Functional identification of gene cluster for the ...

ARTICLES Chinese Science Bulletin 2005 Vol. 50 No. 15 1612 1616

Functional identification of gene cluster for the aniline metabolic pathway mediated by transposable element 1

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LIANG Quanfeng , CHEN Ming , XU Yuquan , 1 1 1 ZHANG Wei , PING Shuzhen , LU Wei , 1 1 1 SONG Xianlong , WANG Weiwei , GENG Lizhao , 2 1 Takeo Masahiro & LIN Min 1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 2. Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2201, Japan Correspondence should be addressed to Lin Min (email: linmin57@ vip.163.com)

Abstract A convenient and widely applicable method has been developed to clone aniline metabolic gene cluster in this study. Three positive recombinant plasmids pDA1, pDB2 and pDB11 were cloned from genomic library of aniline degradation strain AD9. The result of aniline dioxygenase (AD) activity and catechol 2,3-oxygenase (C23O) activity assay showed that pDA1 and pDB11 contain aniline dioxygenase genes and catechol 2,3-dioxygenase genes, respectively. The sequence analysis of the total 24.7-kb region revealed that this region contains 25 ORFs, of which 17 genes involve metabolism of aniline. In the gene cluster, the first five genes (tadQTA1A2B) and the subsequent gene (tadR1) were predicted to encode a multi-component aniline dioxygenase and a LysR-type regulator, respectively, while the others (tadD1C1D2C2EFGIJKL) were expected to encode metacleavage pathway enzymes for catechol degradation. The gene cluster was surrounded by two IS1071 sequences. Keywords: aniline, biodegradation, transposable element , gene cluster. 　 DOI: 10.1360/982005-732

A large quantity of anilines are used in the manufacture of pesticides, herbicides, dyes, plastics, and pharmaceuticals[1,2]. Many aniline-degrading bacteria are able to de― grade aniline and/or its derivatives[3 5]. Aniline oxygenase is the enzyme necessary for the first step in the initial catabolism of aniline under aerobic conditions. Some bacterial aniline dioxygenase (AD) genes cloned by various cloning strategies were used by different groups. Fumiyasu cloned AD gene from UCC22 by using Ani- mutant as host strain[6]; Shuichiro and Urata fished out AD gene from Delftia acidovorans Strain 7N by Southern blotting probed with PCR amplified fragment which is conserved in bacteria[7,8]. All of these methods have their own limitations. Therefore, aniline dioxygenase gene clusters have been cloned and sequenced just from Pseudomonas putita UCC22[6], Acinetobacter sp. YAA[9] , Frateuria species 1612

ANA-18[7] and D. acidovorans 7N[8]. In these strains, gene clusters responsible for the complete conversion of aniline to TCA-cycle intermediates have been cloned only from the aniline degradative plasmids pTDN1 of P. putida UCC22[10] and pYA1 of Acinetobacter sp. YAA[11]. A highly efficient aniline degradation strain AD9 was isolated from activated sludge drained from a textile dying plant. Strain AD9 could grow under the concentration of 4500 mg/L aniline, and could degrade 1000 mg/L aniline completely within 18 h. This study developed a convenient and widely applicable method to clone aniline metabolic gene cluster. 1 1.1

Experimental Strain

Highly efficient aniline degradation strain was isolated from the activated sludge drained from a textile dying plant in Guangzhou, China. 1.2

Construction of the genomic library of strain AD9

The genomic DNA of strain AD9 was obtained by the method of Wilson[12]. The genomic DNA obtained was partially digested by HindIII or EcoRI. DNA fragments from 10 to 20 kb were purified from the gel using a QIAEXII Gel Extraction Kit (QIAEXII) and ligated to pUC19 digested by HindIII or EcoRI, and introduced into E. coli JM109. 1.3

Enzyme assays

Aniline oxygenase activity was measured using a Clark-type oxygen electrode[9]. Catechol 2,3-dioxygenase (C23O) activity was measured spectrophotometrically by increasing absorbance at 375 nm concomitant with the formation of 2-hydroxymuconic semialdehyde[13]. 2

