International Biodeterioration & Biodegradation 120 (2017) 192e202

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International Biodeterioration & Biodegradation

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Degradation of three monochlorobenzoate isomers by different

bacteria isolated from a contaminated soil
Changfeng Xu, Xiaoxia Zang, Xing Hang, Xiaomei Liu, Hongxing Yang, Xiaowei Liu,
Jiandong Jiang*
Department of Microbiology, Key Lab of Environmental Microbiology for Agriculture, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural
University, 210095 Nanjing, People's Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: Three monochlorobenzoate isomer-utilizing bacterial strains, Pseudomonas sp. 2-CBA, Pseudomonas sp. 3-
Received 30 December 2016 CBA, and Hydrogenophaga sp. 4-CBA, were isolated from a haloaromatic-contaminated soil in a disused
Accepted 18 February 2017 chemical factory in Nanjing, China. Compared to strain 3-CBA and strain 4-CBA, which could only utilize
Available online 15 March 2017
its corresponding monochlorobenzoate isomer (3-chlorobenzoate and 4-chlorobenzoate) as the sole
carbon source for growth, strain 2-CBA could utilize both 2-chlorobenzoate and 3-chlorobenzoate for
Keywords:
growth. The three monochlorobenzoate isomers were catabolized via three different dechlorination
Monochlorobenzoate isomer
pathways in the three isolates, respectively. 3-Chlorobenzoate was initially catabolized by the benzoate
Degradation
Dechlorination
1,2-dioxygenase (BenABCD) in strain 3-CBA, whereas 2-chlorobenzoate was catabolized by the hybrid 2-
Dioxygenase halobenzoate 1,2-dioxygenase system in strain 2-CBA, which comprised of the terminal oxygenase
component of 2-halobenzoate dioxygenase system (CbdAB) and the reductase component of benzoate
1,2-dioxygenase system (BenC). For 4-chlorobenzoate, it was catabolized via the classic hydrolytic
dehalogenation pathway in strain 4-CBA. While, strain 4-CBA seemed to have a more compact gene
cluster of fcbABC compared to previously reported strains. This study provides new insights into the
catabolic diversity of structurally similar isomers in a contaminated niche.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction chlorinated benzene ring is very essential for the complete

Halogenated aromatics are widely used in agriculture and in-
dustry as pesticides, solvents and fire retardants (Ogawa and
Miyashita, 1995). Many of them have been identified as priority
organic pollutants by the United Kingdom and the US Environ-
mental Protection Agency (Field and Sierra-Alvarez, 2008). Among
the halogenated aromatics, chlorobenzoates are employed as raw
materials in the formulation of dyes, pharmaceuticals, fungicides
and as preserving agents in adhesives and paints (Ogawa and
Miyashita, 1995; Field and Sierra-Alvarez, 2008). In addition,
chlorobenzoates can also originate as dead-end products during
the metabolism of polychlorinated biphenyls (PCBs) and chlor-
otoluenes and tend to accumulate in PCB-contaminated soils and
sediments (Banta and Kahlon, 2007; Field and Sierra-Alvarez, 2008;
Sunday et al., 2008; Bajaj and Singh, 2015). The degradation of
* Corresponding author.
E-mail address: jiang_jjd@njau.edu.cn (J. Jiang).
mineralization of PCBs by microorganisms (Barrag'an-Huerta et al.,
2007). Due to their high solubility in water, chlorobenzoates show a
significant mobility that is several orders of magnitude higher than
that of PCBs. Chlorobenzoates have been reported to exhibit high
toxicity toward aquatic organisms (Field and Sierra-Alvarez, 2008;
Baggi et al., 2008; Lee and Chen, 2009) and genotoxicity toward
higher plants and possess endocrine-disrupting activity
(Svobodov
a et al., 2009). As a result, the environmental fate of
chlorobenzoates is of great concern. Microbial catabolism is the
main pathway for the dissipation of chlorobenzoates in the envi-
ronment (Gallego et al., 2012; Solyanikova et al., 2015; Yao et al.,
2015). Therefore, the mechanism underlying the microbial catab-
olism of chlorobenzoates is of great interest.
Chlorine removal from chlorobenzoates is very important for
the degradation of chlorobenzoates because it reduces both recal-
citrance to biodegradation and the risk of forming toxic in-
termediates during subsequent metabolic steps (Wang et al., 2010).
Monochlorobenzoates (including 2-chlorobenzoate, 3-
chlorobenzoate, and 4-chlorobenzoate) have been used as model

