Characterization and genome functional analysis of the DDT-degrading bacterium Ochrobactrum sp. DDT-2

STOTEN-22229; No of Pages 7 Science of the Total Environment xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Characterization and genome functional analysis of the DDT-degrading bacterium Ochrobactrum sp. DDT-2 Xiong Pan, Tianheng Xu, Haoyu Xu, Hua Fang ⁎, Yunlong Yu ⁎ Institute of Pesticide and Environmental Toxicology, College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310058, PR China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• The first report on DDT-degrading bacteria from the novel genus Ochrobactrum. • The isolate DDT-2 showed a high degradation ability for DDT. • Potential DDT degradation genes were found in the isolate DDT-2 genome. • A common degradation pathway based on the combined genomic and MS analysis was found.

a r t i c l e

i n f o

Article history: Received 27 December 2016 Received in revised form 5 March 2017 Accepted 6 March 2017 Available online xxxx Editor: Jay Gan Keywords: DDT Genome sequencing Functional annotation KEGG pathway Ochrobactrum sp.

a b s t r a c t A strain of Ochrobactrum sp. DDT-2 that was capable of degrading DDT as the sole carbon and energy source was isolated and sequenced, and its biodegradation characteristics and metabolism mechanism were examined. The genome sequence of the isolate DDT-2 was composed of 4,630,303 bp with a GC content of 55.99% and 4454 coding genes. The degradation rate of DDT by the isolate DDT-2 increased with the increasing substrate concentration (0.1–10 mg/l) and temperature (20–40 °C). The degradation half-life of DDT in the presence of the isolate DDT-2 at pH 7.0 was obviously shorter than those at pH 5.0 and 9.0. Potential DDT degradation genes were found in the isolate DDT-2 genome by a BLASTx search against a DDT degradation genes (DDGs) database. A common biodegradation pathway of DDT was proposed based on the combined analysis of genome annotation and mass spectrometry. DDT was initially dechlorinated to form DDD and DDE. Then, it was transformed into DDMU and DDA via dechlorination and carboxylation, and it may ultimately be mineralized to carbon dioxide. The results suggested that the isolate DDT-2 could be useful for the bioremediation of DDT and its metabolite residues. © 2016 Elsevier B.V. All rights reserved.

1. Introduction 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) is a chlorinated aromatic compound that has been used to control agricultural pests and protect against malaria and typhus. Though DDT was officially banned in China in 1983, the residues of DDT and its metabolites in ⁎ Corresponding authors. E-mail addresses: [email protected] (H. Fang), [email protected] (Y. Yu).

some natural soils, sediments, and contaminated sites are still high (Liu et al., 2015). Furthermore, the use of dicofol and antifouling paint has continued to contribute to DDT pollution (Guo et al., 2009; Xin et al., 2011). Several studies have revealed the acute toxicity, chronic toxicity, and endocrine disrupting effects, such as endocrine and immunological disorders, nervous system injury and cancer, of DDT and its major metabolites 2,2-bis(p-chlorophenyl)-1,1-dichlorethylene (DDE) and 1,1-dichloro-2,2-bis (p-chlorophenyl)ethane (DDD), which pose a serious threat to the environment and human health (Jin et al., 2013;

http://dx.doi.org/10.1016/j.scitotenv.2017.03.052 0048-9697/© 2016 Elsevier B.V. All rights reserved.

Please cite this article as: Pan, X., et al., Characterization and genome functional analysis of the DDT-degrading bacterium Ochrobactrum sp. DDT-2, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2017.03.052

2

X. Pan et al. / Science of the Total Environment xxx (2016) xxx–xxx

Mrema et al., 2013; Pavlikova et al., 2015). Therefore, exploring efficient DDT remediation pathways is essential. Three remediation methods, physical remediation, chemical remediation, bioremediation, have been studied for the removal of environmental DDT contamination (Han et al., 2015; Zhu et al., 2016). Physical remediation, such as soil washing, is costly, and chemical remediation, such as by thiosulfate, can cause secondary pollution (Cai et al., 2007; Thangavadivel et al., 2011). Bioremediation using DDT-degrading bacterium offers a more efficient, economic, and safe method to remove contamination. A few strains capable of utilizing DDT as a sole carbon resource have been documented, such as Alcaligenes eutrophus A5, Alcaligenes sp. KK, Alcaligenes sp. DG-5 and Chryseobacterium sp. PYR2, and their biodegradation mechanisms have also been further clarified (Gao et al., 2011; Xie et al., 2011; Qu et al., 2015; Erdem and Cutright, 2016). Although the biodegradation pathway of DDT has been revealed by mass spectrometry in previous studies, a comprehensive genome annotation related to all DDT degradation steps is lacking (Cui et al., 2014; Crombie et al., 2015). Presently, both genome sequencing and functional annotation are promising approaches to explore the degradation genes encoding for the enzymes involved in the biotransformation and biodegradation of contaminants (Kube et al., 2013). To the best of our knowledge, the integration of genomic and traditional mass spectrometry methods will give us a new insight into biodegradation mechanism of DDT. In the present study, a highly efficient degrading bacterial strain DDT-2, which could utilize DDT as the sole source of carbon and energy, was isolated and identified. The objectives of this study were: 1) to determine the physiological characterization of the isolate DDT-2; 2) to sequence and analyze the genome of the isolate DDT-2; 3) to examine the effects of substrate concentration, pH, and temperature on the biodegradation of DDT; and 4) to explore the potential biodegradation pathway using genome function annotation and mass spectrometric analysis. The study aims to elucidate the biodegradation mechanism of DDT in the new insight.

