of the wild-type strain



Table 3. BhbA substrate preference.

Substrate

Product

Relative activity (%)a

Product


–

0

–

0

22.7

–

0

15.3

–

0

6.2

–

0

21.4

–

0

Substrate

7.6

100

a. BhbA activities with different substrates were determined in the standard enzyme assay system but with different halogenated substrates (at a concentration of 0.2 mM). The relative activity of reductive dehalogenation of DBHB to BHB was set at 100%.

7D-2 grown on bromoxynil or HB were also detected and the appearance of a yellow ring fission product, an indication of PCA-4,5-dioxygenase activity, was also observed. The specific activities of HB-3-monooxygenase and PCA-4,5-dioxygenase were determined to be 35.8 ± 5.5 and 406.3 ± 18.9 U mg−1 when strain 7D-2 grown on bromoxynil and to be 23.6 ± 3.5 and 718.5 ± 64.7 U mg−1 when grown on HB. Combined with the fact that strain 7D-2 also utilized HB as the sole carbon source for growth, HB-3-monooxygenase and PCA-4,5-dioxygenase were shown to be indeed involved in HB metabolism in strain 7D-2.

Discussion Brominated aromatics are widely used as flame retardants, fumigants, dyes, agrochemicals, pharmaceuticals

and herbicides (Alaee, 2003; Wagner et al., 2012), but brominated aromatics are also naturally produced, notably in marine environments (Gribble, 2010). The brominated aromatic herbicide bromoxynil is widely used and commonly detected in the environment. Debromination is the critical step for the mineralization of bromoxynil because the product bearing fewer halogen substituents is more susceptible to aerobic biodegradation (Cupples et al., 2005). Bromoxynil has been reported to be reductively dehalogenated to as far as the product 4hydroxybenzonitrile by the organohalide respiring bacterium of D. chlororespirans under anaerobic conditions (Cupples et al., 2005). However, under aerobic conditions, both in soils and in enrichment and pure cultures, the products of bromoxynil frequently retain the bromine groups (Topp et al., 1992; Rosenbrock et al., 2004). It was originally thought that aerobes rarely support reductive



dehalogenation, and reductive dehalogenation is an exception rather than the rule in aerobes (Janssen et al., 2001; van Pee and Unversucht, 2003). In our study, the aerobic strain 7D-2 was capable of reductively debrominating bromoxynil under aerobic conditions with the release of two equivalents of bromides. Furthermore, we found full-length bhbA homologues exist in the genomes of numerous proteobacterial strains; nearly all of them are aerobes (Fig. 7). Our findings indicate that, in fact, reductive dehalogenation occurs extensively in aerobes. The occurrence of aerobic bacteria capable of reductive dehalogenation in nature indicates that they have evolved to preferably adapt to organohalides and have important ecological roles in the biogeochemical cycle of halogen, which was not fully considered in the past. Although several aerobes/facultative aerobes have been accidentally found to catalyse reductive dehalogenation reactions (Xun et al., 1992; Romanov and Hausinger, 1996; Egland et al., 2001; Pandey et al., 2011), little is known regarding the biochemical mechanisms and molecular biology involved. The glutathione S-transferase family dehalogenase is the only reported RDase in aerobes to date (Xun et al., 1992; Miyauchi et al., 1998). BhbA, characterized in this study, is clearly different from the TeCH and 2,5-DCHQ glutathione S-transferases. The presence of several redox clusterbinding domains and its association with the cell membrane suggests that BhbA is involved in using its brominated substrates as terminal electron acceptors (Thibodeau et al., 2004). To the best of our knowledge, BhbA is the second type of RDase reported in aerobes. Although many putative homologous RDases deduced from genome sequencing have been deposited in the GenBank database (Fig. 7), none has been functionally confirmed until now. Our current study is the first to confirm the reductive dehalogenation function of this type of RDase. Interestingly, BhbA, found in the strict aerobe, shares some key features of respiratory RdhAs that are primarily found in strict anaerobes, such as the presence of two Fe-S cluster-binding motifs and an association with a potential anchor protein. Although they share some features, BhbA also differs from the respiratory RdhAs for halogenated aromatics (e.g. chlorophenol dehalogenases of Desulfitobacterium spp. (Christiansen et al., 1998; van de Pas et al., 1999; Krasotkina et al., 2001) and the CHB dehalogenase of D. chlororespirans Co23) due to its oxygen stability and large molecular weight. The organohalide respiring bacteria thus far described are strictly anaerobic, with only three exceptions. One exception is the facultative anaerobic bacterium, Anaeromyxobacter dehalogenans 2CP-1, which was reported to grow with chlorophenols as well as 2-bromophenol, nitrate, fumarate and oxygen as terminal electron acceptors (Sanford et al., 2002). The other two exceptions are

