Isolation and Characterization of 2,4-D Butyl Ester Degrading

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Isolation and characterization of 2, 4-D butyl ester degrading Acinetobacter sp. ZX02 from a Chinese ginger cultivated soil Lin Xiao, Hai-Fei Jia, In-Hong Jeong, Young-Joon Ahn, and Yong-Zhe Zhu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02140 • Publication Date (Web): 03 Aug 2017 Downloaded from http://pubs.acs.org on August 6, 2017

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Journal of Agricultural and Food Chemistry

RESEARCH ARTICLE

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Agricultural and Environmental Chemistry

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Isolation and Characterization of 2,4-D Butyl Ester Degrading

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Acinetobacter sp. ZX02 from a Chinese Ginger Cultivated Soil

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Lin Xiao1, Hai-fei Jia1, In-Hong Jeong2, Young-Joon Ahn3 and Yong-Zhe Zhu1*

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Changcheng Rd, Chengyang district, Qingdao, Shandong, 266-109, China

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Development Administration, Jeonju 55365, Jeollabuk-do, Republic of Korea

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Republic of Korea

College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University,

Division of Crop Protection, National Institute of Agricultural Science, Rural

Department of Agricultural Biotechnology, Seoul National University, Seoul 08826,

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Running title:

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Isolation and characterization of Acinetobacter sp. ZX02

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ACS Paragon Plus Environment


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■ ABSTRACT: Strain ZX02 was isolated from Chinese ginger cultivated soil

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contaminated with various pesticides, which could utilize 2,4-dichlorophenoxyacetic

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acid butyl ester (2,4-D butyl ester) as the sole carbon source. Based on the sequence

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analysis of 16S rRNA gene as well as the morphological, biochemical and

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physiological characteristics of strain ZX02, the organism belonged to Gram-negative

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bacterium and was identified as Acinetobacter sp. ZX02. The strain ZX02 showed a

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remarkable performance in 2,4-D butyl ester degradation (100% removal in 20 mm; intermediate, zone

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diameter 13–19 mm; resistant, zone diameter 9–12 mm; and no inhibition.34

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Plasmid Detection and Curing. Plasmid of strain ZX02 and its derivative

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bacterium were isolated and detected by using a pLASmix-minipreps kit (Amersham,

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Uppsala, Sweden) according to the manufacturer’s instructions. Electrophoresis in 1%

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agarose was carried out at 90 V for 0.5 h, and plasmid DNA was visualized by

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ethidium bromide staining. 9



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For plasmid curing, strain ZX02 was seeded onto LB broth with ethidium bromide

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(500 mg/L) and shaken （160 r/min, 40 ℃）for 24h.35 The culture diluted with 0.85%

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sodium chloride solution and plated onto LB agar. After incubation of 1 day, colonies

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was subjected to plasmid isolation and visualized in 1% agarose gels. The physical

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loss of plasmid in the cured derivative was confirmed using agarose gel

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electrophoresis of the plasmid DNA preparation of respective cultures, as described

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by Lavanya et al.36 A bacterium with loss of plasmid was selected.

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■ RESULTS

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Strain Isolation and Identification. Ten bacterial strains capable of utilizing

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2,4-D butyl ester as sole source of carbon were isolated after enrichment and

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purification procedures. Strain ZX02 with good growth and high activity of 2,4-D

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butyl ester degradation was selected for further study. The morphological features

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of ZX02 were determined using the scanning electron micrography. Colonies on

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LB plates grew rapidly with a diameter of 4–6 mm. The milky white colonies were

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round, edge neat, smooth surface, and opaque. This strain was a nonmotile,

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Gram-negative, and nonspore-forming bacterium with a morphology columnar

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(1.5–2.5 μm in length and 0.5–0.75 μm in width). Biochemically, the strain was

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oxidase negative, gelatinase negative and urease negative, catalase positive and

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lysine decarboxylase positive. It metabolized glucose oxidatively and reduced

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nitrate. Strain ZX02 was able to grow utilizing glucose, mannitol, melibiose,

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sorbitol, amygdalin, and arabinose as a sole carbon source. However, this organism

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was not able to grow on inositol. Full details of biochemical characteristics of strain 10


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ZX02 are given in Table 1. The genomic G+C content of strain ZX02 was 52  0.2

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mol%. The BLAST results showed that the sequence of 1440 bp of the 16S rRNA

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gene from strain ZX02 exhibited 99% similarity with A. baumannii strain DSM 30007

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(GenBank accession number NR_117677) which lie in the same clade in the

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phylogenetic tree (Figure 2). Based on the characteristics described above, the strain

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ZX02 belongs to the genus Acinetobacter and was designated as Acinetobacter sp.