2. Materials and methods 2.1. Chemicals 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT, purity ≥ 99.5%), 2,2-bis(p-chlorophenyl)-1,1-dichlorethylene (DDE, purity ≥ 98.5%), 1,1dichloro-2,2-bis (p-chlorophenyl)ethane (DDD, purity ≥ 98%), 1-chloro2,2-bis(4′-chlorophenyl)ethylene (DDMU, purity ≥ 99.0%), 2,2bis(4′-chlorophenyl)ethanol (DDOH, purity ≥ 99.5%), and bis(4′chlorophenyl)acetate (DDA, purity ≥ 99.0%) were provided by Dr. Ehrenstorfer GmbH (Augsburg, Germany). Analytical grade sodium chloride, anhydrous sodium sulfate, acetone, and n-hexane were provided by Shuanglin Chemical Co., Hangzhou, China.

2.3. Identification of the DDT-degrading strain The morphological characteristics were determined using a transmission electron microscope (TEM, Hitachi H-7650, Japan) after incubation for 1 d at 30 °C. The BIOLOG GN system was used to assess the carbon substrate utilization of the isolate DDT-2. DNA was extracted using the pyrolysis method (Wilson, 1997). The 16S rDNA was amplified via PCR using the universal primer pair 27F (5′-AGAGTTTGATC CTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The amplification was performed under the following conditions: initial denaturation at 94 °C for 5 min, 34 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 45 s and extension at 72 °C for 90 s, and the final extension at 72 °C for 5 min. The PCR products were purified and sequenced by the Invitrogen Biotechnology Limited Company, Shanghai, China. The obtained 16S rDNA sequence was submitted and compared to the NCBI GenBank database using the BLAST homology search (http://www.ncbi.nlm.nih.gov/BLAST.cgi) to identify the isolate. The phylogenetic tree was constructed by the analysis of a single copy sequence using the software MEGA version 4.0 (Tamura et al., 2007). 2.4. Genome sequencing Approximately 5 μg of the genomic DNA was extracted and purified using the QIAamp kit (Qiagen, Germany). The quantity of the DNA was measured using a NanoDrop Spectrophotometer 2000 (Equl-Thermo Scientific, USA). The DNA samples were sent to Novegene (Beijing, China) and pooled by mechanically fragmenting for a shotgun library construction. The library was finally PCR amplified, clustered and sequenced on an Illumina HiSeq 2500 platform to generate paired-end (2 × 250-bp) reads. Approximately 1,63 G of raw sequences were obtained with N 93% coverage. 2.5. Bioinformatic analysis The DDT degradation genes (DDGs) database contained 12 subdatabases for the complete metabolic pathway, which was constructed based on the method of Fang et al. (2014). The protein database from each sub-database was retrieved from the KEGG pathway maps (http://www.genome.jp/kegg/). All the raw reads were filtered using a self-written script, and the clean reads were assembled them by Velvet version 1.0.12 (www.ebi. ac.uk/~zerbino/velvet) (Zerbino and Birney, 2008). The inner gaps that emerged in the scaffold were filled using the Gap Closer version 1.012. The scaffolds were then uploaded to the CGView Server to plot the graphical circular genome map (http://stothard.afns.ualberta.ca/ cgview_server/) (Grant and Stothard, 2008), and they were aligned against the DDGs database using BLASTx with an E-value cut-off at 10−10 (Qin et al., 2010). The genes were predicted using Glimmer version 3.0 and annotated in the Clusters of Orthologus Groups (COGs), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO), respectively (Delcher et al., 2007).

2.2. Enrichment and isolation of the DDT-degrading strain 2.6. Degradation of DDT by the isolate DDT-2 in a pure culture The DDT-contaminated soil samples were collected from a vegetable field located in Cixi, Zhejiang, China and were homogenized and divided into three subsamples (10.0 g each). Each subsample was transferred into a 250-ml Erlenmeyer flask containing 100 ml of mineral salts medium (MSM, MgSO4·7H2O, 0.40 g; FeSO4·7H2O, 0.002 g; K2HPO4, 0.20 g; (NH4)2SO4, 0.20 g; CaSO4, 0.08 g; H2O, 1000 ml; pH 7.0) supplemented with 10 mg/l of DDT, and the samples were incubated at 30 °C on a rotary shaker operating at 150 rpm. After one week, 1 ml of the soil suspension was inoculated in a 100 ml Erlenmeyer flask containing 20 ml of fresh MSM supplemented with 10 mg/l of DDT as the sole carbon source and energy. This acclimatization process was repeated six times with increasing substrate concentrations from 10 to 50 mg/l until pure cultures were obtained.