http://dx.doi.org/10.1016/j.ibiod.2017.02.020
0964-8305/© 2017 Elsevier Ltd. All rights reserved.
 C. Xu et al. / International Biodeterioration & Biodegradation 120 (2017) 192e202
compounds to study the mechanisms involved in the microbial
dechlorination of chlorinated aromatics (Furukawa et al., 1979).
Several bacterial strains capable of utilizing 4-chlorobenzoate as
the sole carbon source for growth have been isolated (Ruisinger
et al., 1976; Hoskeri et al., 2011). Interestingly, hydrolytic dehalo-
genation appears to be the only conserved pathway by which 4-
chlorobenzoate is catabolized in aerobes (Ruisinger et al., 1976).
In aerobes, the initial step in the degradation of 2- and 3-
chlorobenzoates is usually dihydroxylation of the substrate, a re-
action that is catalyzed by multicomponent non-heme iron dioxy-
genase systems. The 2-halobenzoate 1,2-dioxygenase system,
which was first identified in Pseudomonas cepacia 2CBS, catalyzes
the double hydroxylation of 2-halobenzoate with the concomitant
release of halide and CO2, yielding catechol (Fetzner et al., 1992).
This dioxygenase system comprises a Rieske-type [2Fee2S] ter-
minal oxygenase component and an NADH:acceptor reductase
component (Fetzner et al., 1992). 3-Chlorobenzoate is initially
converted to chlorobenzoate dihydrodiol (catalyzed by a benzoate
1,2-dioxygenase system) by the insertion of O2 into the aromatic
ring. The intermediate is then converted to chlorocatechol by the
1,6-dihydroxycyclohexa-2,4-diene-1-carboxylate dehydrogenase.
Although the catabolic mechanism of separate mono-
chlorobenzoate isomer has been reported, the diversity involved in
the catabolism of different monochlorobenzoate isomers in a
contaminated niche has not been studied.
In this study, three bacterial strains capable of separately uti-
lizing the three monochlorobenzoate isomers as their sole carbon
source for growth were isolated from a haloaromatic-contaminated
soil. The different pathways use to catabolize each mono-
chlorobenzoate isomer were revealed, and the genes responsible
for each monochlorobenzoate catabolism were also elucidated
based on metabolite identification and functional gene expression
analyses. A hybrid 2-halobenzoate 1,2-dioxygenase system and a
more compact gene cluster of fcbABC were found. Our work pro-
vides insights into the diversity of species, pathways, and genes
that are involved in the catabolism of monochlorobenzoate isomers
at one haloaromatic-contaminated site as revealed at the physio-
logical and biochemical levels.
2. Materials and methods
2.1. Chemicals, media, and bacterial strains
Catechol, 2-chlorobenzoate (98%), 3-chlorobenzoate (99%), 4-
chlorobenzoate (99%), 3-chlorocatechol (99%), and 4-
hydroxybenzoate (99%) were purchased from J&K Chemical
(Shanghai, China). All other reagents used in this study were of
analytical reagent grade. Minimal salt medium (MM, 1.0 g NH4NO3,
1.6 g K2HPO4, 0.5 g KH2PO4, 0.2 g MgSO4, and 1.0 g NaCl per liter of
water, pH 7.0) or Luria Bertani medium (LB) supplemented with
monochlorobenzoate isomers were used for cell culture.
Phosphate-buffered saline (PBS; 8.0 g NaCl, 0.2 g KCl, 1.44 g
Na2HPO4, 0.24 g KH2PO4 per liter of water, pH 7.4) was used when
measuring the degradation activity of recombinants.
The bacterial strains and plasmids used in this study are listed in
Table 1. Escherichia coli DH5a was used as a host for the expression
of different recombinant pUC vectors. When appropriate, antibi-
otics were added to the media at the following concentrations:
kanamycin (Km), 50 mg l
1; ampicillin (Amp), 100 mg l
1.
2.2. Isolation and characterization of monochlorobenzoate isomer-
degrading strains
Monochlorobenzoate isomer-degrading strains were isolated
using a conventional enrichment culture technique (Liu et al.,
193
2014). Soil samples were collected from a disused chemical fac-
tory in Liuhe, Nanjing, China. The soil sample was collected from an
area that been contaminated with halogenated aromatics for more
than ten years. Each enrichment culture comprised 100 ml of MM,
5 g of soil sample, and 0.5 mM of 2-chlorobenzoate, 3-
chlorobenzoate, or 4-chlorobenzoate. Enrichments were incu-
bated at 30  C in a rotary shaker at 150 rpm for a week. The final
enrichment cultures were plated on MM agar supplemented with
0.5 mM of the tested monochlorobenzoate isomer. Colonies from
different enrichment cultures were tested for their ability to
degrade monochlorobenzoate isomers. Strains 2-CBA, 3-CBA, and
4-CBA, which degraded 2-chlorobenzoate, 3-chlorobenzoate, and
4-chlorobenzoate, respectively, were isolated. These strains were
identified based on their morphological, physiological, and
biochemical properties using Bergey's Manual of Determinative
Bacteriology and 16 S rRNA gene sequence analysis as references
(Lane, 1991).
The specific primers E1 (50 -atgtaagctcctggggattcac-30 ) and E2
(5 -aagttagtgactggggtgagcg-30 ) were used in enterobacterial re-
0
petitive intergenic consensus (ERIC)ePCR (Ulton et al., 1991) to
distinguish species belonging to the same genus. In brief, the re-
action contained 20 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM
MgCl2, 200 mM dNTPs, 10 pmol of each primer, 0.5 U of Taq DNA
polymerase, 50 ng DNA template and Millipore H2O (to bring the
final volume to 50 ml). The programming conditions were set as
follows: initial denaturation at 95  C for 5 min, followed by 35
cycles of 94  C for 10 s (denaturation), 92  C for 40 s, 49  C for 8 s,
51.5  C for 60 s and extension at 72  C for 5 min; final extension was
performed at 72  C for 10 min. The amplified products were
separated by electrophoresis in 0.75% w/v agarose gels after
staining with ethidium bromide.
2.3. Degradation of monochlorobenzoate isomers in liquid medium
The strains 2-CBA, 3-CBA, and 4-CBA were pre-cultured in LB at
30  C for 24 h before being washed and re-suspended in MM. To
measure the dynamics of monochlorobenzoate isomer degrada-
tion, cells of the strains 2-CBA, 3-CBA, and 4-CBA were inoculated
separately into 100 ml of MM (containing 0.5 mM of each type of
chlorobenzoate isomer) to obtain a final OD600 nm value of 0.2. The
inoculum was incubated at 30  C on a shaker set at 160 rpm.
Samples were collected periodically to determine the concentra-
tion of each chlorobenzoate isomer and to monitor cell growth. The
residual chlorobenzoate isomers were assayed as described below.
Cell growth was determined by monitoring the optical density
(OD600 nm) of the culture. The chloride ions released during
degradation were detected and quantified according to the method
described by Bergmann and Sainik (1957). All experiments were
carried out in triplicate.
2.4. Monochlorobenzoate isomer detection and metabolite assays
The monochlorobenzoate isomers that were present in the
liquid media were extracted in an equal volume of dichloro-
methane. The extract was dried over anhydrous Na2SO4 and
evaporated using a vacuum rotary evaporator at room temperature.
The resulting residue was dissolved in 500 ml of methanol and then
analyzed using high-pressure liquid chromatography (HPLC, 600
controller, Rheodyne 7725i manual injector and 2487 Dual l
Absorbance Detector; Waters, Milford, MA). Kromasil 100-5 C18 was
used as the stationary phase in the separation column (internal
diameter, 4.6 mm; length, 250 mm). The mobile phase was aceto-
nitrile: water: acetic acid (50:50:0.5/v:v:v), and the flow rate was
1.0 ml min
1. The compounds 2-chlorobenzoate (RT, 5.5 min), 3-
chlorobenzoate (RT, 6.9 min), and 4-chlorobenzoate (RT, 7.1 min)
194 C. Xu et al. / International Biodeterioration & Biodegradation 120 (2017) 192e202