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ZX02 (GenBank database accession number KM893861).

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Bacterial Growth and 2,4-D Butyl Ester Degradation. The time course for

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bacterial growth and degradation of 2,4-D butyl ester by strain ZX02 in MSM

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containing 300 mg/L of 2,4-D butyl ester are given in Figure 3. Bacterial growth

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reached the maximum cell density at 96 h and remained a relatively constant

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concentration after then. 2,4-D butyl ester was degraded by ZX02, 55% decrease of

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2,4-D butyl ester in MSM within 48 h. The concentration of 2,4-D butyl ester

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decreased from 300 mg/L to an undetected level in 96 h. No degradation of 2,4-D

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butyl ester was observed in uninoculated controls. These results indicated that the

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concentration of a metabolite increased with the degradation of 2,4-D butyl ester by

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strain ZX02, and 2,4-D butyl ester was almost completely degraded in 96 h. The strain

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ZX02 was able to utilize 2,4-D butyl ester as a carbon and energy source (Figure 3).

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Identification of the Metabolite. An additional peak (retention time, 40.77 min)

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was detected in the samples taken from 24–168 h on HPLC. However, the

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intermediate metabolite was not seen in the control samples. HPLC-MS analysis also

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revealed the presence of one metabolite with 40.77 min of retention time and m/z 11



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161.2 (100%) of mass spectrum. The metabolite was identified as 2,4-D (Figure 4),

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compared with the authenticated sample by HPLC-MS. As shown in Figure 3, 2,4-D

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was observed at 24 h, it reached a peak and remained at a relatively constant

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concentration after 96 h. However, the organism could not degrade 2,4-D further, it

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might utilize the degradation product n-butanol as the sole carbon source for growth

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(Figure 3).

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Effect of temperature and pH on growth of strain ZX02. Growth was pH and

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temperature dependent. Strain ZX02 grew between 25 and 35 °C (optimum, 29 °C),

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but growth was negligible at 25 and 35 °C (Figure 5A). The organism grew

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between pHs 5.0 and 10.0 (optimum, 7.0), could grow well in 2,4-D butyl ester as a

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sole carbon source (Figure 5B).

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Plasmid Detection and Curing. A plasmid of approximately 23 kb in size

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(Figure 6, lane 2) was detected in ZX02. To determine whether the 2,4-D butyl

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ester degrading activity was controlled by the plasmid, curing study was conducted

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by growing ZX02 in LB medium with 500 mg/L ethidium bromide at 37 °C for 48

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h. The plasmid cured bacterium of ZX02 was selected and designated ZX021,

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which showed absence of plasmid on agarose gel electrophoresis clearly to confirm

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plasmid elimination of ZX02 (Figure 6, lane 1). The ability of ZX021 to degrade

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2,4-D butyl ester was confirmed by investigating the bacterial growth in MSM

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containing 2,4-D butyl ester at concentration of 300 mg/L (Figure 7). Growth of the

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cured bacterium was completely inhibited and strain ZX021 was not able to utilize

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2,4-D butyl ester in MSM. Our finding supports loss of the plasmid as the basis for 12


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the loss of 2,4-D butyl ester degradation activity. Presumably, the gene which

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degraded 2,4-D butyl ester to 2,4-D was located on the plasmid.

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Antibiotic Sensitivity. The sensitivity of strain ZX02 and its cured bacterium

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ZX021 to the eight test antibiotics was elucidated using paper disc diffusion. The

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growth-inhibiting responses varied according to compound and concentration tested

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(Table 2). At 30 μg/disc, strain ZX02 was susceptible to tetracycline and resistant to

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amoxicillin, trimethoprim and chloramphenicol, while the cured ZX021 was

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intermediate to amoxicillin, polymixin B sulfate, trimethoprim, and chloramphenicol.