The isolate DDT-2 was pre-incubated in 100 ml of LB medium at 30 °C and 150 rpm. After incubation for 1 d, the cells were collected by centrifugation (8000 × g, 10 min), washed thrice with NaH2PO4Na2HPO4 buffer (0.1 mol/l, pH 7.0), and then suspended in the MSM as the inoculum. To determine the effect of the DDT concentration on the biodegradation, the MSM (pH 7.0) was fortified with DDT at three different initial concentrations (0.1, 1, and 10 mg/l), respectively. To measure the effect of pH on DDT biodegradation, the medium was prepared at pH 5.0, 7.0, and 9.0 with a buffer (0.1 mol/l NaH2PO4-Na2HPO4) and fortified with DDT at a concentration of 1 mg/l. To confirm the effect of temperature on DDT biodegradation, the medium fortified with DDT at a concentration of 1 mg/l was incubated at 20, 30, and 40 °C,

Please cite this article as: Pan, X., et al., Characterization and genome functional analysis of the DDT-degrading bacterium Ochrobactrum sp. DDT-2, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2017.03.052

X. Pan et al. / Science of the Total Environment xxx (2016) xxx–xxx

3

respectively. In this study, all treatments were performed in 100-ml flask containing 20 ml of sterile MSM supplemented with DDT as the sole carbon and energy source. Each flask was inoculated with a suspension of the isolate DDT-2 to reach an initial inoculum level of OD600 = 0.2. All flasks were incubated in a shaker at 30 °C and 150 rpm in the dark. The whole culture was taken, and the DDT residues were determined after 3 d, 7 d, 14 d, and 28 d, respectively. The extraction method for DDT and the metabolites in MSM is shown in the Supplementary Information, and the recovery study of DDT is summarized in Table S2. Each treatment was performed in triplicate. The uninoculated treatment was conducted as the control under the same conditions.

including RNA processing and modification, chromatin structure and dynamics, amino acid transport and metabolism, nucleotide transport and metabolism, carbohydrate transport and metabolism, coenzyme transport and metabolism, secondary metabolites biosynthesis, transport and catabolism, were revealed by the genome functional annotation of the isolate DDT-2 against the COGs database (Fig. 1b). Meanwhile, the cellular component, molecular function and biological process of the isolate DDT-2 were also classified and revealed by genome functional annotation against the GO database (Fig. 1c). Additionally, a graphical circular genome map of the isolate DDT-2 is shown in Fig. S3.

2.7. GC–MS analyses of DDT and its metabolites

3.3. Degradation of DDT by the isolate DDT-2

To identify and determine DDT and its metabolites formed by the isolate DDT-2, a GC–MS-QP2010 plus (Shimadzu Corporation, Kyoto, Japan) equipped with a VF1701 silica capillary column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies, USA) was employed. The flow rate of the carrier gas (He) was 1 ml/min. The temperatures of the injector and detector were 270 °C and 280 °C, respectively. The oven temperature was initially set to 100 °C and subsequently increased to 210 °C at 15 °C/min, held for 2 min, raised to 270 °C at 7 °C/min and held for 1 min, raised to 280 °C at 15 °C/min, and held for 5 min. The temperatures of the ion source and transfer line were set at 230 °C and 260 °C, respectively. The ionization mode electron was set at 70 eV.

Degradation of DDT by the isolate DDT-2 at different initial concentrations, temperatures and pH values is shown in Fig. 2, and the kinetic data are summarized in Table 2. After incubation for 28 d, the hydrolysis rate of DDT in all the controls without inoculation was b13%, except for pH 9.0 (20%). In all the inoculated treatments, the degradation rates of DDT by the isolate DDT-2 at concentrations of 0.1, 1.0, and 10.0 mg/l were 0.003, 0.019, and 0.042 mg/l/d, respectively (Fig. 2a–c). The corresponding degradation rates of DDT at a concentration of 1.0 mg/l were 0.013, 0.019, and 0.022 mg/l/d at 20, 30, and 40 °C (Fig. 2d) and 0.003, 0.019, and 0.013 mg/l/d at pH 5.0, 7.0, and 9.0 (Fig. 2e), respectively. In this study, the degradation of DDT by the isolate DDT-2 followed the pseudo first-order kinetics model. As shown in Table 2, the degradation half-life of DDT at concentrations of 0.1, 1.0, and 10.0 mg/l by the isolate DDT-2 was 3.66, 18.24, and 102.39 d, respectively, and the corresponding value of DDT at 1.0 mg/l was 33.60, 17.77, and 14.65 d at 20, 30, and 40 °C. The ANOVA analysis showed that the degradation halflife of DDT increased with the increasing initial concentration, but it decreased with the increase in temperature. Additionally, the degradation half-life of DDT (1.0 mg/l) by the isolate DDT-2 was 18.24, 85.47, and 23.34 d at pH 5.0, 7.0, and 9.0, respectively. The ANOVA analysis showed that the degradation half-life of DDT was significantly (p ≤ 0.05) shorter at pH 7.0 than at pH 5.0 and 9.0, which indicated that neutral condition was the most suitable for the biodegradation of DDT. The degradation rate of DDT by the isolate DDT-2 increased with increasing initial concentration and temperature, and the favorable pH condition was neutral. Similar results were reported in some DDTdegrading bacteria, such as Alcaligenes sp. KK, and Alcaligenes sp. DG-5 (Gao et al., 2011; Xie et al., 2011). However, the degradation capability of Alcaligenes sp. KK and Alcaligenes sp. DG-5 for DDT was inhibited when the DDT concentration exceeded 20 mg/l. Wang et al. (2013) reported that a temperature of 40 °C was not beneficial for the acetamiprid biodegradation by Ochrobactrum sp.D-12.