Results and discussion

A genomic library was constructed with the HindIIIdigested total DNA of AD9 and introduced into competent E. coli JM109. When transformants were screened on LB plates containing X-gal, IPTG and Ap (50 µmL), about 10000 colonies containing inserted fragment, were obtained, and then the genomic library of strain AD9 was constructed. In this study, we first cloned AD gene from AD9 by using a new strategy. Briefly, recombinants containing AD gene cluster that indicates the accumulation and auto-oxidation of catechol formed from aniline are able to form brown colonies. By constructing partial genomic DNA library and screening positive colonies with the method described above, we successfully cloned AD gene from AD9. The incompletely degradation products of aniline show different colors. When the step of catechol degradation is blocked, the accumulation and auto-oxidation of catechol formed from aniline show brown color; when C23O overexpresses or the step of HMS degradation is blocked, Chinese Science Bulletin

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ARTICLES the accumulation of HMS shows brilliant yellow color. Therefore, we had developed a convenient and widely applicable method to clone aniline degradation gene clusters. We screened three positive clones containing aniline metabolic gene cluster from genomic libraries: (i) When transformants were screened on LB plates containing aniline (300 mg/L) and Ap (50 µg/mL), positive colony showed a brown color on the plates, indicating accumulation of catechol resulting from aniline oxidation (Fig. 1(a)). A recombinant plasmid was extracted from one of the positive colonies and analyzed using restriction enzymes. The restriction analysis revealed that the recombinant plasmid, designated pDA1, has a 9.3-kb HindIII insert in the vector pUC19. (ii) The transformants were screened again by spraying their colonies with 0.1 mol/L catechol solution (in 10 mmol/L phosphate buffer, pH7.0). One colony showed a brilliant yellow color on the plate, indicating C23O activity (Fig. 1(b)). The C23O positive strain contained a recombinant plasmid, designated pDB11, which had a 15.4-kb HindIII insert fragment. Restriction analysis showed that the insert fragment of pBD11 did not overlap with that of pDA1. (iii) To confirm whether both insert fragments were situated next to each other, the transformants obtained from another library constructed with the EcoRI-digested AD9 DNA were screened by the catechol spray. Consequently, one C23O positive strain was obtained. The strain contained a recombinant plasmid, which had an 8.2-kb EcoRI insert fragment and was designated pDB2. Restriction analysis of pDB2 revealed that, as shown in Fig. 2, the insert fragment of pDB2 overlapped with both inserts of pDA1 and pDB11 and that the insert of pDB11 was next to that of pDA1.

The E. coli cells harboring pDA1 showed apparent aniline oxygenase activity (32±3 mg O2· g dry wt−1·h−1). To evaluate the C23O activity, the cell extract of the recombinant strain pDB2 was prepared and used for the measurement of C23O activity. The results showed 3.2 units/mg crude protein of C23O activity and pDA1 and pDB11 contained AD genes and C23O genes, respectively. The DNA fragments cloned in pDA1, pDB2, and pDB11 were sequenced, and finally, the nt sequence of the total 24.7-kb region was determined (GenBank accession number AY940090). Homology searches were carried out in order to find gene candidates. It was found that the region contained 25 ORFs (Table 1), at least of which tadQTA1A2BR1D1C1D2C2EFGIJKL were expected to be involved in the complete metabolism of aniline to TCA-cycle intermediates shown in Fig. 3. It was found that the gene cluster was surrounded by two IS1071 sequences (Table 1 and Fig. 2). tadR1 was expected to encode a LysR-type regulator. tadQTA1A2B, which are controlled by a promoter, encoded a multi-component ADs. tadD1C1D2C2EFGIJKL, which were controlled by a promoter, were expected to encode meta-cleavage pathway enzymes for catechol degradation. The pathway contained two sets of plant-type ferredoxin (TadD1 and TadD2) and C23O (TadC1 and TadC2) genes for ring fission of catechol. The remaining tadEFGIJKL gene cluster was required for the degradation of the ring fission product 2-hydroxymuconic semialdehyde (HMS) to pyruvate and acetyl CoA. The cluster contained genes encoding HMS dehydrogenase (HMSD), HMS hydrolase (HMSH), 2-oxopent-4-dienoate (OE) hydratase (OEH),

Fig. 1. Function-based cloning of aniline metabolic gene cluster of strain AD9. (a) pDA1, a positive clone containing AD genes and showing a brown color on the plate containing aniline; (b) pDB11, a positive clone containing C23O genes showing a brilliant yellow color by spraying catechol.