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These findings indicated that loss of the plasmid led to the loss of resistant to the

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polymixin B sulfate, and the encoding gene of resistance to polymixin B sulfate may

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be located on the plasmid.

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■ DISCUSSION

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An Acinetobacter sp., designated as strain ZX02, was isolated and identified as

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2,4-D butyl ester degrading bacterium from a 2,4-D butyl ester contaminated Chinese

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ginger cultivated soil. Strain ZX02 exhibited similarity with members of the A.

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baumannii group and lied in same clade in the phylogenetic tree and taxonomical and

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biochemical characterization. However, morphological character of A. baumannii and

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the strain ZX02 were completely different; A. baumannii was a typically short, almost

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round, and rod-shaped (coccobacillus), while the strain ZX02 was typically

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long-shaped bacillus. A. baumannii strains have been reported to degrade various

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chemicals, such as petroleum hydrocarbon of crude oil,

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phenol,39 4-chloroaniline,40 and diclofop-methyl.41

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waste car engine oil,38

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A number of strains that could degrade 2,4-D had been reported, although 2,4-D

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butyl ester degrading bacterial strains are rarely reported. For example, Cupriavidus

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gilardii T-1 degraded 2,4-D 3.4 times faster than the model strain Cupriavidus necator 13



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JMP134, and the degradation pathway of 2,4-D was as follows: 2,4-D underwent

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decarboxymethyl group to form 2,4-dichlorophenol (2,4-DCP), 2,4-DCP was then

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degraded to 3,5-dichlorocatechol (3,5-DCC) via a hydrolysis pathway; 3,5-DCC

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followed by ortho-cleavage of the catechol ring to cis-2-dichlorodiene lactone (CDL),

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and CDL formed presumably through hydrolysis reaction to chloromaleylacetic acid

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(CMA); and CMA might participate in tricarboxylic acid cycle, and finally

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mineralized 2,4-D.42 Mycobacterium sp. YC-RL4 and Camelimonas sp. were capable

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of degrading phthalate acid esters.43,44

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In the current study, strain ZX02 could grow well in 2,4-D butyl ester and might

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utilize this compound as the sole carbon source for growth. This strain was capable of

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totally degrading 2,4-D butyl ester into 2,4-D, although it could not degrade 2,4-D

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further. Presumably, the gene which degraded 2,4-D butyl ester to 2,4-D was located

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on the plasmid. It was able to utilize 2, 4-D butyl ester in a high concentration as a

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sole carbon source, without the supplementation of other co-substrates. Strain ZX02

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was potentially used in bioremediation because of its good growth rate and high

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biodegradation efficiency toward 2,4-D butyl ester. 2,4-D butyl ester is an extremely

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toxic compound to various fresh water and estuarine or marine species.45 Our findings,

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along with previous studies, indicate that stain ZX02 and strain T-142 combination

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could degrade 2,4-D butyl ester effectively and thoroughly.

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Bacterial plasmids plays an important role in the degradation of exogenous

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chemicals. A few of phenoxyacetic herbicides degrading bacteria were found to carry

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plasmids encoding the gene because the degradation some phenoxyacetic herbicide

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such as 2,4-D were identified on the plasmids.29,46 It is known that A. baumannii

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harbors plasmids of different sizes, enabling researchers to identify strains by plasmid

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content.47 Certain plasmids play an important role in the adaptation of natural 14


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microbial populations to petroleum hydrocarbon of crude oil.48 Several bacteria which

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were isolated from different soil samples from Estonian agricultural enterprises, were

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all harbored 2,4-D-degradative plasmids (30–100 kb) containing tfd genes of 2,4-D

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degradation.49,50 In the current study, strain ZX02 harbored a single plasmid of

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approximately 23 kb in size and plasmid curing indicated the single plasmid of

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approximately 23 kb was found to be responsible for carrying genes for 2,4-D butyl

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ester degradation in the strain ZX02. Plasmid curing confirmed that the gene for 2,4-D

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butyl ester in ZX02 was plasmid-borne. Qiu et al.26 studied the isolation of a methyl

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parathion (MP)-degrading bacterium and localization of the responsible degrading

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genes. They reported that Ochrobactrum sp. B2 harbors a plasmid encoding the ability

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to degrade p-nitrophenol, whereas MP-hydrolyzing activity is encoded on the

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bacterial chromosome. This strain could be used in future cell or plasmid-mediated

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bioaugmentation tests (once its plasmid is isolated and characterized) with better

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chances to survive in soil than exogenous bacteria.