2.8. Accession number The 16S rDNA sequence of the isolate DDT-2 was submitted to the NCBI GenBank database under the accession number KY328283. The whole genome sequence of the isolate DDT-2 was deposited under the accession number LKAD00000000 (Submission ID SUB1105029, BioProject ID PRJNA296453, and BioSample ID SAMN04099172). 3. Results and discussion 3.1. Isolation and identification of the isolate DDT-2 The strain DDT-2 capable of utilizing DDT as its sole carbon and energy source was isolated from the collected soil samples. The growth curve of the isolate DDT-2 in pH 7.0 of MSM supplemented with DDT at a concentration of 1 mg/l is shown in Fig. S1. The cells were gramnegative, capsular, flagellated, and non-spore forming rods (0.5– 0.7 μm × 0.7–0.9 μm, Fig. 1a). The colonies were pallid, smooth, and moist with bacterial opacity when cultured on LB plate for 1 d at 30 °C. The isolate could grow in the MSM containing 1 mg/l of DDT at 20–40 °C and at pH 5.0–9.0. The metabolisms of 95 different substrates after inoculation for 24 h in BIOLOG GN2 are shown in Table S1, which illustrated the likelihood that the isolate DDT-2 was related to the genus Ochrobactrum with a similarity of 0.74 and a matching degree of 3.93. The sequences of the 16S rDNA also indicated that the isolate DDT-2 belonged to the genus Ochrobactrum with 99% similarity. Additionally, a phylogenetic tree was also constructed based on the single copy protein sequences (Fig. S2). According to the results above, the isolate was designated Ochrobactrum sp. DDT-2. 3.2. Genome characteristics As revealed in Table 1, all clean reads of the isolate DDT-2 were assembled into 68 contigs with the contig N50 of 206,085 bp. The genome sequence of the isolate DDT-2 was composed of 4,630,303 bp with a GC content of 55.99%. Approximately 4454 coding genes and 47 repeated sequences were predicted, and 3513, 3149, 2306, and 3144 genes were annotated against the COGs, GO, KEGG, and Swiss-Pro databases, respectively. The isolate DDT-2 genome consisted of 35 genes in a unique gene family and 38 pseudogenes. Twenty-five function classes,

3.4. Identification of DDT metabolites by mass spectrometry Total ion chromatogram of neutral extracts from the cultures after inoculation for 14 d with 10 mg/l of DDT is shown in Fig. S4. Four major DDT metabolites were found and their retention times were 13.25, 14.13, 14.75, and 14.97 min, respectively. Through the analyses for known standard compounds and comparisons against mass spectra in the NIST library, the metabolites were identified as DDD at m/z 320 (C14H10Cl4, Fig. S5), DDE at m/z 318 (C14H8Cl4, Fig. S6), DDMU at m/z 266 (C14H11Cl3, Fig. S7), and DDA at m/z 282 (C14H10Cl2O2, Fig. S8), respectively. In this study, the fast growth of the isolate DDT-2 (Fig. S1) and no accumulation of the DDT metabolites were observed throughout the whole incubation, which deduced that the degradation of DDT by the isolate DDT-2 may be a mineralization pathway. Among the four metabolites, DDE and DDD were common major metabolites of DDT and they were previously detected in the studies of Alcaligenes eutrophus A5 and Chryseobacterium sp. PYR2 (Qu et al., 2015; Erdem and Cutright, 2016). In our study, no accumulations of DDE and DDD were observed. Thus, DDE was not considered a dead-

Please cite this article as: Pan, X., et al., Characterization and genome functional analysis of the DDT-degrading bacterium Ochrobactrum sp. DDT-2, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2017.03.052

4

X. Pan et al. / Science of the Total Environment xxx (2016) xxx–xxx

Fig. 1. Scanning electron micrograph of the isolate DDT-2 (a), functional annotation of the isolate DDT-2 genome against the Clusters of Orthologous Groups (COG) database (b), and functional classification of the isolate DDT-2 genome against Gene Ontology (GO) database (c).