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Fig. 2. Genetic organization of the aniline degradation tad gene cluster of D. tsuruhatensis AD9. pDA1, inserted HindIII fragment containing AD genes; pDB11, inserted HindIII fragment containing C23O genes; pDB2, inserted EcoRI fragment containing C23O genes.

acetaldehyde Dehydrogenase (ADA), 4-hydroxy-2oxovalerate (HO) aldolase (HOA), 4-oxalocrotonate (OC) decarboxylase (OCD) and OC tautomerase (OCT). As shown in Table 1, among 25 ORFs in the tad genes, 20 ORFs shared striking identity to those of the plasmid-encoded tdn genes of P. putida UCC22. The remaining 5 ORFs in tad gene cluster had not ever been found in tdn gene cluster. orf11 was expected to encode a LysR-type regulator; however, the function of the gene product has not been characterized as yet. orf10 was expected to encode an unknown protein. In the region downstream of tadL, there are three ORFs, orf22, 23, 24, whose Table 1 Gene products of aniline metabolic gene cluster strain of AD9 and homology with corresponding proteins of P. putita UCC22　 Homologous proteins ORF Putative function (sequence identity) 1 Transposase TnpA (96%) 2

Amino group transfer

3

Amino group transfer

TdnQ (94%) TdnT (84%)

4

Large subunit of terminal dioxygenase

TdnA1 (96%)

5

Small subunit of terminal dioxygenase

TdnA2(92%)

6

Electron transfer protein

TdnB (84%)

7

LysR-type regulator

TdnR (91%)

8

Plant-type ferredoxin

TdnD (73%)

9

Catechol 2,3-dioxygenase

TdnC (97%)

10

Unknown product

11

LysR-type regulator

12

Plant-type ferredoxin

TdnD2(83%)

13

Catechol 2,3-dioxygenase

TdnC2(93%)

14

Unknown product

ORF4(76%)

15

2-HMS dehydrogenase

TdnE(94%)

16

2-HMS hydrolase

TdnF(89%)

17

2-Oxopent-4-dienoate hydratase

TdnG(87%)

18

Acetaldehyde dehydrogenase

TdnI(93%)

19

4-Hydroxy-2-oxovalerate aldolase

TdnE(93%)

20

4-Oxalocrotonate decarboxylase

TdnK(93%)

21

4-Oxalocrotonate tautomerase

TdnL(100%)

22

Transcriptional regulator

23

Hydrolases or acyltransferases

24

Muconate cycloisomerase

25

Transposase

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TnpA (100%)

gene products shared considerable similarity to MarR-type regulators, â-ketoadipate enol-lactone hydrolases, and muconate cycloisomerases, respectively (Table 1). The tad pathway possesses two routes for conversion of HMS to OE: one is the hydrolytic branch catalysed by HMSH and the other is OC branch catalysed by HMSD, OCT and OCD. The enzymatic steps that covert the intermediate OE to TCA-cycle intermediates pyruvate and acetyl CoA are catalysed by OEH, HOA, ADA (Fig. 3). Many aniline-degrading bacteria have been isolated. ― Bacterial species of Alcaligenes[14], Pseudomonas[15 17], [18] [19,20] [21] Acinetobacter , Rhodococcus , Frateuria , Morax― ella[22], Nocardia[23], and Delftia[24 26] are able to degrade aniline and/or its derivatives. The 16S rDNA sequence homology comparison (GenBank accession number　 AY89912), the G+C content and the hybridization rate between AD9 and the type strain, together with the result of phenotypic characteristics, AD9 was identified as a strain of D. tsuruhatensis. Furthermore, the PFGE and Southern hybridization revealed that tad gene cluster is located on the AD9 chromosome (data not shown). D. tsuruhatensis was established as a new bacterial species, and D. tsuruhatensis T7 (the type strain of D. tsuruhatensis) is not able to degrade aniline[27]. Gene clusters responsible for the complete conversion of aniline to TCAcycle intermediates have been cloned only from the aniline degradative plasmids[10,11]. Transposons that widely exist in the biodegradation xenobiotics have great ability to adapt to environmental pollutants. Catabolic transposons that allow the horizontal spreading of degradative genes among microbial communities in different ways and mediate genetic rearrangements, such as insertions, deletions, duplications and inversions, are attributable to the presence of elements that possess the ability to mobilise the catabolic genes. This greatly expands the substrate range of the microorganisms in the environment and enhances the evolution of novel degradation pathways. This enhanced metabolic versatility can be exploited and is believed to play a major part in the bioremediation of polluted environments[28]. The tad gene cluster showed significant similarity in nucleotide sequence and genetic organization to the plasmid-encoded aniline degradation gene cluster of P. putida UCC22 (Table 1). The similarities in genetic organization and nucleoChinese Science Bulletin