Table 1
Strains and plasmids used in this study.

Strain and plasmid Characteristic Reference or

source

Strain
Pseudomonas sp. 2-CBA Ampr, Chloramphenicol (Cm)r, capable of utilizing 2-chlorobenzoate or 3-chlorobenzoate as the sole carbon source for This study
growth
Pseudomonas sp. 3-CBA Ampr, Kmr, capable of utilizing 3-chlorobenzoate as the sole carbon source for growth This study
Hydrogenophaga sp. 4- capable of utilizing 4-chlorobenzoate as the sole carbon source for growth This study
CBA
E. coli DH5a F
recA1 endA1 thi-1 supE44 relA1 deoR D(lacZYA-argF)U169F 80dlacZD M15 Invitrogen
Plasmid
pUC18 Ampr, the vector contains a multiple cloning site (MCS) for DNA cloning TaKaRa
pUC18-fcbBAC Ampr, A 3.1-kb fragment containing fcbBAC genes obtained from strain 4-CBA inserted into the Xbal/HindIII site of pUC18 This study
pUC19 Ampr, the vector contains a multiple cloning site (MCS) for DNA cloning TaKaRa
pUC19-benABCD Ampr, A 3.8-kb fragment containing benABCD genes from strain 3-CBA inserted into the Xbal/HindIII site of pUC19 This study
pBBR1MCS-2 Cmr, a broad-host-range cloning vector Kovach et al., 1995
pBBR1MCS2-cbdAB Cmr, a 2.2-kb fragment containing cbdAB genes from strain 2-CBA inserted into the BamHI/Xbal site of pBBR1MCS-2 This study
pRK600 Cmr, a helper plasmid for triparental conjugation Finan et al., 1986
pET29a-cbdAB Kmr, a 2.0-kb fragment containing cbdAB genes from strain 2-CBA inserted into pET29a This study
pET29a-benC Kmr, a 1.2-kb fragment containing benC gene from strain 3-CBA inserted into pET29a This study
pET29a-catA Kmr, a 0.9-kb fragment containing catA gene from strain 3-CBA inserted into pET29a This study