end metabolite. Similar to our results, Fang et al. (2010) reported the further degradation of DDE and DDD by the strain Sphingobacterium sp. D-6, and their secondary metabolites were identified to be DDMU, DDNS, DDA, and DBP. Eganhouse and Pontolillo (2008) found DDMU in sediment core from microbial dechlorination of DDE during an 11year period. In addition, Heberer and Dünnbier (1999) pointed out that the reductive dechlorination of DDD could ultimately produce small amounts of DDA. Therefore, the detection of DDA may be unusual. 3.5. DDT degradation genes revealed by genome annotation In the 4454 genes predicted in strain DDT-2 genome by a BLASTx search against the DDGs database, potential DDT degradation genes encoding for dehydrochlorinase and dehalogenase were found. As

shown in Table S3, the dhc gene carried in BSP002540 played a part in the transformation of DDT → DDE and DDD → DDMU. The rdh gene harbored in BSP002831 was involved in the transformation of DDT → DDD, while the dhg gene harbored in BSP002205 was responsible for the degradation step of DDMS → DDOH. The reductive dehalogenase encoded by those genes was essential for the mineralization of DDT. Lovecka et al. (2015) affirmed the presence of the dehydrochlorinase gene (linA) in the DDT-contaminated soil and noted that dechlorination was the first degradation step. The hdt gene harbored in BSP003659 was found to be involved in the degradation step of DDNU → DDOH via hydrolyzation, and DDOH could be converted into DDA by further carboxylation. The dcl gene carried in BSP003442 that encoded for decarboxylase was another dominant gene in DDT biodegradation and played a part in the transformation of DDA → DDM. Fang et al. (2014) found similar DDT degradation genes

Please cite this article as: Pan, X., et al., Characterization and genome functional analysis of the DDT-degrading bacterium Ochrobactrum sp. DDT-2, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2017.03.052

X. Pan et al. / Science of the Total Environment xxx (2016) xxx–xxx Table 1 Genome characteristics of the isolate DDT-2. Characteristic

Genome

Characteristic

Genome

Raw reads size (bp)

1,633,643,132

6177

Sequencing depth Clean reads size (bp) kmer depth Genome size (bp) GC content (%) Contig number Contig N50 (bp) Gene

326.7 1,426,076,736 304 4,630,303 55.99 68 206,085 4454

Gene total size (bp) Gene mean length (bp) Gene GC content (%) Repeat sequence number

3,853,518 865

Repetitive sequence length (bp) miRNA rRNA tRNA Coding gene annotated Coding gene assigned to COGs Coding gene assigned to KEGG Coding gene assigned to GO Coding gene assigned to Swiss-Prot Gene family number Unique gene family

56.58 47

Gene in unique gene family Pseudogene

5 7 49 4392 3513 2306 3149 3144 3274 17 35 38

that involved these multistep degradation processes in freshwater and marine sediments using metagenomic analyses. 3.6. Biodegradation pathway of DDT

shown in Fig. 3, DDT may be transformed into DDE and DDD by the function of dhc gene and rdh gene, respectively. The sds and dhg genes may degrade DDD to DDMU, and then converted into DDOH or first into DDNU and then to DDOH by the dht gene. Subsequently, DDOH may be carboxylated to DDA and then decarboxylated to DDM, and may ultimately be mineralized to carbon dioxide. A similar potential complete biodegradation pathway of DDT was also reported by Fang et al. (2014) using metagenomic analysis, they found a multistep process involving reductive dechlorination, hydrogenation, dioxygenation, hydroxylation, decarboxylation, hydrolysis, and meta-ring cleavage reactions. 3.6.2. Pathway based on mass spectrometry Based on the structures of the identified metabolites, a proposed biodegradation pathway of DDT is presented in Fig. 3. DDT could first be transformed into DDD and DDE via dechlorination at the trichloromethyl group, subsequently converted to the metabolite DDMU via further dechlorination, and then form DDA via carboxylation. Finally, DDA may be mineralized to carbon dioxide. Similar to our results, DDD, DDE, DDMU, and DDA had been previously detected as DDT metabolites by some bacterial strains, such as Aeromonas hydrophila HS01, Chryseobacterium sp. PYR2, and Pseudomonas putida T5 and so on (Cao et al., 2012; Rangachary et al., 2012; Qu et al., 2015). Barragán-Huerta et al. (2007) reported that the members of Pseudomonas aeruginosa and Flavimonas oryzihabitans could transform DDT into DDE, DDMU and DDOH in broth medium. Fang et al. (2010) reported a mineralization process of DDT by the strain Sphingobacterium

0.12 0.1mg/l(control)

0.1mg/l(strain DDT-2)

1.0

0.08 0.06 0.04 0.02 0.00 -0.02

a 0

7

14

21

28

Day after treatment Concentration of DDT (mg/L)

1.2

1mg/l(control)

1mg/l(strain DDT-2)

1.0

0.8

0.6

0.4

°C °C °C

0.2

°C °C

d

°C

0.0

0.8

0

7

0.6

14

21

28

Day after treatment

0.4

1.0 0.2 0.0

b 0

7

14

21

28

Day after treatment 12

Concentration of DDT (mg/L)

Concentration of DDT(mgL-1)

0.10

10mg/l(control)

10mg/l(strain DDT-2)

10 8 6 4 2 0

c 0

Concentration of DDT(mgL-1)

Concentration of DDT (mg/L)

3.6.1. Pathway based on genome annotation All potential DDT degradation genes found by a BLAST search against the DDGs database inferred the biodegradation pathway of DDT. As

5

0.8

0.6

0.4

pH5(control) pH7(control) pH9(control)

0.2

pH5(strain DDT-2) pH7(strain DDT-2) pH9(strain DDT-2)

e

0.0 7

14

21

Day after treatment

28

0

7

14

21

28

Day after treatment

Fig. 2. Biodegradation of DDT by the isolate DDT-2 in the mineral salts medium at different initial concentrations (a–c), temperatures (d), and pH values (e).