facultative aerobic bacteria, Enterobacteriaceae strain MS-1 and Enterobacter agglomerans biogroup 5 (ATCC 27993), which have been reported to transform PCE via trichloroethene (TCE) to cis-1,2-dichloroethene by organohalide respiration (Sharma and McCarty, 1996). However, reductive dehalogenations by these organisms require strictly anaerobic conditions (the absence of thermodynamically more favourable electron acceptor oxygen) (Jagnow et al., 1977). The differences between the dehalogenation catalysed by strain 7D-2 and that of other known organohalide respiring bacteria are that (i) strain 7D-2 can conduct reductive dehalogenation in the presence of oxygen in agreement with the presumption, based on thermodynamic considerations, that the reductive dehalogenation process itself may not always require strictly anoxic, low-potential conditions (Haggblom and Bossert, 2003), and (ii) strain 7D-2 cannot grow with DBHB or BHB as electron acceptors under anaerobic conditions, although weak dehalogenation was observed, indicating that it cannot exploit reductive dehalogenation as an energy-generating reaction. The weak dehalogenation activity of strain 7D-2 under anaerobic conditions was proposed to be the insufficient production of electrons for the reductive debromination process or lack of energy for the transportation of the substrate through the cytoplasmic membrane in the absence of oxygen. Electron transfer phosphorylation in anaerobes usually requires an association between the proteins involved and the cytoplasmic membrane. Commonly, respiratory RdhA genes (rdhA) are found to be linked with the orf designated rdhB coding for a hydrophobic protein (RdhB) with two or three transmembrane helices (Neumann et al., 1996). It has been speculated that the RdhB is active in anchoring the respiratory RdhA, which is predicted to have no transmembrane helices and to be translocated into or across the cytoplasmic membrane by the TAT signal peptide; however, no biochemical evidence to support this hypothesis has been presented to date. The only indication of a functional RdhB is the coexpression of rdhA and rdhB for the CprA of Desulfitobacterium dehalogenans (Smidt et al., 2000) and the PceA of Dehalospirillum multivorans (Neumann et al., 1998). In our study, BhbA, which is predicted to have no transmembrane helices, was shown to be membrane associated. The absence of a TAT signal peptide indicates that BhbA is not located on the outer but rather on the inner face of the cytoplasmic membrane. The gene bhbA is closely associated with bhbB, which resembles extracytoplasmic binding receptor genes. BhbB was predicted to be a hydrophobic protein with two transmembrane helices (Fig. 6B) and was confirmed to be membrane located (Fig. S2). BhbB expression significantly enhanced the dehalogenation activity in resting cells and contributed more than 90% of BhbA activity to the membrane fraction