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Fig. 3.

Fig. 4.

Putative aniline metabolic enzymes and pathway of strain AD9.

Catechol is a central intermediate in the degradation pathways of various aromatic compounds.

tide sequence suggest that the aniline metabolic gene clusters are located on transposons . Takeo et al. found that culture medium (LB plus aniline) of subclone containing AD gene cluster of Acinetobacter sp．　strain YAA is brownish[11]. Catechol is a central intermediate in the degradation pathways of various aromatic compounds such as phenol, naphthalene, mandelic acid and salicylic acid (Fig. 4)[29,30]. This study provides a possible functional screening strategy. Convenient and widely applicable it can be used to clone genes that encode the key enzyme of other aromatic compounds degradation pathway, such as phenol hydroxylase, naphthalene dioxygenase, etc. Chinese Science Bulletin

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Acknowledgements This work was supported by 863 Project (Grant No. 2005AA226030) of the Ministry of Science and Technology and the National Natural Science Foundation of China (Grant Nos. 30470047 & 30200007).

References 1. Meyer, U., Biodegradation of synthetic organic colorants, in Microbial Degradation of Xenobiotics and Recalcitrant Compounds (eds. Leisinger, T., Hütter, R., Cook, A. M. et al.), London: Academic Press, 1981, 371―385. 2. Kearney, P. C., Kaufman, D. D., Herbicides: Chemistry, Degradation, and Mode of Action, 2nd ed., New York: Marcel Dekker, Inc., 1975. 3. Bollog, J. M., Blattmann, P., Laanio, T., Adsorption and transformation of four substituted anilines in soil, J. Agric. Food Chem.,

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ARTICLES 1978, 26: 1302―1306. 4. Lyons, C. D., Katz, S., Bartha, R., Mechanisms and pathways of aniline elimination from aquatic environment, Appl. Environ. Microbiol., 1984, 48: 491―496. 5. Lyons, C. D., Katz, S., Bartha, R., Persistence and mutagenic potential of herbicide-derived aniline residues in pond water, Bull. Environ. Contam. Toxicol., 1985, 35: 696―703. 6. Fukymori, F., Saint, C. P., Nucleotide sequences and regulational analysis of involved in conversion of aniline to catechol in Pseudomonas putida UCC22 (pTDN1), J. Bacteriol.,1997, 179: 399― 408. 7. Murakami, S., Hayashi, T., Maeda, T. et al., Cloning and functional analysis of aniline dioxygenase gene cluster, from Frateuria species ANA-18, that metabolizes aniline via an ortho-cleavage pathway of catechol, Biosci. Biotechnol. Biochem., 2003, 67: 2351― 2358. 8. Urata, M., Uchida, E., Nojiri, H. et al., Genes involved in aniline degradation by Delftia acidovorans strain 7N and its distribution in the natural environment, Biosci. Biotechnol. Biochem., 2004, 68: 2457―2465. 9. Fujii, T., Takeo, M., Maeda, Y., Plasmid-encoded genes specifying aniline oxidation from Acinetobacter sp. strain YAA, Microbiology, 1997, 143: 93―99. 10. Fukymori, F., Saint, C. P., Complete nucleotide sequence of the catechol metabolic region of plasmid pTDN1, J. Gen. Appl. Microbiol., 2001, 47: 329―333. 11. Takeo, M., Fujii, T., Maeda, Y., Sequence analysis of the genes encoding a multicomponent dioxygenase involved in oxidation of aniline and o-toluidine in Acinetobacter sp. strain YAA, J. Ferment. Bioeng., 1998, 85: 17―24. 12. Wilson, K., Preparation of genomic DNA from bacteria, in Current protocols in molecular biology (eds. Ausubel, F. M., Brent, R., Kingston, R. et al.), New York: Wiley, 1987, 2.4.1―2.4.5. 13. Nakazawa, T., Yokota, T., Benzoate metabolism in Pseudomonas putida (arvilla) mt-2, demonstration of two benzoate pathways, J. Microbiol., 1973, 115: 262―267. 14. Rhodes, M. E., Aniline utilization by Alcaligenes faecalis: A taxonomic reappraisal, J. Appl. Bacteriol., 1970, 33: 714―720. 15. Anson, J. G., Mackinnon, G., Novel Pseudomonas plasmid involved in aniline degradation, Appl. Environ. Microbiol., 1984, 48: 868―869. 16. Aoki, K., Kodama, N., Murakami, S. et al., A high level of accumulation of 2-hydroxymuconic 6-semialdehyde from aniline by the transpositional mutant Y-2 of Pseudomonas species AW-2, Microbiol. Res., 1997, 152: 129―35.

