1 Department of Microbiology, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 12 zhongguancun Nandajie, Beijing 100081, P. R. China
2 Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2201, Japan
Correspondence
Min Lin
linmin57{at}vip.163.com
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers of the sequences of the aniline degradation gene cluster and the 16S rDNA cloned from strain AD9 are AY940090 and AY89912, respectively.
A supplementary table summarizing the morphological, physiological and biochemical characteristics of strain AD9, Delftia acidovorans and Delftia tsuruhatensis is available with the online version of this paper.
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INTRODUCTION |
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In order to determine biodegradability and mechanisms of biodegradation of anilines, many aniline-degrading bacteria have been isolated. Species of Alcaligenes (Rhodes, 1970), Pseudomonas (Anson & Mackinnon, 1984
; Aoki et al., 1997
; Travkin et al., 2003
), Acinetobacter (Kim et al., 1997
), Rhodococcus (Aoki et al., 1983
; Fuchs et al., 1991
), Frateuria (Aoki et al., 1984
), Moraxella (Zeyer et al., 1985
), Nocardia (Bachofer et al., 1975
) and Delftia (Kahng et al., 2000
; Boon et al., 2001
; Liu et al., 2002
) are able to degrade aniline and/or its derivatives. Moreover, plasmid-dependent aniline degradation has been reported in several bacterial strains (Anson & Mackinnon, 1984
; Latorre et al., 1984
; McClure & Venables, 1987
; Fujii et al., 1997
; Boon et al., 2001
). However, 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 (Fukumori & Saint, 1997
) and pYA1 of Acinetobacter sp. YAA (Fujii et al., 1997
). These gene clusters are very similar in their genetic organization; both contain genes encoding a multi-component aniline dioxygenase (AD), a LysR-type regulator, and several meta-cleavage pathway enzymes. The ADs encoded by these gene clusters consist of five proteins, two of which are homologous to glutamine synthetase and glutamine amidotransferase, suggesting that they are involved in the transfer of the amino group of aniline. Similarities in the sequences of the other three proteins to other aromatic compound dioxygenases suggest that they function as the large and small subunits of the oxygenase component and the reductase component in the AD enzyme systems (Fukumori & Saint, 1997
; Takeo et al., 1998
). Recently, AD genes of the same type were cloned from Frateuria sp. ANA-18 (Murakami et al., 2003
) and Delftia acidovorans 7N (Urata et al., 2004
). The AD genes of the former strain are located over 1·7 kb upstream of a catechol ortho-cleavage pathway gene cluster (the cat1 gene cluster) encoded on the chromosome, while those of the the latter strain are located just upstream of a catechol 2,3-dioxygenase gene. However, other meta-cleavage pathway enzyme genes have not been reported.
Here we report a chromosome-encoded gene cluster responsible for the complete conversion of aniline to TCA-cycle intermediates cloned from a Delftia strain. Interestingly, the gene cluster, surrounded by two IS1071 sequences, is almost identical to that encoded on plasmid pTDN1 of P. putida UCC22 (Fukumori & Saint, 1997), which is one of the representative aniline degradation gene clusters.
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METHODS |
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Construction of the genomic library of strain AD9.
The genomic DNA of strain AD9 was obtained by the method of Wilson (1987) employing SDS-proteinase K lysis and selective precipitation of cell debris and polysaccharides with cetyltrimethylammonium bromide. The genomic DNA obtained was partially digested by HindIII or EcoRI and separated in a 0·6 % (w/v) agarose gel. DNA fragments of 920 kb were purified from the gel using a QIAEXII Gel Extraction Kit (Qiagen), ligated to pUC19 digested by HindIII or EcoRI, and introduced into E. coli JM109 by electroporation using a Gene pulser cell (Bio-Rad) according to the manufacturer's instructions.
DNA sequencing and computer sequence analysis.
Cloned DNA fragments were sequenced by TaKaRa Bio (Kyoto), and nucleotide and amino acid sequences were analysed using DNAman software (Lynnon Biosoft). Sequence comparisons were made against the sequences in the GenBank using the BLAST program (Altschul et al., 1990). Phylogenetic trees were constructed using GENETYX-WIN Version 5.1 (Genetyx Co.).
Aniline degradation tests and aniline oxygenase assay.
