Front. Environ. Sci. Engin. China 2008, 2(1): 69–72 DOI 10.1007/s11783-008-0019-7

RESEARCH ARTICLE

Isolation of a Pseudomonas Stutzeri strain that degrades 1,2,4-trichlorobenzene and characterization of its degradative plasmid Lei SONG, Hui WANG ( ), Hanchang SHI, Hongying HU Environmental Simulation and Pollution Control State Key Joint Laboratory, Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, China

© Higher Education Press and Springer-Verlag 2008

Abstract The genetic information encoding metabolic pathways for xenobiotic compounds in bacteria often resides on catabolic plasmids. The aim of the present work was to know the location of the genes for degrading 1,2,4trichlorobenzen. In this paper a 1,2,4-trichlorobenzenedegrading strain THSL-1 was isolated from the soil of Tianjin Chemical Plant using 1,2,4-trichlorobenzene as the sole carbon source. The strain was identified as Pseudomonas stutzeri through morphologic survey and 16S rDNA sequence determination. A plasmid was discovered from strain THSL-1 by using the alkali lysis method. When the plasmid was transformed into E. coli. JM109 by the CaCl2 method, the transformant could grow using 1,2,4-trichlorobenzene as the sole carbon source and had the degradation function of 1,2,4-trichlorobenzene. Therefore, it could be deemed that the plasmid carried the degradative genes of 1,2,4trichlorobenzene. The average size of the plasmid was finally determined to be 40.2 Kb using selectively three kinds of restricted inscribed enzymes (HindIII, BamHI, and XholI) for single cutting and double cutting the plasmid pTHSL-1, respectively. Keywords 1,2,4-trichlorobenzene, 16S rDNA, degradative plasmid, Pseudomonas sp.

biologically toxic and structurally highly stable, the residues of 1,2,4-TCB accumulates in the environment [1], which leads to a series of environmental problems. Due to its chemical stability and unique symmetrical structure, biodegradation becomes the most important means to remove 1,2,4-TCB from the environment [2]. Some bacteria have been reported to be able to use 1,2,4TCB as the sole carbon source and energy substrate, including Pseudomonas aeruginosa RH01 [3], Pseudomonas sp. strain PS12 [4], Pseudomonas sp. strain P51 [5], Burkholderia sp. strain PS14 [4], and Dehalococcoides strain CBDB1 [6]. However, most studies in this field were limited to the metabolic properties of bacteria and degradation pathways for 1,2,4-TCB. There has been no report on the degradation mechanism at the molecular level of trichlorobenzene except the study by Van der Meer [5] in 1991, which showed that a 110 Kb plasmid which was able to degrade trichlorobenzene was isolated from Pseudomonas sp. strain P51. Here, we report the isolation of a strain of Pseudomonas stutzeri bacteria, named as THSL-1, using 1,2,4-TCB as the sole carbon source, from the chemical-polluted soil. From the bacteria strain, a new degradative plasmid was discovered with an approximate size of 40.2 Kb. The preliminary study of the plasmid properties is also reported in this paper.

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Introduction

1,2,4-trichlorobenzene (1,2,4-TCB), which is an important industrial chemical in the production of dyes and pharmaceuticals, is widely used as solvent and insecticide. As it is Translated from China Environmental Science, 2005, 25(4): 385–388 [䆥㞾: Ё೑⦃๗⾥ᄺ] E-mail: [email protected]

Materials and methods

Reagents and media 1,2,4-TCB, analytical grade, was purchased from Shanghai Reagent Company. Media included Luria Bertani broth (LB) and mineral salt medium. The mineral salt medium consisted of (per liter of distilled water) 7.0 g of Na2HPO4 · 2H2O, 2.0 g of KH2PO4, 500 mg of (NH4)2SO4, 200 mg of MgSO4 · 7H2O, and 1.0 mL of trace-element solution. The trace-element solution consisted