(Fig. 4A and B). Furthermore, the detection of BhbB peptides in gel slices with RDase activities (Table S1), which was in agreement with the results from Tang and Edwards (2013), also indicated the interaction between BhbA and BhbB. From these data, BhbB is concluded to be a membrane-anchoring subunit for the attachment of BhbA to the inner face of cytoplasmic membrane. This is the first experimental evidence to confirm the membraneanchoring function of RdhBs. The low dehalogenase activity found in cytoplasmic fractions was deduced to be due to the escape of BhbA from the membrane during cell breaking processes. The finding of low dehalogenase activity in the membrane fraction from strain 2B-bhbA (only expressing bhbA) is slightly perplexing. It is possible that other extracytoplasmic binding receptors in Comamonas sp. strain 2B could attach BhbA to the membrane with low efficiency. The presence of a corrinoid cofactor in BhbA is made unlikely by these facts: (i) BhbA is not inhibited by sulphite or light-reversibly inhibited by iodopropane; (ii) BhbA lacks a cobalamin binding site motif; and (iii) the dehalogenation rate of the resting cells is not enhanced by supplementation with vitamin B12. A hydroxyl group ortho to the halogen atom was found to be required during BhbA dehalogenation. A similar phenomenon was reported in the orthochlorophenol RDase from D. chlororespirans (Krasotkina et al., 2001) and the TeCH RDase (glutathione Stransferase) from S. chlorophenolicum (Xun et al., 1992; Habash et al., 2002). The ortho-hydroxyl group has been proposed to be required for substrate binding or participation in the chemistry of the dehalogenation reaction. The faster dehalogenation of brominated aromatics compared with their chlorinated counterparts is in agreement with studies of abiotic chemical dehalogenation in which the aryl-bromine bond was shown to be weaker than the aryl-chlorine bond (Brown and Krishnamurthy, 1969). More interestingly, all bacterial strains, except the Hydrogenophaga sp. PBC (Gan et al., 2012), harbouring the putative RDases homologous to BhbA, are marine microorganisms (Gordon and Betty Moore Foundation Marine Microbial Genome Sequencing Project; Lee et al., 2007; Moran et al., 2007; Kang et al., 2011) (Fig. 7). These findings indicate a marine origin of the bhbA gene. Although the dehalogenating functions of these homologous BhbA have not been confirmed, it is possible that these marine microorganisms have evolved the bhbA analogous genes in response to the natural production of halogenated compounds in the marine environment (Ahn et al., 2003; Futagami et al., 2009). Due to the horizontal transfer of such genes, terrestrial microorganisms such as Comamonas sp. 7D-2 and Hydrogenophaga sp. PBC gained the genes and facilitated their adaptation to halogenated compound-contaminated environments. It is difficult to determine whether the bhbA analogous gene was

transferred to an ancestor of these two strains or transferred around the Comamonadaceae family in betaproteobacteria once it got out of the alpha- and gammaproteobacterial marine microorganisms. The acquisition of the dehalogenase gene through horizontal transfer is also suggested by the fact that the gene is located on a wild-type plasmid and surrounded by transposase genes (Fig. 1B). In addition, the best BhbA substrate is not the xenobiotic herbicide bromoxynil but DBHB (Table 3), which has been reported to be naturally produced in marine environments (green alga Ulva lactuca) (Flodin and Whitfield, 1999; Gribble, 2010). These results indicate that the evolution of the bhbA gene was not due to the selection pressure of bromoxynil and indicate that bhbA was horizontally transferred from marine microorganisms. The nitrilase gene bxn2 is also closely associated with a transposase gene, indicating that it might also have been gained through horizontal transfer. The acquisition of the bxn2 gene facilitates the debromination process of the host by hydrolysing bromoxynil to its best debromination substrate. The marine microorganisms harbouring the homologous BhbA are intensively distributed in the Roseobacter clade (including the genera of Roseobacter, Phaeobacter, Ruegeria, Planktotalea, Silicibacter, Jannaschia, Leisingera, Loktanella, Nautella, Oceanibulbus, Sulfitobacter and Thalassiobium), which was reported to constitute up to 25% of the total marine bacterial community and process a significant portion of the total carbon in the marine environment (Buchan et al., 2005; Brinkhoff et al., 2008). The finding indicates that Roseobacter clade microorganisms also play important roles in the biogeochemical cycle of halogen in marine system. The discovery of the clustering of RDase and ringcleavage oxygenase genes in a wild-type plasmid of the aerobic strain 7D-2 demonstrates that the aerobe has uniquely evolved. These types of aerobes have great potential in the bioremediation of halogenated compoundcontaminated sites that are aerobic systems; they have a competitive advantage against other aerobes by using a thermodynamically favourable reaction to reductively dehalogenate halogenated compounds and against anaerobes by using a typical oxidative ring-cleavage pathway to mineralize aromatic metabolites.