Please cite this article as: Pan, X., et al., Characterization and genome functional analysis of the DDT-degrading bacterium Ochrobactrum sp. DDT-2, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2017.03.052

6

X. Pan et al. / Science of the Total Environment xxx (2016) xxx–xxx

Table 2 Degradation kinetic data for DDT with the isolate DDT-2 at different substrate concentrations, temperatures, and pH values. Concentration (mg/l)

Temperature (°C)

pH

Kinetic function

0.1 1.0 10.0 1.0 1.0 1.0 1.0 1.0 1.0

30 30 30 20 30 40 30 30 30

7.0 7.0 7.0 7.0 7.0 7.0 5.0 7.0 9.0

C C C C C C C C C

= = = = = = = = =

0.0932e−0.1894 ∗ t 0.9977e−0.03801 ∗ t 9.9173e−0.00677 ∗ t 0.9897e−0.02063 ∗ t 0.96938e−0.03899 ∗ t 0.98209e−0.04730 ∗ t 1.06417e−0.00811 ∗ t 0.9977e−0.03801 ∗ t 0.9559e−0.0297 ∗ t

sp. D-6. DDT could be initially dechlorinated to form DDD and DDE, further dechlorinated to DDMU, and deoxygenated to form DDA. The aromatic ring in DDA may be cleaved, and the product may ultimately be oxidized to carbon dioxide. Bajaj et al. (2014) also reported that the strain Rhodococcus sp. IITR03 could degrade DDT to DDE, DDD, and DDMU in minimal media. 3.6.3. Common pathway based on genome annotation and mass spectrometry In this study, each biodegradation step of DDT revealed using mass spectrometry had a corresponding DDT degradation gene found using genome functional annotation. As shown in Fig. 3, a common biodegradation pathway based on both genomic and GC–MS analysis was proposed. DDT may be dechlorinated to DDE and DDD, further dechlorinated to DDMU, and then converted into DDA, and may

Degradation Rate(mg/l/d)

DT50a (d)

r2

0.003 0.019 0.042 0.013 0.019 0.022 0.003 0.019 0.013

3.66 18.24 102.39 33.6 17.77 14.65 85.47 18.24 23.34

0.995 0.998 0.969 0.989 0.976 0.984 0.917 0.998 0.989

ultimately be mineralized to carbon dioxide. Genome annotation could comprehensively explore the DDT degradation genes carried in the isolate DDT-2, but not all of the annotated degradation genes had degradation activity for DDT. Nevertheless, the DDT metabolites (DDD, DDE, DDMU, and DDA) identified using mass spectrometry analysis could be used to efficiently screen the target genes capable of expressing DDT degradation activity from the above annotated degradation genes. Therefore, the combined genome annotation and mass spectrometry analysis could rapidly confirm the common DDT degradation genes harbored in the isolate DDT-2, which was chosen to verify the expression activity of the DDT degradation function. 3.7. Verification of the DDT degradation pathway To verify the degradation pathway of DDT, especially the degradation pathway annotated by genomic analysis, degradation experiments with four DDT metabolites at an initial concentration of 10.0 mg/l by the isolate DDT-2 were conducted in MSM at the inoculation level of OD600 = 0.4 under the conditions of pH 7.0, 150 rpm, and 20 °C. After incubation for 7 d, the degradation percentages of DDE, DDD, DDMU, and DDA were 25.11%, 7.27%, 28.54%, and 100%, respectively, compared to their corresponding uninoculated controls (Table 3). These results indicated that these four metabolites were efficiently degraded by the isolate DDT-2. 4. Conclusions The results obtained in this study indicate that DDT can be degraded or detoxified rapidly by the isolate DDT-2 in MSM. The genome of the isolate DDT-2 contained 4,630,303 bp, 55.99% GC content, and 4454 coding genes. The degradation half-life of DDT by the isolate DDT-2 in MSM increased with the increasing substrate concentration and decreased with the increasing temperature, and the optimal pH for biodegradation was 7.0. The isolate DDT-2 could degrade DDT into the metabolites DDE, DDD, DDMU, and DDA via dechlorination, hydroxylation, and carboxylation based on both the genomic and GC–MS analyses. In this study, the isolate DDT-2 showed a high biodegradation ability for DDT that was similar to the previously reported DDTdegrading bacterial strains, such as Aeromonas hydrophila HS01, Alcaligenes sp. KK, Alcaligenes sp. DG-5, Alcaligenes eutrophus A5, and Chryseobacterium sp. PYR2, and furthermore, the degradation of DDT by the isolate DDT-2 may be a mineralization process. Therefore, this strain may be a promising biological resource to completely remove

Fig. 3. Degradation pathway of DDT by the isolate DDT-2 based on both genomic and GC–MS analyses. Solid line represents the degradation pathway of DDT based on genome annotation. The shaded area represents the degradation pathway of DDT based on GC–MS analysis, which also represents the common degradation pathway of DDT based on both the genomic and GC–MS analyses. DDT: 1,1,1-trichloro-2,2bis(p-chlorophenyl)ethane; DDD: 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane; DDE: 2,2-bis(p-chlorophenyl)-1,1-dichlorethylene; DDMU: 1-chloro-2,2-bis(4′chlorophenyl)ethylene; DDNU: unsym-bis(4′-chlorophenyl)ethylene; DDMS: 1chloro-2,2-bis(4′-chlorophenyl)ethane; DDOH: 2,2-bis(4′-chlorophenyl)ethanol; DDA: bis(4′-chlorophenyl)acetate; and DDM: bis(4′-chlorophenyl)methane.