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of (per liter of distilled water) 600 mg of Ca(NO3)2 · 4H2O, 40 mg of CuSO4 · 5H2O, 200 mg of FeSO4 · 7H2O, 20 mg of ZnSO4 · 7H2O, 20 mg of MnSO4 · 4H2O, 3 mg of H3BO3, and 4 mg of NaMoO4 · 2H2O. Enrichment and isolation The soil samples were collected from Tianjin Chemical Plant, China. For the bacteria strain enrichment, the diluted soil sample was directly plated on the LB agar plates which contained 1,2,4-TCB. The plates were incubated at 30°C. After 4–5 days, colonies grown on the plates were picked up and inoculated into the mineral salt medium. We used 100 mL serum bottles to which 40 mL of LB medium or mineral salt medium was added. 1,2,4-TCB was added to the bottles after sterilization in amounts of 10–20 μL per 40 mL medium. The bottles were incubated at 30°C on a rotary shaker at 150 r/m. Upon growth, subcultures were made and serial dilutions were plated on the mineral salt medium in which 1,2,4-TCB was the sole carbon source and incubated at 30°C. Colonies grown on the plates were isolated and transferred to the serum bottles as described above. Repeated twice like this, the single colony that can grow on 1,2,4-TCB as the sole carbon source was isolated. Analytical method The growth of strain was determined by measuring the optical density at 600 nm and the concentration of chloride ions in the culture medium during the growth was measured by ion-chromatography. Before measuring, the samples were filtrated with 0.45 μm membrane. Identification of bacteria strain The strain was identified by analyzing its 16S rDNA sequence. A single colony was selected and dissolved in 50 mL Tris-EDTA for 15 min in 95°C water bath.PCR primers Pf (5p-AGAGT-TTGATCCTGG-CTCAG-3p) and Pr(5p-TACGGCTACCTTGT-TACGACTT-3p) were used to amplify the fragment of bacteria 16S rDNA. The reaction mixtures (50 μL) for PCR contained 10xbuffer, 1.5 mmol/L MgCl2, 150 μmol/L dNTPs, 75 pmol/L of each primer, and Taq polymerase. The PCR was performed with the following standard thermocycle programme: 94°C for 5 min; 30 cycles of 94°C for 1 min, 46°C for 1 min, 72°C for 2 min, and the last cycle of 72°C for 10 min. Purification, ligation, and transformation of the product of PCR was performed as described by Sambrook et al. [7]. Product sequencing was analyzed by Shanghai Sheng Gong Biological Engineering Company. Finally, the 16S rDNA sequence of the strain was compared with other 16S rDNA sequences reported in GenBank database using the software Vector NTI. Extraction and transformation of plasmid The plasmid DNA was isolated from the strain cultivated on 1,2,4TCB, using the method described by Sambrook et al. [7]. The plasmid was then transformed into E. coli. JM109 according to the method described by Sambrook et al. [7]. The determination of size of plasmid The plasmid size was estimated using single or combination of three restriction enzyme (HindIII, BamHI, and XholI) digestion for single and double enzyme digestion followed by electrophoresis analysis on agarose.

Lei SONG, et al.

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Results and discussion

Isolation and identification of bacteria strain With 1,2,4TCB as the carbon source in the culture as described in “enrichment and isolation”, a strain named as THSL-1 that could grow on 1,2,4-TCB as the sole carbon source and energy was isolated. It was a gram-negative, motile, and rod-shaped organism and has a regular, circular and smooth colony when the strain was cultivated on an LB plate. Compared with other 16S rDNA sequences of the most-similar organism in GenBank database, the 16S rDNA sequence of strain THSL-1 showed 99% similarity to the 16S rDNA of Pseudomonas stutzeri family. Taking all the characteristics together, the strain THSL-1 was tentatively classified as Pseudomonas stutzeri. It has been reported that the strains in Pseudomonas stutzeri family can utilize xylene [8], naphthalene, and methylnaphthalene in crude oil [9], petroleum polyethylene glycol [10], trichloroethylene, and 1,1-acetylendichloride [11] as the carbon source, but not 1,2,4-TCB, which is first reported here. Degradation of 1,2,4-TCB by strain THSL-1 To quantify the ability of the degradation of 1,2,4-TCB, the strain THSL-1 was inoculated into the mineral salt medium containing 1,2,4-TCB as the sole carbon source and incubated at 30°C on a rotary shaker at 150 r/m. The initial concentration of 1,2,4-TCB was 4x10−3 mol/L. Determinations of bacteria density and chloride concentration were performed daily. Due to the release of chloride and the degradation of 1,2,4-TCB that take place synchronously for the chlorinated aromatic compounds, the changes of released chloride can reflect the ability of strain THSL-1 in the degradation of 1,2,4-TCB. The experimental results are shown in Fig. 1. After fiveday culture, the bacteria reached an optical density (OD) of 0.27 while 3.95 mmol/L chloride ions were released into the medium.

U, OD600; O, chloride ion concentration Fig. 1 Growth of strain THSL-1 and release of chloride ion

Detection and transformation of the plasmid The genetic information encoding metabolic pathway for xenobiotic compounds in bacteria often resides on degradative plasmids [5]. In our case, a plasmid named as pTHSL-1 was extracted from strain THSL-1. In order to study the

Isolation of a Pseudomonas Stutzeri strain that degrades 1,2,4-trichlorobenzene and characterization of its degradative plasmid

functional genes on the plasmid, the extracted plasmid was then transformed into E. coli. JM109. As shown in Fig. 2, the transformant has a plasmid DNA band with the same size as pTHSL-1 DNA.