were detected at 250 nm (Fetzner et al., 1992). The concentration of
these compounds was determined from the peak area (relative to
individual standard calibration curves).
The metabolites resulting from monochlorobenzoate isomer
degradation were identified using a high-performance liquid
chromatography (HPLC)-mass spectrometry (MS) instrument
(UHPLC-LTQ-Orbitrap, Thermo) equipped with an electrospray
ionization source and operated in the negative polarity mode. For
UV chromatography involving HPLC-MS, all compounds were
detected at 250 nm. Full-scan signals were recorded within an m/z
range of 100e350 m/z at a 60,000 resolution setting in the negative
mode. The auto gain control mode was used to optimize the in-
jection time. The mobile phase was acetonitrile: water: acetic acid
(50:50:0.5/v: v: v), and the flow rate was 1.0 ml min
1.
2.5. Genome sequencing, bioinformatic analysis and the cloning of
genes responsible for monochlorobenzoate isomer degradation
Draft genomes of the three isolates were sequenced using an
Illumina HiSeq 2000 system at Shanghai Majorbio Bio-pharm
Technology Co., Ltd. (Shanghai, China). The draft genome
sequence was assembled using SOAP de novo software (version
1.05; http://soap.genomics.org.cn/soapdenovo.html). De novo gene
prediction was conducted using the Glimmer system (version 3.0;
http://ccb.jhu.edu/software/glimmer/index.shtml). Functional
annotation was accomplished based on the BLAST analysis of pro-
tein sequences in the Non-Redundant protein database, Cluster of
Orthologous Groups, and the Kyoto Encyclopedia of Genes and
Genomes database; an E-value cutoff of 1E
5 was used. The draft
genome sequences of strains 2-CBA, 3-CBA, and 4-CBA have been
deposited in the GenBank database under accession numbers
KY288018, KY288019, and KY288020, respectively.
For pure cultures, the PCR template was obtained by extracting
total genomic DNA according to standard procedures. The fcbBAC
genes that function in 4-chlorobenzoate dehalogenation (Savard
et al., 1986) were amplified using the primers PS2eF (50 -cccaagct-
tagcaacagtccgcagcaattcgt-30 ) and PS2-R (50 -tgctctagaattgcgga-
gattgcccccatt-30 ). The benABCD genes that function in 3-
chlorobenzoate dihydroxylation (Neidle et al., 1987) were ampli-
fied using the primers 3x-18benA (50 -cgagctcatgtccctgggattcgac-
tac-30 ) and 3X-18BenD (50 -tgctctagatcagccgaggtcgccgcccccta-30 ).
The cbdAB genes that function in 2-chlorobenzoate dihydroxylation
were amplified using the primers cbdAB-F (50 -
cgcggatccgcccgcccctcagacaacgt-30 ) and cbdAB-R (50 -tgctctaga-
gaatcaacaagtgaagcatg-30 ). PCR was performed using a Perkin-Elmer
9600 thermal cycler as follows: 94  C for 5 min, followed by 30
cycles of 94  C for 30 s, 64  C for 30 s, and 72  C for 4 min. A final
extension step was carried out at 72  C for 10 min.
The amplified genes involved in the degradation of 3-
chlorobenzoate and 4-chlorobenzoate were digested using Xbal/
HindIII and were ligated separately into pUC18 to generate re-
combinant plasmids. The recombinant plasmids were then trans-
formed into competent E. coli DH5ɑ cells using standard
procedures. The cbdAB genes involved in the degradation of 2-
chlorobenzoate were amplified and ligated into the BamHI/Xbal
sites of the broad-host-range vector pBBR1MCS-2; then, the re-
combinant pBBR1MCS2-cbdA2B2 vector was mobilized from E. coli
DH5ɑ to strain 3-CBA with the aid of the plasmid pRK600. The
functions of the enzymes encoded by these genes were confirmed
by determining the degradative capacities of resting recombinant
cells that heterologously expressed these genes. Resting cells were
prepared as described by Prakash et al. (2011). Growth character-
istics were determined by inoculating the recombinants into
20 ml MM of liquid medium containing 0.5 mM of each mono-
chlorobenzoate isomer and 20 mM sodium pyruvate. The cultures
were shaken (160 rpm) at 30  C. E. coli containing the empty vector
was used as the negative control in all experiments.
The gene catA was cloned from strain 3-CBA and ligated to pET-
29a. The primers used were pET29a-catA-BamHI (50 -cgcggatccat-
gaccgtgaaaatttcccac-30 ) and pET29a-catA-SacI (50 -
0
cgcgagctcggcctcctgcaacgcccgcgg-3 ). The recombinant plasmid was
transformed into E. coli BL21 (DE3). The degradation of catechol/
chlorocatechol and the new product was detected spectrophoto-
metrically by the increase in A260 (Hayaishi et al., 1957).
The complementation of BenC to the function of CbdC was also
confirmed in vitro. The genes benC and cbdAB were amplified using
the primer pairs pET29a-benC-BamHI/pET29a-benC-SacI (50 -
cgcggatccatgagctaccagatcgcactg-3’/50 -cgagctccggatgaaggaaat-
caggccg-30 ) and pET29a-cbdAB-BamHI/pET29a-cbdAB-SacI (50 -
cgcggatccatgagtaccccactcattgca-3’/50 -cgagctctaagcacctactttc-
gaaaat-30 ), respectively. These amplified fragments were ligated to
pET29a and then transformed into competent BL21 (DE3). The host
strains BL21 (DE3) were cultured in LB at 37  C. When the OD600
reached 0.4, 0.2 mM isopropyl-b-D-thiogalactopyranoside (IPTG)
was added and cultured at 16  C for 6 h. The cells were harvested by
centrifugation, and washed twice with 20 mM sodium phosphate
 C. Xu et al. / International Biodeterioration & Biodegradation 120 (2017) 192e202
buffer (pH7.2). The cell suspension was sonicated and centrifuged
at 10,000g for 1 h at 4  C to yield the crude extracts. The assay of
2-chlorobenzoate dioxygenase activity was carried out in a reaction
system containing 500 ml pET29a-benC crude extract, 500 ml
pET29a-cbdAB crude extract, 1 mM Fe(NH4)2(SO4)2, 2 mM flavin
adenine dinucleotide, 2 mM NADH, and 0.3 mM 2-chlorobenzoate
at 30  C. After 1 h, the reaction was terminated by adding sulfuric
acid to a final concentration of 0.5 mM. The concentrations of the
substrate 2-chlorobenzoate and the product catechol were detec-
ted by HPLC. The same reaction system without pET29a-benC crude
extract was used as the negative control.
3. Results
3.1. Isolation of monochlorobenzoate isomer-degrading strains
After one week of enrichment of the soil collected from the
disused chemical factory, no 2-chlorobenzoate, 3-chlorobenzoate
or 4-chlorobenzoate was detectable by HPLC in the corresponding
enrichment cultures. Then, each enrichment culture was plated on
MM agar supplemented with the corresponding mono-
chlorobenzoate isomer as the sole carbon and energy source. After