Table 3 Degradation of four DDT metabolites at an initial concentration of 10.0 mg/l by the isolate DDT-2 in mineral salts medium at pH 7.0, 150 rpm and 20 °C (OD600 = 0.4). DDT metabolites

Control without inoculation

Treatment with inoculation

Removal percentages

DDE DDD DDMU DDA

10.18 ± 0.19 9.39 ± 0.10 9.86 ± 0.34 6.45 ± 0.43

7.62 ± 0.61 8.70 ± 0.24 7.05 ± 0.24 ND

25.11% 7.27% 28.54% 100%

Please cite this article as: Pan, X., et al., Characterization and genome functional analysis of the DDT-degrading bacterium Ochrobactrum sp. DDT-2, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2017.03.052

X. Pan et al. / Science of the Total Environment xxx (2016) xxx–xxx

and detoxify DDT and its metabolite residues in contaminated soil. Further studies should be conducted to verify the function of the DDT degradation genes at protein levels. Acknowledgements This work was supported by the National Key Research and Development Program of China (2016YFD0200201), the National Natural Science Foundation of China (No. 21477112, 41271489), and the National High Technology R&D Program of China (No. 2012AA06A204). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2017.03.052. References Bajaj, A., Mayilraj, S., Mudiam, M.K.R., Patel, D.K., Manickam, N., 2014. Isolation and functional analysis of a glycolipid producing Rhodococcus sp. strain IITR03 with potential for degradation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT). Bioresour. Technol. 167, 398–406. Barragán-Huerta, B.E., Costa-Pérez, C., Peralta-Cruz, J., Barrera-Cortés, J., Esparza-García, F., Rodríguez-Vázquez, R., 2007. Biodegradation of organochlorine pesticides by bacteria grown in microniches of the porous structure of green bean coffee. Int. Biodeterior. Biodegrad. 59 (4), 239–244. Cai, X., Sheng, G., Liu, W., 2007. Degradation and detoxification of acetochlor in soils treated by organic and thiosulfate amendments. Chemosphere 66 (2), 286–292. Cao, F., Liu, T.X., Wu, C.Y., Li, F.B., Li, X.M., Yu, H.Y., Tong, H., Chen, M.J., 2012. Enhanced biotransformation of DDTs by an iron- and humic-reducing bacteria Aeromonas hydrophila HS01 upon addition of goethite and anthraquinone-2,6-disulphonic disodium salt (AQDS). J. Agric. Food Chem. 60 (45), 11238–11244. Crombie, A.T., Khawand, M.E., Rhodius, V.A., Fengler, K.A., Miller, M.C., Whited, G.M., McGenity, T.J., Murrell, J.C., 2015. Regulation of plasmid-encoded isoprene metabolism in Rhodococcus, a representative of an important link in the global isoprene cycle. Environ. Microbiol. 17 (9), 3314–3329. Cui, C.Z., Li, P.P., Liu, G., Tang, H.Z., Lin, K.F., Luo, Q.S., Liu, S.S., Xu, P., Liu, Y.D., 2014. Genome sequence of Martelella sp. strain AD-3, a moderately halophilic polycyclic aromatic hydrocarbon-degrading bacterium. Genome Announc. 2 (1), 7687–7693. Delcher, A.L., Bratke, K.A., Powers, E.C., Salzberg, S.L., 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23 (6), 673–679. Eganhouse, R.P., Pontolillo, J., 2008. DDE in sediments of the Palos Verdes shelf, California: in situ transformation rates and geochemical fate. Environ. Sci. Technol. 42 (17), 6392–6398. Erdem, Z., Cutright, T.J., 2016. Biodegradation potential of 1,1,1-trichloro-2,2-bis(pchlorophenyl) ethane (4,4′-DDT) on a sandy-loam soil using aerobic bacterium Alcaligenes eutrophus A5. Environ. Eng. Sci. 33 (3), 149–159. Fang, H., Dong, B., Yan, H., Tang, F.F., Yu, Y.L., 2010. Characterization of a bacterial strain capable of degrading DDT congeners and its use in bioremediation of contaminated soil. J. Hazard. Mater. 184, 281–289. Fang, H., Cai, L., Yang, Y., Ju, F., Li, X.D., Yu, Y.L., Zhang, T., 2014. Metagenomic analysis reveals potential biodegradation pathways of persistent pesticides in freshwater and marine sediments. Sci. Total Environ. S470–471, 983–992. Gao, B., Liu, W.B., Jia, L.Y., Xu, L., Xie, J., 2011. Isolation and characterization of an Alcaligenes sp. strain DG-5 capable of degrading DDTs under aerobic conditions. J. Environ. Sci. Health B 46 (3), 257–263.