Fig. 2 Electrophoresis analysis on agarose gel for plasmids isolated from THSL-1 and the transformant

E. coli. JM109 used for the plasmid transformation contained no plasmid and could not grow on 1,2,4-TCB. The results for the growth of pTHSL-1-transformated E. coli. JM109 are shown in Table 1. It showed that E. coli. JM109 that harbored pTHSL-1 could grow not only on the LB medium but also on the mineral salt medium plate that only contained 1,2,4-TCB as the sole carbon source, whereas E. coli. JM109 (no plasmid) in control 2 could only grow on the LB plate but not on the mineral salt medium plate. The plasmid that was not transformed into the host strain in control 1 could not grow on either the LB plate or the mineral salt medium plate. It clearly indicated that the transformant has acquired the ability of utilizing 1,2,4-TCB. It also suggested that host strains for the degradative plasmid pTHSL-1 might be generic [12], which facilitates the gene modification in the future. Table 1 Transformation of pTHSL-1 (E. coli. JM109 was host strain) No.

plasmid DNA

test THSL-1 control 1 THSL-1 control 2 distilled water

host strain

mineral salt medium (added 1,2,4trichlorobenzene)

E. coli. JM109 distilled water E. coli. JM109

scarce colonies no colony no colony

luria bertani broth (LB) medium crowed colonies no colony crowed colonies

Degradation of 1,2,4-TCB by pTHSL-1-harbored strain pTHSL-1-transformed E. coli. JM109 was cultivated

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at 37°C on a rotary shaker at 150 r/m with 1,2,4-TCB at the concentration of 4x10−3 mol/L as the sole carbon source. As shown in Fig. 3, after four days culture, the bacteria reached an OD of 0.34 and chloride ions were released into the medium of 2.34 mmol/L (Fig. 3). The relevance of chloride ion concentration in the medium with the transformant growth illustrated that pTHSL-1-transformed E. coli. JM109 was able to degrade 1,2,4-TCB, indicating that the pTHSL-1 carried the gene(s) that could degrade 1,2,4-TCB. Therefore, the isolated plasmid, pTHSL-1, from strain THSL-1 was a degradative plasmid.

U, OD600; O, chloride ion concentration Fig. 3 Growth of E. coli. JM109 (pTHSL-1) and release of chloride ions

Determination of the size of pTHSL-1 The plasmid pTHSL-1 was digested with three different individual restriction endonuclease enzymes (HindIII, BamHI, and XholI) or two enzymes combined, respectively, followed by electrophoresis analysis. The results are shown in Table 2. The numbers of DNA fragments from double enzyme digestion should be equal to the sum of fragment numbers by two single same enzyme digestions. Figure 4 shows the results for the electrophoresis of enzyme-digested plasmid on an agarose gel. Based on these results, the size of pTHSL-1 was Table 2 Fragments and their size of plasmid DNA digested by single and double endonucleases (Kb) No.

HindIII

BamHI

HindIII /BamHI

XholI

1 2 3 4 5 6 7 8 9 10 11 12 13 14 total

22.42 11.18 5.10 1.40 1.30

21.48 16.96 2.46

15.60 11.30 5.00 3.30 2.03 1.40 1.30 0.70

22.42 3.53 3.53 3.02 2.60 2.20 1.10 0.90 0.85

41.40

40.90

40.63

40.15

XholI /HindIII

XholI /BamHI

9.74 4.50 4.19 3.46 3.25 3.00 2.45 2.12 1.80 1.27 1.18 1.02 0.90 0.80 39.68

21.30 3.53 3.53 3.02 2.60 1.52 0.90 0.89 0.70 0.68

38.67

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Lei SONG, et al.

Fig. 4 Digestion of pTHSL-1

estimated as 40.2 Kb (Table 2), which differed from the size of pP51 (110 Kb) reported by Van der Meer [5]. It suggested that the genetic message on pTHSL-1 may be different from that on the reported 110 Kb plasmid. Taken together, pTHSL-1 was able to be replicated and expressed in a generic host, E. coli. JM109; therefore, further characterization of the plasmid at the molecular level might provide an insight into its application.

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Conclusions

A 1,2,4-TCB-degrading strain, named as THSL-1, was isolated from the contaminated soil sample using 1,2,4-TCB as the sole carbon source. The strain was identified as Pseudomonas stutzeri by its morphology and 16S rDNA sequence comparison. The correlation of strain growth and the concentration of released chloride ions reflected the degradation of 1,2,4-TCB by the strain THSL-1. The strain E. coli. JM109 transformed with the plasmid, pTHSL-1, extracted from strain THSL-1 could grow on the mineral medium plate containing 1,2,4-TCB as the sole carbon and energy source and was observed capable of degrading 1,2,4-TCB in the four-day culture experiments. It indicated that the pTHSL-1 was a 1,2,4-TCB-degradative plasmid which was able to inhabit in a generic host. The plasmid pTHSL-1 was analyzed by restriction enzyme digestion followed by gel electrophoresis and its size was estimated as 40.2 Kb. Acknowledgements This work was supported by the National High Technology Research and Development Program of China (863 Program) (Grant Nos. 2002AA601170 and 2002A A601150).

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