Experimental procedures Chemicals, bacterial strains, plasmids and culture conditions Bromoxynil (purity, 98%) was purchased from the Pesticide Research Institute (Shanghai, China). Standard compounds of DBHB, BHB, HB and PCA were purchased from Sigma-Aldrich (St. Louis, USA); DCHB, CHB, 3,5dichloro-4-hydroxyl-nitrobenzene, 3,5-dibromobenzoate, 2,6dibromophenol, 2-bromophenol, 2,3-, 2,4- and 2,5-DCP,



2-chlorobenzoate and 4-chlorobenzoate were purchased from J&K Chemical (Shanghai, China). The bacterial strains and plasmids used in this study are listed in Table S2 (Supporting information). Comamonas sp. strain 7D-2 was isolated from a bromoxynil octanoatecontaminated soil sample collected from Repont Pesticide Factory (Jintan, Jiangsu) using a conventional enrichment culture technique. The medium used for enrichment was MM (1.0 g NH4NO3, 1.6 g K2HPO4, 0.5 g KH2PO4, 0.2 g MgSO4 and 1.0 g NaCl per litre of water, pH 7.0) supplemented with 0.3 mM bromoxynil octanoate, and the enriched culture was transferred three times. A consortium containing two bacterial strains of 7D-2 and OB-3, which was capable of mineralizing bromoxynil octanoate was isolated. Strain 7D-2 showed the ability to completely degrade bromoxynil, the product of bromoxynil octanoate transformed by strain OB-3. Strain 7D-2 has been deposited in China General Microbiological Culture Collection Center (CGMCC) under the Accession No. 6931. Strain 7D-2 was identified as Comamonas sp. based on its morphological, physiological and biochemical properties with reference to Bergey’s Manual of Determinative Bacteriology and 16S rRNA gene sequence analysis. Comamonas sp. strains were grown at 30°C in LB or MM supplemented with substrates (0.2–0.3 mM bromoxynil, DBHB or BHB). To determine whether strain 7D-2 could use bromoxynil as the sole carbon and nitrogen sources for growth, MM without NH4NO3 and NaCl (to avoid the interference for bromides detection) was used. For determination of the ammonia released from nitrile hydrolysis, E. coli cells harbouring pUC-bxn2 were cultured in MM (without NH4NO3) with bromoxynil as the substrate. When culturing strains carrying antibiotic resistance markers, the medium was supplemented with 100 mg l−1 ampicillin (Amp), 50 mg l−1 kanamycin (Km) or 80 mg l−1 gentamicin (Gm) as necessary.

Plasmid isolation, curing and sequencing Routine plasmid isolation was performed with a Plasmid Miniprep Kit (Shanghai Generay Biotech). To isolate the large wild-type plasmid pBHB from strain 7D-2, the alkaline lysis method (Thomas et al., 1988; Ma et al., 2007) was used with minor modifications. The plasmid pBHB was sequenced with a Roche 454 GsFLX, Illumina GAIIx (Shanghai Majorbio Biopharm Technology). The wild-type plasmid was cured by subculturing strain 7D-2 in LB without bromoxynil for several generations.

Bromoxynil and its metabolite detection, determination and identification Bromoxynil and its metabolites were detected and analysed using HPLC (600 controller, Rheodyne 7725i manual injector and 2487 Dual λ Absorbance Detector; Waters, Milford, MA). Kromasil 100-5 C18 was used in the separation column as the stationary phase (4.6 mm internal diameter and 250 mm length). The mobile phase was acetonitrile : water : acetic acid (50:50:0.5/v : v : v), and the flow rate was 0.8 ml min−1. Bromoxynil, DBHB and BHB were detected at 221 nm, and HB and PCA were detected at 255 nm. The concentration of these compounds was determined from the peak area ratio relative to individual standard calibration curves.

The upper pathway metabolites produced from bromoxynil degradation such as DBHB, BHB and HB were first identified by the comparison to the HPLC profiles of standard compounds and further identified using LC-MS (LC-MSD-TrapSL, USA) equipped with an electrospray ionization source and operated in the negative polarity mode. For UV chromatography in LC-MS, all compounds were detected at 255 nm. Full scan signals were recorded within an m/z range of 200– 400 m/z. Auto gain control mode was used to optimize injection time. The lower pathway metabolites produced from HB degradation, such as PCA and CHMS, were identified by the comparison to the standard compounds and by tandem mass spectrometry (MS/MS) on a Finnigan TSQ Quantum Ultra AM instrument (Thermal, USA). The metabolites were confirmed by standard MS using negative electrospray ionization and scanned in the normal mass range from 100 m/z to 200 m/z. Characteristic fragment ions were detected by second-order MS. The bromine ions released during dehalogenation were detected and quantified according to the method of Bergmann and Sainik (1957). The ammonia released from nitrile hydrolysis was detected and quantified using the Nessler reagent colorimetric method (Krug et al., 1979).