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A. baumannii can cause nosocomial infections such as pneumonia, septicaemia,

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urinary tract infections and wound infections, and complicates the treatment of

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nosocomial infections owing to its resistance to a wide range of antibiotics.51,52 The

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sensitivity to eight antibiotics of strain ZX02 was detected. The strain ZX02 was

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susceptible to tetracycline, resistant to amoxicillin, trimethoprim and chloramphenicol,

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while the plasmid cured derivative ZX021 was intermediate to amoxicillin, polymixin

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B sulfate, trimethoprim and chloramphenicol. Plasmid curing of strain ZX02 resulted

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in the loss of resistant to polymixin B sulfate. This finding indicates that the gene of

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resistant to antibiotics of polymixin B sulfate location is on the detected plasmid of

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ZX02. Similar results were obtained by Higgins et al.53 who found that strain A.

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baumannii resisitant to carbapenem of is encoded on a single plasmid of 15



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approximately 30 kb. Resistance to amphotericin maybe the necessary condition of

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survival in the soil contaminated by fungicides for the strain ZX02.

345 346

AUTHOR INFORMATION

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Corresponding Author

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*Phone: +86-532-8608-0523. Fax: +86-532-8608-0640. E-mail: [email protected]

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Funding

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This study was carried out with the support of "Research Program for Agricultural Science

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& Technology Development (Project No. PJ012094)", National Institute of Agricultural

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Sciences, Rural Development Administration, Republic of Korea and the High-Level

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Personnel Research Foundation of Qingdao Agricultural University (No. 631431).

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Notes

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The authors declare no competing financial interest.

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Lin Xiao and Hai-fei Jia contributed equally to this work.

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enriched from soil. FEMS Microbiol. Lett. 2007, 2, 209–216.

477

(41) Smith-Grenier, L. L.; Adkins, A. Isolation and characterization of soil

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microorganisms capable of utilizing the herbicide diclofop-methyl as a sole source of

479

carbon and energy. Can. J. Microbiol. 1996, 3, 21–26.

480

(42) Wu, X.; Wang, W.; Liu, J.; Pan, D.; Tu, X.; Lv, P.; Wang, Y.; Cao, H.; Wang, Y.;

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Hua, R. Rapid biodegradation of the herbicide 2,4-dichlorophenoxyacetic acid by

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Cupriavidus gilardii T-1. J. Agric. Food Chem. 2017, 65, 3711–3720.

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(43) Chen, X.; Zhang, X.; Yang, Y.;Yue, D.; Xiao, L.; Yang, L. Biodegradation of an

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endocrine-disrupting chemical di-n-butyl phthalate by newly isolated Camelimonas sp.

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and enzymatic properties of its hydrolase. Biodegradation 2015, 26,171–182.

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(44) Ren, L.; Jia, Y.; Ruth, N.; Qiao, C.; Wang, J.; Zhao, B.; Yan, Y. Biodegradation

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of phthalic acid esters by a newly isolated Mycobacterium sp. YC-RL4 and the

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bioprocess with environmental samples. Environ. Sci. Pollut. Res. Int. 2016, 16,

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16609–16619.

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(45) Meehan, W. R.; Norris, L.A.; Sesrs, H. S. Toxicity of various formulations of

491

2,4-D to salmonids in southeast Alaska. J. Fish. Res. Board Can. 1974, 4, 480–486.

492

(46) Don, R. H.; Pemberton, J. M. Genetic and physical map of the

493

2,4-dichlorophenoxyacetic acid-degradative plasmid pJP4. J. Bacteriol. 1985, 1, 466–

494

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495

(47) Dorsey, C. W.; Tomaras, A. P.; Actis, L. A. Sequence and organization of pMAC, 22


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497

resistance. Plasmid 2006, 2, 112–123.

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nitrifying bacteria isolated from oil-impacted mangrove sediment in the Niger Delta

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of Nigeria. Bull. Environ. Contam. Toxicol. 2012, 6, 1020–1026.