7

Grant, J.R., Stothard, P., 2008. The CGView server: a comparative genomics tool for circular genomes. Nucleic Acids Res. 36, W181–W184. Guo, Y., Yu, H.Y., Zeng, E.Y., 2009. Occurrence, source diagnosis, and biological effect assessment of DDT and its metabolites in various environmental compartments of the Pearl River Delta, South China: a review. Environ. Pollut. 157 (6), 1753–1763. Han, Y.L., Shi, N., Wang, H.F., Pan, X., Fang, H., Yu, Y.L., 2015. Nanoscale zerovalent ironmediated degradation of DDT in soil. Environ. Sci. Pollut. Res. 23 (7), 1–11. Heberer, T., Dünnbier, U., 1999. DDT metabolite bis(chlorophenyl)acetic acid: the neglected environmental contaminant. Environ. Sci. Technol. 33 (14), 2346–2351. Jin, X.T., Song, L., Zhao, J.Y., Li, Z.Y., Zhao, M.R., Liu, W.P., 2013. Dichlorodiphenyltrichloroethane exposure induces the growth of hepatocellular carcinoma via, Wnt/β-catenin pathway. Toxicol. Lett. 225 (1), 158–166. Kube, M., Chernikova, T.N., Al-Ramahi, Y., 2013. Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica. Nat. Commun. 4 (2), 113–115. Liu, L., Bai, L.P., Man, C.G., Liang, W.H., Li, F.S., Meng, X.G., 2015. DDT vertical migration and formation of accumulation layer in pesticide-producing sites. Environ. Sci. Technol. 49 (15), 9084–9091. Lovecka, P., Pacovska, I., Stursa, P., Vrchotova, B., Kochankova, L., Demnerova, K., 2015. Organochlorinated pesticide degrading microorganisms isolated from contaminated soil. New Biotechnol. 1 (32), 26–31. Mrema, E.J., Rubino, F.M., Brambilla, G., Moretto, A., Tsatsakis, A.M., Colosio, C., 2013. Persistent organochlorinated pesticides and mechanisms of their toxicity. Toxicology 307 (1416), 74–88. Pavlikova, N., Smetana, P., Halada, P., Kovar, J., 2015. Effect of prolonged exposure to sublethal concentrations of DDT and DDE on protein expression in human pancreatic beta cells. Environ. Res. 142, 257–263. Qin, J., Li, R., Raes, J., Arumugam, M., Burgdorf, K.S., Manichanh, C., et al., 2010. A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464 (7285), 59–65. Qu, J., Xu, Y., Ai, G.M., Liu, Y., Liu, Z.P., 2015. Novel Chryseobacterium sp. PYR2 degrades various organochlorine pesticides (OCPs) and achieves enhancing removal and complete degradation of DDT in highly contaminated soil. J. Environ. Manag. 161, 350–357. Rangachary, L., Rajagopalan, R.P., Singh, T.M., Krishnan, M.H., 2012. Purification and characterization of DDT-dehydrohalogenase from Pseudomonas putida T5. Prep. Biochem. Biotechnol. 42 (1), 60–76. Tamura, K., Dudley, J., Nei, M., 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24 (8), 1596–1599. Thangavadivel, K., Megharaj, M., Smart, R.S.C., 2011. Ultrasonic enhanced desorption of DDT from contaminated soils. Water Air Soil Pollut. 217 (1), 115–125. Wang, G.L., Chen, X., Yue, W.L., Zhang, H., Li, F., Xiong, M.H., 2013. Microbial degradation of acetamiprid by Ochrobactrum sp. D-12 isolated from contaminated soil. PLoS One 8 (12), e82603. Wilson, I.G., 1997. Inhibition and facilitation of nucleic acid amplification. Appl. Environ. Microbiol. 63 (10), 3741–3751. Xie, H., Zhu, L.S., Xu, Q.F., Wang, J., Liu, W., Jiang, J.H., Meng, Y., 2011. Isolation and degradation ability of the DDT-degrading bacterial strain KK. Environ. Earth Sci. 62 (1), 93–99. Xin, J., Liu, X., Liu, W., Jiang, L., Wang, J., Niu, J., 2011. Production and use of DDT containing antifouling paint resulted in high DDTs residue in three paint factory sites and two shipyard sites, China. Chemosphere 84 (3), 342–347. Zerbino, D.R., Birney, E., 2008. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18, 821–829. Zhu, F., Li, L.W., Ma, S.Y., Shang, Z.F., 2016. Effect factors, kinetics and thermodynamics of remediation in the chromium contaminated soils by nanoscale zero valent Fe/Cu bimetallic particles. Chem. Eng. J. 302, 663–669.

Please cite this article as: Pan, X., et al., Characterization and genome functional analysis of the DDT-degrading bacterium Ochrobactrum sp. DDT-2, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2017.03.052