One loop of strain AD9 was inoculated into LB medium and the culture was incubated overnight at 30 °C. The cells were harvested by centrifugation (4000 g, 4 °C, 10 min), washed with 10 mM potassium phosphate buffer (pH 7·0) twice, and suspended in MS medium to give an OD600 of 1·0. Aniline degradation tests were started by adding aniline to the cell suspension at various concentrations up to 5500 mg l1. The cell suspension was shaken on a rotary shaker at 180 r.p.m., at 30 °C, and samples of the suspension were taken at specific intervals. After removing the cells from the samples by centrifugation (8000 g, 4 °C, 10 min), aniline in the supernatant was detected using the diazo-coupling method (Snell, 1954).
Aniline oxygenase activity was measured using a Clark-type oxygen electrode (YSI 5100, Yellow Springs Instruments). Fresh cells, pre-grown in 40 ml LB medium, were harvested by centrifugation (4000 g, 4 °C, 10 min) and washed with the phosphate buffer (10 mM, pH 7·0). The cells were suspended in 100 ml MS medium containing aniline (600 mg l1) or succinate (10 mM), and incubated at 30 °C with rotary shaking (180 r.p.m.) for 24 h to obtain aniline-induced cells or non-aniline-induced (succinate-grown) cells. The cells were harvested, washed twice with the phosphate buffer, and suspended in the same buffer. Oxygen uptake was measured polarographically at 30 °C. The reaction mixture contained 100 mg aniline l1 and washed cells in phosphate buffer. Endogenous respiration was measured in the absence of aniline, and the oxygen uptake rates obtained were corrected for the endogenous respiration.
Expression of AD genes in E. coli.
Recombinant E. coli strains were cultivated in 6 ml LB medium containing Ap. When the cultures reached early exponential phase, IPTG was added at a final concentration of 1 mM. After 4 h incubation, the cells were harvested, washed with phosphate buffer, and suspended in 0·8 % (w/v) sodium chloride solution. Finally, cell suspensions with an OD660 of 1018 were prepared. The aniline oxygenase activity was measured polarographically as described above.
Measurement of catechol 2,3-dioxygenase (C23O) activity.
Recombinant E. coli strains were cultivated at 37 °C in 3 ml LB medium containing Ap and IPTG (1·0 mM) until the OD660 reached approximately 1·0. The cells were harvested, washed with phosphate buffer, and suspended in 1·6 ml 50 mM sodium chloride solution containing 10 % (v/v) ethanol. A crude cell extract was prepared by sonication of the cell suspension using an ultrasonic disruptor (TOMY UD-200; power 6, 2 min, three times on ice). Cell debris was removed by centrifugation (8000 g, 4 °C, 10 min). C23O activity was measured spectrophotometrically by the increase in absorbance at 375 nm concomitant with the formation of 2-hydroxymuconic semialdehyde (Nakazawa & Yokota, 1973). The reaction mixture contained 0·1 mM catechol and the cell extract in 50 mM sodium phosphate buffer (pH 7·5) and the reaction was carried out at 24 °C. The quantity of protein in the cell extract was determined by the Lowry method, using bovine serum albumin as the standard. One unit of activity was defined as the amount of enzyme required to produce 1 µmol product min1.
Southern hybridization analysis.
The genomic DNA of strain AD9 was digested by HindIII, EcoRI or PstI. After electrophoresis in a 1·0 % (w/v) agarose gel, the digested fragments were transferred onto a Hybond-N+ nylon membrane (Amersham). A 526 bp DNA fragment and a 580 bp DNA fragment, which correspond to parts of the AD genes (tadQ region, see Fig. 1) and the transposase (TnpA) gene of the IS1071 region (tnpA-L1 region, see Fig. 1
), respectively, were amplified from pDA1 by PCR using the primer sets for the tadQ gene (TadQF, 5'-ACGATGGTGCTGTTCCGCAA-3', and TadQR, 5'-TATGAGGCAGGATGGTGACG-3') and for the TnpA gene sequence (TnpAF, 5'-GCCATTGAAGGTGTCATCCG-3', and TnpAR, 5'-AGGTATTCCACGCCATCACG-3'), respectively. The PCR mixture contained 5 µl 10x ExTaq buffer (TaKaRa), 4 µl dNTPs (2·5 mM each), 100 pmol of each primer, 1·25 U ExTaq (TaKaRa), 2·5 ng of the template already described, and sterile distilled water to adjust the total volume to 50 µl. PCR was carried out using a PCR Express machine (Thermo Hybaid) and the following temperature programme: 94 °C for 3 min; 30 cycles consisting of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min; and 72 °C for 5 min. The PCR products were labelled with digoxigenin using a DIG-High Prime DNA Labelling and Detection Starter Kit II (Boehringer Mannheim Biochemicals) and used as gene probes. Southern hybridization was carried out according to the protocol for the labelling and detection kit.