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(49) Vedler, E.; Vahter, M.; Heinaru, A., The completely sequenced plasmid

502

pEST4011 contains a novel IncP1 backbone and a catabolic transposon harboring tfd

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genes for 2,4-dichlorophenoxyacetic acid degradation. J. Bacteriol. 2004, 186, 7161–

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7174.

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(50) Itoh, K.; Tashiro, Y.; Uobe, K.; Kamagata, Y.; Suyama, K.; Yamamoto, H., Root

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nodule Bradyrhizobium spp. Harbor tfdAα and cadA, homologous with genes

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encoding

508

Microbiol. 2004, 70, 2110–2118.

509

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antimicrobial resistance, and treatment options. Clin. Infect. Dis. 2008, 46, 1254–

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1263.

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(52) Héritier, C.; Dubouix, A.; Poirel, L.; Marty, N.; Nordmann, P. A nosocomial

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outbreak of Acinetobacter baumannii isolates expressing the carbapenem-hydrolysing

514

oxacillinase OXA-58. J. Antimicrob. Chemother. 2005, 1, 115–118.

515

(53) Higgins, P. G.; Poirel, L.; Lehmann, M.; Nordmann, P.; Seifert, H. OXA-143, a

516

novel carbapenem-hydrolyzing class D-lactamase in Acinetobacter baumannii.

517

Antimicrob. Agents Chemother. 2009, 12, 5035–5038.

2,4-dichlorophenoxyacetic

acid-degrading

proteins.

518 23


Appl.

Environ.


519

Figure captions

520

Figure 1. Structure of 2,4-D butyl ester.

521

Figure 2. Phylogenetic tree based on the 16S rRNA gene sequences of 2,4-D butyl

522

ester degrading strain ZX02. GenBank accession numbers are given in parentheses.

523

The scale bar indicates 0.2 substitutions per nucleotide position.

524

Figure 3. Bacterial growth and corresponding degradation of 2,4-D butyl ester as a

525

sole carbon source for Acinetobacter sp. ZX02. The error bars indicate standard

526

deviations of the means (n = 3). ◆ The optical density (OD) of culture; ▲ 2,4-D

527

butyl ester concentration in mineral salts basal medium (MSM); ● 2,4-D butyl ester

528

concentration in a noninoculated control; ■ 2,4-D concentration in MSM.

529

Figure 4. Mass spectra of a metabolite of 2,4-D butyl ester degradation and 2,4-D

530

standard.

531

Figure 5. Bacterial growth at various temperatures (A) and pHs (B). The error bars

532

indicate standard deviations of the means (n = 3).

533

Figure 6. Agarose gel electrophoresis profile of plasmid isolated from strain

534

Acinetobacter sp. ZX02 and plasmid-cured strain ZX021. Lanes 1, Acinetobacter sp.

535

strain ZX02; 2, plasmid isolated from strain ZX02, Approximately 23 kb plasmid in

536

size was observed; 3, λ DNA Hind Ⅲ digest.

537

Figure 7. Growth pattern of Acinetobacter sp. ZX02 (solid triangle) and its plasmid

538

cured bacterium ZX021 (solid square) in mineral salts basal medium supplemented

539

with 300 mg/L 2,4-D butyl ester. The error bars indicate standard deviations of the

540

means (n = 3).

541

24


Page 24 of 41

Page 25 of 41

542 543


Figure 1.

544 545 546 547 548

25



549

Page 26 of 41

Figure 2.

550

94

100 Acinetobacter sp. ZX02 (KM 893861) Acinetobacter baumannii strain DSM 30007 (NR 117677) Acinetobacter calcoaceticus PHEA-2 strain PHEA-2 (NR 102826)

55

Acinetobacter tjernbergiae strain 7N16(NR 028850.1) Acinetobacter baylyi strain B2 (NR 028848)

100

82

56

Acinetobacter towneri strain AB1110 (NR 028849) Enhydrobacter aerosaccus strain G (NR 029005)

Acinetobacter schindleri strain LUH5832 (NR 025412)

93

99

Acinetobacter lwoffii DSM 2403 (NR 026209)

Acinetobacter radioresistens strain FO-1(NR 026210) Acinetobacter junii strain DSM 6964 (NR 026208) 71

551

Acinetobacter johnsonii strain ATCC 17909 (NR 044975) 0.1

552

26


Page 27 of 41

553


Figure 3.