Functional confirmation of bxn2, bhbDE, bhbF, bhbD2E2 and bhbF2 genes in E. coli DH5α Primer pairs were designed to amplify the bxn2, bhbDE, bhbF, bhbD2E2 and bhbF2 genes from the plasmid pBHB with PrimeSTAR Polymerase (Takara, Dalian) (see Supplementary Table S3). BamHI (in the forward primer) and HindIII (in the reverse primer) restriction sites were introduced to ensure the correct direction of gene insertion. For functional expression of the genes, a Shine–Dalgarno sequence was included before the start codon (ATG) of each orf. The amplified gene fragments were digested with restriction enzymes and ligated into the BamHI–HindIII sites of pUC18 to generate the recombinant plasmids pUC-bxn2, pUC-bhbDE, pUC-bhbF, pUC-bhbD2E2 and pUC-bhbF2 respectively (Table S2). The recombinant plasmids were then transformed into competent E. coli DH5α cells using standard procedures. Gene function was confirmed by performing resting cell assays on E. coli heterologously expressing the genes. Resting cells were prepared as described by Prakash et al. (2011) with minor modifications. The resting cells were washed three times with PBS (8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 per litre of water, pH 7.4) and resuspended in 10 ml PBS. Bromoxynil nitrilase activity was confirmed by the decrease in bromoxynil and increase in DBHB and ammonium ions. HB-3-monooxygenase activity was confirmed by the production of PCA from the standard compound HB. PCA-4,5-dioxygenase activity was confirmed by the production of the yellow compound CHMS from the standard compound PCA. In all experiments, E. coli cells carrying empty pUC18 were used as a negative control. HB-3-monooxygenase and PCA-4,5-dioxygenase activities in the wild-type strain 7D-2 were also investigated. The cells grown until the exponential phase with 0.2 mM bromoxynil or HB as the sole carbon source were harvested by centrifugation (10 000 g for 10 min), washed twice with PBS



and disrupted using an ultrasonic cell disruptor at 4°C. The cell lysates were clarified by centrifugation (15 000 g for 15 min) at 4°C. The HB-3-monooxygenase activity was assayed by detecting the decrease in absorbance at 340 nm due to the substrate-dependent oxidation of NADPH at 30°C (Seibold et al., 1996). The PCA 4,5-dioxygenase activity was assayed by measuring the increase in the absorbance at 410 nm derived from CHMS (Ono et al., 1970; Kamimura et al., 2010). The appearance of a yellow product was also used as an indication of PCA-4,5-dioxygenase activity (Adriaens et al., 1989). One unit of the activities of HB-3monooxygenase and PCA-4,5-dioxygenase was defined as the amount of enzyme that transforms 1 nmol of HB and PCA, respectively, per min at 30°C. Specific activity was expressed in units per milligram of protein. The protein concentration was determined using the Bradford method (Bradford, 1976) with bovine serum albumin as a standard.

Functional confirmation of the RDase gene Because the RDase gene was not functionally expressed in E. coli, and knockout of genes located on an antibiotic resistance-free wild-type plasmid is not feasible, the function of the RDase gene cluster was confirmed through complementation experiments. A 7 kb fragment containing the bhbABCDEF gene cluster was cloned into the BamHI–XbaI sites of the broad-host-range vector pBBR1MCS2 (Kovach et al., 1995) to generate pMCS2-7k, which was then introduced into the plasmid-cured strain 2B by triparental mating with the helper strain E. coli HB101 (pRK2013). The empty vector pBBR1MCS2 was also introduced into strain 2B as a negative control. To further clarify the specific gene(s) involved in reductive dehalogenation, a series of recombinant plasmids containing different fragments of the bhbABC gene cluster was constructed. First, a 517 bp fragment (named Q2) upstream of bhbF was cloned into the BamHI–HindIII sites of pBBR1MCS2 to generate pMCSQ2. In fragment Q2, a promoter sequence (5′-atgcggttgaatttcctatccgttcgtgccatacttg gcctcatgcggtt-3′, with a promoter score of 0.92) was identified by Neural Network Promoter Prediction software (http://www .fruitfly.org/seq_tools/promoter.html). The bhbABC, bhbAB, bhbAC and bhbA gene fragments, all of which contained Shine–Dalgarno sequences, were cloned into the BamHI– XbaI sites of pMCSQ2 to generate pMCSQ2-bhbABC, pMCSQ2-bhbAB, pMCSQ2-bhbAC and pMCSQ2-bhbA respectively. To further investigate the effect of the C-terminal region (NAD(P)H-dependent oxidoreductase domain) of BhbA on RDase activity, the recombinant plasmids pMCSQ2RD800, pMCSQ2-RD880 and pMCSQ2-RD1020, which contain bhbB and different lengths of bhbA, were constructed similarly as described above. The primer sequences used are listed in Table S3 (Supporting information). These recombinant plasmids were then introduced into strain 2B by triparental mating. A 1 ml aliquot of resting strain 2B cells containing different recombinant plasmids was inoculated into 50 ml PBS supplemented with 10 mM sodium succinate and 0.2 mM DBHB or BHB and incubated at 30°C. Then, 2 ml of sample was collected and centrifuged at 12 000 g at 4°C for 5 min. The concentrations of DBHB, BHB and HB in the supernatant