3e4 days of incubation, colonies grown on the agar that exhibited
unique morphologies were selected and tested for their ability to
degrade monochlorobenzoate isomers. Finally, three bacterial
strains (designated as 2-CBA, 3-CBA, and 4-CBA, showing the
highest degradation activity toward 2-chlorobenzoate, 3-
chlorobenzoate, and 4-chlorobenzoate, respectively) were
obtained.
These isolated strains were identified based on their physio-
logical and biochemical properties and 16 S rRNA gene sequence
analysis. The phylogenetic tree of the 16 S rRNA gene sequences
showed that strains 2-CBA, 3-CBA, and 4-CBA were related to the
Pseudomonas, Pseudomonas and Hydrogenophaga species lineages,
respectively, and were closely clustered with Pseudomonas taiwa-
nensis BCRC17751T, Pseudomonas taiwanensis BCRC17751T, and
Hydrogenophaga atypica BSB41.8T, with sequence similarity scores
of 99.93%, 99.93% and 98.69%, respectively (Fig. S1). Based on their
phenotypic characteristics and phylogenetic analyses, strains 2-
CBA, 3-CBA, and 4-CBA were identified as Pseudomonas, Pseudo-
monas and Hydrogenophaga species, respectively. Strains 2-CBA and
3-CBA were further distinguished using ERIC-PCR (Fig. S2) and a
draft genome sequence comparison; the results showed that the
isolated strains were different species in the genus Pseudomonas
though they exhibited high 16 S rRNA gene similarities.
3.2. Degradation of monochlorobenzoate isomers by strains 2-CBA,
3-CBA, and 4-CBA
Each of the three isolates could utilize the corresponding
monochlorobenzoate isomer as the sole sources of carbon and
energy for growth, suggesting that these strains are capable of
mineralizing these monochlorobenzoate isomers (Fig. 1). Strains 2-
CBA, 3-CBA, and 4-CBA degraded 0.5 mM 2-chlorobenzoate, 3-
chlorobenzoate, and 4-chlorobenzoate within 27 h, 27 h, and 21 h
as the OD600nm value increased from 0.033, 0.028, and 0.042 to
0.105, 0.085, and 0.108, respectively. Interestingly, in addition to 2-
chlorobenzoate, strain 2-CBA could also utilize 3-chlorobenzoate as
the sole carbon and energy sources for growth and could degrade
0.5 mM 3-chlorobenzoate within 72 h as the OD600nm value
increased from 0.023 to 0.060. Compared to strain 3-CBA, strain 2-
CBA was less efficient at degrading 3-chlorobenzoate (Fig. 1).
Strains 3-CBA and 4-CBA only degraded 3-chlorobenzoate and 4-
chlorobenzoate, respectively, and did not degrade any other mon-
ochlorobenzoate isomers. The qualitative release of chloride ions
195
during the degradation of monochlorobenzoate isomers was
qualitatively detected by silver nitrate titration with nitric acid
acidification (Fig. S3), showing that dehalogenation occurred dur-
ing the degradation of the monochlorobenzoate isomers. Equiva-
lent amounts of chloride ions were released during the degradation
of each monochlorobenzoate isomer by quantitative detection
(Fig. 2), showing that the dehalogenation was complete regardless
of whether it occurred before or after ring cleavage.
3.3. Identification of genes involved in the catabolism of
monochlorobenzoate isomers
After genome sequencing and annotation (http://www.ncbi.
nlm.nih.gov/BLAST/), the degradation pathways for the three
monochlorobenzoate isomers and the enzymes involved in the
degradation were proposed, which were similar with previous re-
ports. The functions of the proposed enzymes in strains 2-CBA, 3-
CBA, and 4-CBA (Table S1) were further confirmed by heteroge-
neous expression and enzyme assays.
The FcbABC encoded by fcbABC genes in strain 4-CBA showed
91%e99% identities to the 4-chlorobenzoate-CoA ligase (FcbA), 4-
chlorobenzoyl-CoA dehalogenase (FcbB), and 4-hydroxybenzoyl-
CoA thioesterase (FcbC) from Pseudomonas sp. DJ-12 (Chae et al.,
2000) and Alcaligenes sp. AL3007 (Gulick et al., 2004) (Fig. 3) and
were suspected to catalyze the hydrolytic dehalogenation of 4-
chlorobenzoate. The fcbABC genes were amplified from strain 4-
CBA and ligated into the Xbal-HindIII site of pUC18 to generate
the recombinant plasmid pUC-fcbBAC. The recombinant plasmids
were then transformed into E. coli DH5a to construct recombinant
E-BAC to overexpress FcbBAC. It is found that 0.5 mM 4-
chlorobenzoate was completely degraded in 12 h in 20 ml MM
supplemented with 1 ml LB, releasing an equivalent amount of
chloride ion (Fig. 4). Only a small fraction (<0.1 mM) of 4-
hydroxybenzoate was detected because it could be utilized by the
host strain. The production of 4-hydroxybenzoate was also
confirmed by the HPLC-MS analysis (Fig. 5C). Based on these data, it
appeared that strain 4-CBA catabolized 4-chlorobenzoate to 4-
hydroxybenzoate via the hydrolytic dehalogenation pathway,
which was sequentially catalyzed by FcbA, FcbB, and FcbC (Fig. 6C).
Though the pathway and the genes involved in the degradation of
4-chlorobenzoate are conserved in many strains (Lo €ffler and
Muller, 1991; Zhuang et al., 2003), the fcbABC gene cluster in
strain 4-CBA seemed to be more compact and well evolved than
that in strains DJ-12 and AL3007 (Fig. 3).
The BenABCD encoded by benABCD genes in strain 3-CBA
showed over 99% identities to the large subunit of the hydroxy-
lase (BenA), the small subunit of the hydroxylase (BenB), the
reductase (BenC) of benzoate 1,2-dioxygenase system, and the 1,6-
dihydroxycyclohexa-2,4-diene-1-carboxylate dehydrogenase
(BenD) from Pseudomonas plecoglossicide NyZ12 (Li et al., 2015),
Pseudomonas monteilii SB3101 (Denholm et al., 2014), and Pseudo-
monas putida DLL-E4 (Hu et al., 2014) (Fig. 3) and were speculated
to catalyze the dihydroxylation of 3-chlorobenzoate. The benABCD
genes in strain 3-CBA were cloned and ligated into the Xbal-HindIII
site of pUC19 to generate the recombinant plasmid pUC19-
benABCD, which was then transformed into E. coli DH5a to obtain
the recombinant E-ABCD to overexpress BenABCD. After 6 days,
20% of 0.5 mM 3-chlorobenzoate was degraded by the recombinant
E-ABCD, and the products 3- and 4-chlorocatechols were also
detected (Fig. 5). Although 3-chlorobenzoate was not completely
degraded, the decrease in the level of 3-chlorobenzoate and the
production of chlorocatechols showed that strain 3-CBA catabo-
lized 3-chlorobenzoate to chlorocatechols using the benzoate 1,2-
dioxygenase system. The low 3-chlorobenzoate-degrading activity
in the expressed E. coli strain might be due to inhibition by the
196 C. Xu et al. / International Biodeterioration & Biodegradation 120 (2017) 192e202