554 555 556

27



557 558

Figure 4.

559 560 561

28


Page 28 of 41

Page 29 of 41

562 563 564


Figure 5.

565 566 567

29



568

Figure 6.

569 570

30


Page 30 of 41

Page 31 of 41

571 572


Figure 7.

573 574 575

31



576 577

Table 1. Biochemical characteristics of strain ZX02 Characteristics

578

Response

β-Galactosidase

+

Arginine dihydrolase

+

Lysine decarboxylase

+

Ornithine decarboxylase

+

Citrate utilization

+

H2S production

-

Urease

-

Tryptophane deaminase

-

Indole production

-

Acetoin production

+

Gelatinase

-

Glucose

+

Mannitol

+

Inositol

-

Sorbitol

+

Rhamnose

+

Sucrose

+

Melibiose

+

Amygdalin

+

Arabinose

+

Cytochrome-oxidase

-

Catalase

+

NO2 production

+

“+”, positive; “-”, negative.

579

32


Page 32 of 41

Page 33 of 41


580

Table 2. Zone diameter of growth inhibition of strain ZX02 and its cured bacteria

581

ZX021 against eight antibiotics tested Antibiotics

Zone diameter (mm) of at each concentration (g/disc) ZX02

582

ZX021

30

10

6

30

10

6

Amoxicillin

12

NIa

NI

15

12

NI

Amphotericin

NI

NI

NI

NI

NI

NI

Metronidazole

NI

NI

NI

NI

NI

NI

Polymixin B sulfate

NI

NI

NI

14

12

NI

Tetracycline

21

21

18

23

23

21

Trimethoprim

11

11

NI

14

14

12

Vancomycine

NI

NI

NI

NI

NI

NI

Chloramphenicol

11

11

11

14

13

11

a

No inhibition.

583

33



584 585

586 587 588 589

TOC Graphic

590 591 592

34


Page 34 of 41

Page 35 of 41


Figure 1. Structure of 2,4-D butyl ester. 83x32mm (300 x 300 DPI)



Phylogenetic tree based on the 16S rRNA gene sequences of 2,4-D butyl ester degrading strain ZX02. GenBank accession numbers are given in parentheses. The scale bar indicates 0.2 substitutions per nucleotide position. 258x102mm (300 x 300 DPI)


Page 36 of 41

Page 37 of 41


Figure 3. Bacterial growth and corresponding degradation of 2,4-D butyl ester as a sole carbon source for Acinetobacter sp. ZX02. The error bars indicate standard deviations of the means (n = 3). ◆ The optical density (OD) of culture; ▲ 2,4-D butyl ester concentration in mineral salts basal medium (MSM); ● 2,4-D butyl ester concentration in a noninoculated control; ■ 2,4-D concentration in MSM. 133x78mm (300 x 300 DPI)



Figure 4. Mass spectra of a metabolite of 2,4-D butyl ester degradation and 2,4-D standard. 97x105mm (300 x 300 DPI)


Page 38 of 41

Page 39 of 41


Figure 5. Bacterial growth at various temperatures (A) and pHs (B). The error bars indicate standard deviations of the means (n = 3). 97x134mm (300 x 300 DPI)



Figure 6. Agarose gel electrophoresis profile of plasmid isolated from strain Acinetobacter sp. ZX02 and plasmid-cured strain ZX021. Lanes 1, Acinetobacter sp. strain ZX02; 2, plasmid isolated from strain ZX02, Approximately 23 kb plasmid in size was observed; 3, λ DNA Hind Ⅲ digest. 115x127mm (300 x 300 DPI)


Page 40 of 41

Page 41 of 41


Figure 7. Growth pattern of Acinetobacter sp. ZX02 (solid triangle) and its plasmid cured bacterium ZX021 (solid square) in mineral salts basal medium supplemented with 300 mg/L 2,4-D butyl ester. The error bars indicate standard deviations of the means (n = 3). 112x78mm (300 x 300 DPI)


Isolation and Characterization of 2,4-D Butyl Ester Degrading

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