were measured as described above. RDase activity was confirmed by the decrease in DBHB/BHB and the increase in BHB/HB.

Partial purification of BhbA and identification by MALDI-TOF/TOF MS Late exponential-phase cultures of strain 2B-bhbAB were harvested by centrifugation at 15 000 g for 5 min. Approximately 6.0 g of wet cells was resuspended in 30 ml PBS (containing 2 mM dithiothreitol) and then disrupted by sonication at 4°C. The suspension was centrifuged at 200 000 g for 1 h at 4°C. The supernatant was filtered by membrane filtration (pore size, 0.22 μm) and retained as the cytoplasmic fraction. The precipitate was resuspended in 30 ml buffer A [pH 8.0, 20 mM Tris-HCl, 20% glycerol, 0.5% (w/v) Triton X-100 and 2 mM dithiothreitol] and incubated for 1 h at 4°C. The insoluble fraction was removed by centrifugation at 200 000 g for 1 h at 4°C, and the soluble one was retained as the membrane fraction. BhbA was purified from the membrane fraction of strain 2B-bhbAB by fast protein liquid chromatography (Amersham Biosciences) at 4°C. The membrane fraction was loaded on a Mono Q column equilibrated with buffer B [pH 8.0, 20 mM Tris-HCl, 20% glycerol, 0.02% (w/v) Triton X-100 and 1 mM dithiothreitol]. The dehalogenase-active fraction was eluted with a stepwise gradient method: (i) a 10 ml linear gradient from 0 to 150 mM NaCl in buffer B at a flow rate of 1.0 ml min−1, followed by (ii) a 40 ml linear gradient from 150 to 400 mM NaCl in buffer B at a flow rate of 1.0 ml min−1. The dehalogenase-active fraction eluted at a NaCl concentration of 200 mM. The partially purified BhbA was identified by SDS-gel electrophoresis with an 8% polyacrylamide resolving gel and a 5% stacking gel stained with Coomassie Brilliant Blue G250 (Amresco, USA). The Protein Molecular Weight Marker (High) (TaKaRa Biotechnology) was used as the marker. The enriched protein band was excised, digested with trypsin and analysed with an ABI5800 Proteomics Analyser MALDI-TOF/ TOF MS (Applied Biosystems, Foster City, USA) controlled by GPS Explorer V3.6 software (Applied Biosystems, USA) at Shanghai Bio-Tech. Based on the combined MS and MS/MS spectra, the proteins were identified based on a 95% or higher confidence interval of their scores in the MASCOT V2.1 search engine (Matrix Science, London, UK).

Native PAGE, in-gel activity assay and peptide identification A combination of native PAGE, RDase activity assay and MALDI-TOF/TOF MS peptide identification was performed similarly as described by Adrian et al. (2007). The cell membrane fraction from strain 2B-bhbAB was first separated by native PAGE with an 8% polyacrylamide resolving gel and a 5% stacking gel. A 40 μl volume of the membrane fraction was loaded in duplicate and run successively at 100 V for 60 min then at 160 V for 3 h in an ice bath. When electrophoresis was complete, one lane was cut from the rest of the gel and stained with Coomassie Brilliant Blue G250 (Amresco, USA). The remainder of the gel was stored in PBS containing



2 mM DTT at 4°C. For assaying RDase activity, the unstained gel lane was aligned with the Coomassie-stained gel lane and cut into gel slices. Each gel slice was further cut into 1 mm square pieces and transferred to a 2 ml Eppendorf tube with 1 ml PBS containing 0.2 mM NADPH, 0.1 mM DTT and 0.1 mM DBHB. These tubes were incubated at 30°C for 6 h; then samples were collected for HPLC analysis to evaluate the extent of dehalogenation of DBHB. The Coomassiestained bands, corresponding to the parallel unstained bands, were identified by MALDI-TOF/TOF MS as described above.