Fig. 1. The degradation of 2-chlorobenzoate (A), 3-chlorobenzoate (B), and 4-chlorobenzoate (C) in minimal salt medium shown with the cell growth of strains 2-CBA, 3-CBA, and 4-
CBA. The data are represented as the means ± standard deviation for triplicate experiments. When the error bar is not visible, it lies within the data point.

Fig. 2. The degradation of monochlorobenzoate isomers (-) and the equivalent release of chloride ions (C) by strains 2-CBA (A), 3-CBA (B), and 4-CBA (C) when the corresponding
monochlorobenzoate isomer was used as the sole carbon source (in MM not containing NH4NO3 and NaCl) under aerobic conditions. The data are represented as the
means ± standard deviation for triplicate incubations. When the error bar is not visible, it lies within the data point.
 C. Xu et al. / International Biodeterioration & Biodegradation 120 (2017) 192e202 197

Fig. 3. Comparison of the gene clusters responsible for the catabolism of different monochlorobenzoate isomers in different strains. The host strains are shown to the right of the
gene clusters. Homologous genes are shown using identical colors. Identifies between the proteins encoded by these catabolic genes and homologous genes are shown above the
gene symbols. cbdA encodes the large subunit of the terminal oxygenase component of 2-halobenzoate dioxygenase system; cbdB encodes the small subunit of the oxygenase
component of 2-halobenzoate dioxygenase system; cbdC encodes the NADH:acceptor reductase component of 2-halobenzoate dioxygenase system (Haak et al., 1995; Suzuki et al.,
2000); benA encodes the large subunit of the hydroxylase component of benzoate 1,2-dioxygenase system; benB encodes the small subunit of the hydroxylase component of
benzoate 1,2-dioxygenase system; benC encodes the reductase component of benzoate 1,2-dioxygenase system; benD encodes 1,6-dihydroxycyclohexa-2,4-diene-1-carboxylate
dehydrogenase; catA encodes catechol 1,2-dioxygenase; benK and benE encode benzoate transporters (Denholm et al., 2014; Hu et al., 2014; Li et al., 2015); fcbA encodes 4-
chlorobenzoate-CoA ligase; fcbB encodes 4-chlorobenzoyl-CoA dehalogenase; fcbC encodes 4-hydroxybenzoyl-CoA thioesterase (Chae et al., 2000; Gulick et al., 2004).

produced chlorocatechols. In the wild-type strain 3-CBA, the catA
gene (encoding catechol 1,2-dioxygenase, which catalyzes the
further ring cleavage of the produced chlorocatechol) was also
found downstream of the benABCDK cluster. In the reaction system
containing crude extracts of CatA, the degradation of catechol and
4-chlorocatechol was detected, and the products muconate and 3-
chloromuconate were observed (Fig. 7A and B). Because the dihy-
droxylation of 3-chlorobenzoate by benzoate 1,2-dioxygenase sys-
tem did not release chlorine spontaneously, it was proposed that
the dechlorination of 3-chlorobenzoate occurs after aromatic ring
cleavage in strain 3-CBA (Fig. 6B).
The CbdAB encoded by cbdAB genes in strain 2-CBA showed
97%e98% identities to the large subunit of the terminal oxygenase
component (CbdA) and the small subunit of the oxygenase
component (CbdB) of 2-halobenzoate dioxygenase system from
Burkholderia sp. TH2 (Suzuki et al., 2000) and Pseudomonas cepacia
Fig. 4. The degradation of 4-chlorobenzoate (-), release of chloride ion (C), and
production of 4-hydroxybenzoate (:) by recombinant E-BAC (pUC-fcbBAC).
2CBS (Haak et al., 1995) (Fig. 3) and were proposed to be involved in
2-chlorobenzoate degradation. In the genome sequence of strain 2-
CBA, only the cbdAB genes were clustered together; cbdC, which
encodes the NADH:acceptor reductase component (CbdC), was not
found (Fig. 3). The expression of cbdAB genes alone in E. coli did not
result in the degradation of 2-chlorobenzoate. The reductase
component (BenC) of benzoate 1,2-dioxygenase system encoded by
benC gene from the benABC cluster in strain 2-CBA showed 52%
similarity to CbdC. It was speculated that the benC gene might
complement the function of the cbdC gene in strain 2-CBA,
although strain 3-CBA, which contains the benABC genes, did not
degrade 2-chlorobenzoate. The cbdAB genes that were amplified
from strain 2-CBA were ligated to the broad-host-range vector
pBBR1MCS-2, and the recombinant pBBR1MCS2-cbdAB was then
mobilized from E. coli DH5a into strain 3-CBA to obtain the re-
combinant E-AB-C with the aid of the pRK600 plasmid. It was found
that 0.5 mM 2-chlorobenzoate was completely degraded by resting
cells of the recombinant E-AB-C in 3 days. The recombinant ac-
quired the ability to grow on 2-chlorobenzoate as the sole carbon
source because the produced catechol could be further utilized due
to the presence of the catechol ring cleaving gene catA in the
genome of strain 3-CBA (Fig. 3). BenC was also confirmed to com-
plement the function of CbdC in vitro. In the reaction system con-
taining both crude extracts of BenC and CbdAB, it was found that 2-
chlorobenzoate was transformed to catechol. While, in the negative
control (without BenC), 2-chlorobenzoate remained unchanged. In
summary, the strain 2-CBA catabolized 2-chlorobenzoate using the
hybrid 2-halobenzoate 1,2-dioxygenase system CbdAB/BenC, thus
spontaneously dechlorinating the substrate before aromatic ring
cleavage; catechol was then cleaved from the molecule by the
catechol 1,2-dioxygenase, CatA (Fig. 6A).
198 C. Xu et al. / International Biodeterioration & Biodegradation 120 (2017) 192e202

Fig. 5. HPLC and MS identification of the metabolites produced during the degradation of 2-chlorobenzoate (A), 3-chlorobenzoate (B), and 4-chlorobenzoate (C) by recombinants E-
AB-C, E-ABCD and E-BAC, respectively. The mass spectra show prominent protonated molecular ions at m/z ¼ 109.08 [M-H]
(A), m/z ¼ 143.05 [M-H]
(B), and m/z ¼ 137.04 [M-H]

(C), enabling the assignment of the molecular ions [M
] at m/z ¼ 110, m/z ¼ 144, and m/z ¼ 138; these compounds were identified as catechol, 3-chlorocatechol/4-chlorocatechol,
and 4-hydroxybenzoate, respectively.