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17. Travkin, V. M., Solyanikova, I. P., Rietjens, I. M. et al., Degradation of 3,4-dichloro- and 3,4-difluoroaniline by Pseudomonas fluorescens 26-K, J. Environ. Sci. Health B, 2003, 38: 121―132. 18. Kim, S. I., Leem, S. H., Choi, J. S. et al., Cloning and characterization of two catA Genes in Acinetobacter lwoffii K24, J. Bacteriol., 1997, 179: 5226―5231. 19. Aoki, K., Shinke, R., Nishira, H., Metabolism of aniline by Rhodococcus erythropolis AN-13, Agri. Biol. Chem., 1983, 47: 1611―1616. 20. Fuchs, K., Schreiner, A., Lingens, F., Degradation of 2-methylaniline and chlorinated isomers of 2-methylaniline by Rhodococcus rhodochrous strain CTM, J. Gen. Microbiol., 1991, 137: 2033― 2039. 21. Aoki, K., Ohtsuka, K., Shinke, R. et al., Rapid biodegradation of aniline by Frateuria species ANA-18, Agri. Biol. Chem., 1984, 48: 856―872. 22. Zeyer, J., Wasserfallen, A., Timmis, K. N., Microbial mineralization of ring-substituted anilines through an ortho-cleavage pathway, Appl. Environ. Microbiol, 1985, 50: 447―453. 23. Bachofer, R., Lingens, F., Schafer W Conversion of aniline into pyrocatechol by a Nocardia sp.: Incorporation of oxygen-18, FEBS Lett., 1975, 50: 288―290. 24. Boon, N. J., Goris, P. D. V., Verstraete, W., et al., Genetic diversity among 3-chloroaniline- and aniline-degrading strains of the Comamonadaceae, Appl. Environ. Microbiol., 2001, 67: 1107―1115. 25. Liu, Z., Yang, H., Huang, Z. et al., Degradation of aniline by newly isolated, extremely aniline-tolerant Delftia sp. AN3, Appl. Microbiol. Biotechnol., 2002, 58: 679―682. 26. Kahng, H. Y., Kukor, J. J., Oh, K. H., Characterization of strain HY99, a novel microorganism capable of aerobic and anaerobic degradation of aniline, FEMS Microbiol. Lett., 2000, 190: 215― 221. 27. Shigematsu, T., Yumihara, K., Ueda, Y. et al., Delftia tsuruhatensis sp. nov., a terephthalate assimilating bacterium isolated from activated sludge, Int. J. Syst. Bacteriol., 2003, 53: 1479―1483. 28. Top, E. M., Springael, D., The role of mobile genetic elements in bacterial adaptation to xenobiotic organic compounds, Curr. Opin. Biotechnol., 2003, 14: 262―269. 29. Dagley, S., Microbial metabolism of aromatic compounds, in Comprehensive Biotechnology (eds. Moo-Young, M., Bull, A. T., Dalton, H.), Oxford: Pergamon Press, 1985, 483―505. 30. Xu, M., Zhang, A. Q., Han, S. K., 3-D QSAR Study on a set of Nitroaromatic compounds, Chinese Science Bulletin, 2001, 47(3): 189 192. (Received May 10, 2005; accepted July 4, 2005)

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