The BhbA enzyme assay A standard enzyme assay was performed under aerobic conditions in a 7 ml centrifuge tube containing 3.0 ml PBS, 0.1 mM DBHB and 0.2 mM NADPH. The reaction was initiated by the addition of 30 μl of enzyme preparation and incubated at 30°C for 60 min. The reaction was stopped by the addition of 15 μl sodium dithionite (1 M). The mixture was centrifuged at 16 000 g for 10 min and filtered by membrane filtration (pore size, 0.22 μm); then, the DBHB concentration was analysed using HPLC as described above. One unit of BhbA activity was defined as the amount of BhbA that catalyses the reduction of 1 nmol of DBHB per minute. Specific activity was expressed in units per milligram of protein. All assays were performed independently three times, and the means and standard deviations were calculated.

Biochemical characterization of BhbA The optimum temperature for BhbA activity was determined with the above-described standard enzyme assay mixture at 0–60°C, and the pH range was determined at pH values between 4.0 and 10.0. To investigate the effect of potential inhibitors on BhbA activity, various chemical agents (e.g. 50 mM sodium sulphite, 0–10 mM sodium thiosulphate, 0–10 mM sodium dithionite and 5 mM EDTA) were added and pre-incubated at 4°C for 60 min, followed by measurement of enzyme activity. Similarly, to investigate the effect of metal ions on BhbA activity, different metal ions (Ca2+, Fe3+, Al3+, Li+, Zn2+, Mg2+, Co2+, Cd2+, Cu2+, Hg2+ and Mn2+) were added at a final concentration of 0.1 mM. To investigate the effect of cofactors on BhbA activity, different cofactors, including 0.2 mM NADH, 0.1 mM FAD, 0.1 mM FMN, 0.1 mM NAD+, 0.1 mM Fe2+, 0.2 mM GSH (with 0.2 mM ATP) and 0.4 mM methyl viologen (MV) reduced by 0.2 mM sodium dithionite were added to the standard enzyme assay mixture (without NADPH). To study the effect of oxygen on BhbA activity, the enzyme assays were performed under anaerobic and aerobic condition, and the activities were compared. To study the oxygen resistance of BhbA, BhbA was exposed to air at 4°C for 24, 48 or 72 h, and the activity was determined. All assays were performed independently three times. The substrate range of BhbA was determined under standard enzyme assay conditions but with different halogenated substrates (at a concentration of 0.2 mM). In addition, negative controls without added enzyme and positive controls containing 0.2 mM DBHB were performed. The halogenated substrates including halogenated phenols, benzoates and nitrobenzenes were analysed by the same method as described for DBHB assay.

Sequence analysis The orfs predicted by GeneMark software were analysed manually with BLASTp and the NCBI database. The signal peptide sequences were analysed using the SignaIP 4.0 server (Petersen et al., 2011). The BhbB transmembrane region was predicted by TMpred (http://www.ch.embnet.org/ software/TMPRED_form.html) and confirmed by TMHMM (Moller et al., 2001). The amino acid sequences of BhbA and different respiratory RdhAs were aligned with BioEdit 6.0 software. The phylogenetic analysis of the sequences of BhbA and other homologous putative RDases was performed with MEGA 3.1.

Nucleotide sequence accession number The complete sequence of the wild-type plasmid pBHB from Comamonas sp. 7D-2 has been submitted to the GenBank database under the Accession No. KC771559.

Acknowledgements This work was supported by grants from the Chinese National Science Foundation for Excellent Young Scholars (31222003), the Chinese National Natural Science Foundation (31070100), the Program for New Century Excellent Talents in University (NCET-12-0892), the Outstanding Youth Foundation of Jiangsu Province and the National Science and Technology Support Plan (2013AA102804). S. Zinder’s research on haloaromatic degradation was supported by funding by the DuPont Corporation.

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