4. Discussion microorganisms have evolved multiple mechanisms, such as mu-

The strains Pseudomonas sp. 2-CBA and Pseudomonas sp. 3-CBA
had a lower 3-chlorobenzoate degradation efficiency compared
with Pseudomonas putida (Gallego et al., 2012), which degraded
0.64 mM 3-chlorobenzoate in 14 h with a removal efficiency of
92.0%. While, strain Pseudomonas sp. 2-CBA had a higher 2-
chlorobenzoate efficiency compared with Burkholderia cepacia
(Urgun-Demirtas et al., 2003), which degraded 0.5 mM 2-
chlorobenzoate more than 120 h. Hydrogenophaga sp. 4-CBA
which degraded 0.5 mM 4-chlorobenzoate in 21 h was more effi-
cient than strain SK-3 (Kim and Picardal, 2000), which needed 85 h
to degrade the equal substrate. To adapt to environmental stresses,
tation, DNA rearrangement and horizontal gene transfer (Liang
et al., 2012). Adaptive evolution has enabled some microorgan-
isms to utilize a multitude of exogenous chemicals via catabolism,
thus leading to metabolic diversity (Phale et al., 2007). Chlor-
obenzoates, which are widely used as raw materials and occur as
intermediates in PCB and chlorotoluene catabolism, are universally
present in environments, thus posing a selective pressure for mi-
croorganisms to evolve metabolic pathways that catabolize chlor-
obenzoates. The catabolism of 2-, 3-, and 4-chlorobenzoate isomers
under denitrifying, Fe-reducing, sulfidogenic and methanogenic
conditions in River Nile sediments by anaerobic microbial consortia
has been studied (Kazumi et al., 1995). Furthermore, 33 denitrifying
 C. Xu et al. / International Biodeterioration & Biodegradation 120 (2017) 192e202 199

Fig. 6. The catabolic pathways of 2-chlorobenzoate, 3-chlorobenzoate, and 4-chlorobenzoate in strain 2-CBA (A), strain 3-CBA (B) and strain 4-CBA (C), respectively. The enzymes
involved in the catabolic pathways are shown above the reaction equations.

Fig. 7. Spectrophotometry detection of the transformation of catechol (A) and chlorocatechol (B) by the expressed CatA. The crude CatA was extracted from E. coli containing
pET29a-catA. The products muconate and 3-chloromuconate produced from catechol and chlorocatechol respectively were detected spectrophotometrically by the increase in A260.
200 C. Xu et al. / International Biodeterioration & Biodegradation 120 (2017) 192e202
Proteobacteria that can degrade monofluoro-, monochloro-, or
monobromo-benzoates using nitrate as the terminal electron
acceptor were isolated at various geographical and ecological sites
(Song et al., 2000). However, the utilization of three mono-
chlorobenzoate isomers as the sole carbon source for growth by
pure aerobic cultures isolated from one niche has not been re-
ported. In this study, three pure bacterial strains of Pseudomonas sp.
2-CBA, Pseudomonas sp. 3-CBA, and Hydrogenophaga sp. 4-CBA
were isolated from a haloaromatic-contaminated soil and were
found capable of utilizing 2-chlorobenzoate, 3-chlorobenzoate, and
4-chlorobenzoate, respectively, as the sole carbon source for
growth; this finding illustrates the diversity of bacterial strains that
catabolize monochlorobenzoate isomers present in one niche.
Diverse strains that can degrade isomers appear common and have
also been reported to degrade phthalate and hexa-
chlorocyclohexane (HCH) isomers. Pseudomonas aeruginosa PP4,
Pseudomonas sp. PPD, and Acinetobacter lwoffii ISP4 were found to
utilize phthalate isomers and were isolated from a soil using an
enrichment culture technique (Vamsee et al., 2006). In addition, a
consortium comprising nine bacterial strains and a fungal strain
was found capable of mineralizing a-, b-, g-, and d-HCH isomers
(Elcey and Kunhi, 2010). Diverse strains capable of catabolizing
nitrophenol isomers have also been reported. Arthrobacter sp.
strain NyZ415 (Liu et al., 2010), Pseudomonas sp. strain WBC-3 (Wei
et al., 2010a), Pseudomonas sp. strain NyZ402 (Wei et al., 2010b),
Burkholderia sp. strain SJ98 (Min et al., 2014) utilized para-nitro-
phenol (PNP) as the sole sources of carbon and energy. Cupriavidus
necator JMP134 (Yin et al., 2010) utilized meta-nitrophenol as the
sole sources of carbon, nitrogen, and energy. While, Alcaligenes sp.
strain NyZ215 (Xiao et al., 2007) was able to grow on ortho-nitro-
phenol as the sole sources of carbon, nitrogen, and energy. A con-
sortium consisting of Pseudomonas sp. strain WBC-3, Cupriavidus
necator JMP134, and Alcaligenes sp. strain NyZ215 was inoculated
into soil contaminated with three nitrophenol isomers and was
able to remove all nitrophenols in inoculated soils between 2 and
16 days (Chi et al., 2013). In addition, simultaneous biodegradation
of three mononitrophenol isomers could be achieved by a tailor-
made microbial consortium immobilized in sequential batch re-
actors, providing a pilot study for a novel approach for the biore-
mediation of mixture of mononitrophenol isomers (Fu et al., 2016).
These isolated three strains were found to have narrow sub-
strate range for the tested 26 aromatic compounds (Table S2). More
interestingly, strains 3-CBA and 4-CBA could only degrade 3-halo-
substituted benzoate and 4-halo-substituted benzoate, respec-
tively. The strain 2-CBA was more versatile and could degrade both
2- and 3-halo-substituted benzoates. However, none of the isolates
could simultaneously catabolize all three monochlorobenzoate
isomers, showing that different mechanisms are required for the
catabolism of each isomer. Similarly, most of the above-mentioned
33 denitrifying bacteria exhibited a narrow substrate specificity for
monohalobenzoate isomers, even under denitrifying conditions
(Song et al., 2000). The structure of the isomers significantly
affected the enzymatic reaction even though the isomers had
similar structures (Gabriel et al., 2008). Different isomers might
force microbial hosts to evolve different enzymes to adapt to their
individual structures (Lal et al., 2010). Three metabolic pathways
and three types of dioxygenases were previously found to be
involved in the catabolism of phthalate isomers (Vamsee et al.,
2006). In addition, variant enzymes (with low efficiencies) have
evolved to degrade HCH isomers in addition to the major Lin
pathway for the aerobic degradation of HCH in soil (Lal et al., 2010).
In sphingomonads, a wide variety of lin genes degrade HCH isomers
(Lal et al., 2006).
Microorganisms have evolved different pathways to catabolize
monochlorobenzoate isomers (Fig. 6). The strain 2-CBA first
transformed 2-chlorobenzoate to catechol using a hybrid 2-
halobenzoate 1,2-dioxygenase system (comprising of the large
and small subunits of terminal oxygenase component of 2-
halobenzoate dioxygenase system (CbdAB) and the reductase
component of benzoate 1,2-dioxygenase system (BenC)) and then
cleaved the catechol using catechol 1,2-dioxygenase. The substrate
was dechlorinated before ring cleavage (Fig. 6A). Additionally, the
strain 3-CBA converted 3-chlorobenzoate into chlorocatechol using
benzoate 1,2-dioxygenase and then cleaved the chlorocatechol
using the catechol 1,2-dioxygenase, which was similar in strain 2-
CBA. The substrate appeared to be dechlorinated after ring cleav-
age (Fig. 6B). The strain 4-CBA catabolized 4-chlorobenzoate via the
conserved hydrolytic dechlorination pathway. First, the formation
of a CoA thioester of 4-chlorobenzoate (catalyzed by 4-
chlorobenzoate-CoA ligase) was required; sufficient energy is
available at this step to facilitate the replacement of the 4-chlorine
atom with a hydroxyl group (catalyzed by 4-chlorobenzoyl-CoA
dehalogenase). Finally, CoA is removed by 4-hydroxybenzoyl-CoA
thioesterase to form 4-hydroxybenzoate (Fig. 6C). Although the
genes involved in the catabolism of monochlorobenzoate isomers
that were found in strains 2-CBA, 3-CBA, and 4-CBA showed high
similarity (91e99%) with previously reported genes, a wide variety
of metabolic pathways for degrading different mono-
chlorobenzoate isomers was found in a single niche.
Strains 3-CBA and 2-CBA both contained benABCD genes, which
are present in many bacteria (Fig. 3). Interestingly, the strain 2-CBA
also contains cbdAB genes (showing 95e98% similarity to those
found in strains 2CBS and TH2) but lacks the cbdC gene (Haak et al.,
1995). The gene cluster governing the catabolism of 2-
chlorobenzoate in strain 2-CBA was somewhat different from that
found in Pseudomonas cepacia 2CBS. The benC gene in strain 2-CBA
was presumed to complement the function of the cbdC gene,
indicating that strain 2-CBA has evolved a new recombinant
pathway to catabolize 2-chlorobenzoate. Genetic engineering can
be employed to boost the catabolic efficiency of microorganisms for
use in bioremediation. In this way, it is possible to construct novel
pathways that are not found in nature, which may permit the
mineralization of highly recalcitrant compounds and avoid the
accumulation of toxic dead-end products (Ghosal et al., 2016).
The maintenance of metabolic diversity appears beneficial for
microbial strains or consortia. For example, microorganisms with
diverse metabolic capacities will possess adaptive advantages over
other microorganisms in nutritionally infertile (oligotrophic) en-
vironments by enabling them to utilize alternative carbon sources
and in toxic environments by detoxifying the toxic compounds.
Usually, contaminated sites include many different compounds.
Therefore, if only one species with a specific degrading ability for
one compound was present, other compounds would not be uti-
lized, and the bacteria might be sensitive to other toxic compounds.
The association of different strains with diverse catabolic abilities
improves the versatility of consortia such that they can thrive in
environments that are contaminated with mixed compounds; such
associations enable consortia to survive in different environments.
5. Conclusions
In this study, three bacterial strains with the abilities to
mineralize three monochlorobenzoate isomers respectively were
isolated from one haloaromatic-contaminated soil niche. The genes
that encode the key enzymes catalyzing the initial reactions during
the monochlorobenzoates degradation were further identified and
characterized by heterologous expression and biotransformation
assays. The different pathways used to catabolize each mono-
chlorobenzoate isomer were revealed and 2-chlorobenzoate was
catabolized by the new hybrid 2-halobenzoate 1,2-dioxygenase
 C. Xu et al. / International Biodeterioration & Biodegradation 120 (2017) 192e202
system CbdAB/BenC in strain 2-CBA. All these results provide a
comprehensive understanding of the catabolic diversity of struc-
turally similar isomers in a contaminated niche.
Acknowledgments
This work was supported by grants from the National Key
Research and Development Program of China (2016YFD0800203),
the Fundamental Research Funds for the Central Universities
(KYZ201422), and the joint National Natural Science Foundation of
China and the Israel Science Foundation Research Program
(31461143009).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ibiod.2017.02.020.
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