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RESULTS |
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Aniline degradation by strain AD9
Strain AD9 grew on and degraded aniline well at 2037 °C (the optimum temperature was around 30 °C), and it grew well in a wide pH range from 4·0 to 9·0 (the optimum pH was around 7·0). Although it could degrade up to 5000 mg aniline l1 in MS medium, over 1000 mg aniline l1 inhibited growth. The highest concentration (5000 mg l1, 53·8 mM) at which AD9 can grow is identical to that of Delftia sp. AN3 (Liu et al., 2002), which was reported as the most aniline-tolerant aniline degrader. When cells pre-grown in LB medium containing aniline were suspended at an OD600 of 1·0 in MS medium, the culture degraded 1000 mg aniline l1 completely within 18 h (data not shown). This ability to degrade aniline was not lost even after repeated culturing (six times) in LB medium without aniline.
Strain AD9 was also able to utilize m-toluidine and p-toluidine as a sole source of carbon, but not o-toluidine, 4-chloroaniline, 2-chloroaniline, 2,4-xylidine, 3,4-dichloroaniline or 2,4-dichloroaniline.
To examine the aniline oxygenase activity of AD9, oxygen uptake was measured using aniline-induced cells and non-aniline-induced (succinate-grown) cells. The aniline-induced cells showed apparent aniline oxygenase activity of 82±6 mg O2 (g dry wt)1 h1, while the non-aniline-induced cells showed only one-third of this activity [30±2 mg O2 (g dry wt)1 h1]. This result indicates that aniline oxidation in AD9 is inducible.
Cloning of aniline degradation genes from AD9
A genomic library was constructed with HindIII-digested total DNA of AD9 and introduced into competent E. coli JM109. When transformants were screened on LB plates containing Ap and aniline, five colonies showed a brown colour on the plates, indicating accumulation of catechol resulting from aniline oxidation. A recombinant plasmid was extracted from one of the positive colonies and analysed by restriction enzymes. The analysis revealed that the recombinant plasmid, designated pDA1, had a 9·3 kb HindIII insert in the vector pUC19. The transformants were screened again by spraying their colonies with 0·1 M catechol solution (in 10 mM phosphate buffer, pH 7·0). One colony showed a brilliant yellow colour on the plate, indicating C23O activity. 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. To confirm whether the two insert fragments were situated next to each other, the transformants obtained from another library constructed with EcoRI-digested AD9 DNA were screened by the catechol spray. 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. 1(a), the insert fragment of pDB2 overlaps the inserts of both pDA1 and pDB11 and that the insert of pDB11 is adjacent to that of pDA1.
Sequence analysis of cloned fragments
The DNA fragments cloned in pDA1, pDB2 and pDB11 were sequenced, and the nucleotide sequence of the total 24·7 kb region was thereby determined. Homology searches were performed to identify gene function. It was found that the region contains 23 intact ORFs (ORF224, Table 2), at least 17 of which (tadQTA1A2BRD1C1D2C2EFGIJKL) were expected to be involved in the complete metabolism of aniline to TCA-cycle intermediates as shown in Fig. 1(b)
. The first five gene products (TadQTA1A2B) and the subsequent gene product (TadR) showed significant aa sequence identity (8496 %) to multi-component aniline dioxygenases (ADs) and LysR-type regulators, respectively, found in other aniline-degrading bacteria, P. putida UCC22, D. acidovorans 7N and Acinetobacter sp. YAA. The remaining 11 gene products (TadD1C1D2C2EFGIJKL) exhibited considerable identity (73100 %) to meta-cleavage pathway enzymes found in aromatic-compound-degrading and aniline-degrading bacteria (Table 2
).
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Partial sequences encoding the TnpA of IS1071 (Nakatsu et al., 1991) were found at both ends of this 24·7 kb region. Inverted repeat sequences of 110 bp (nt 17441853 and nt 2440324512 in AY940090), which are completely identical to those of IS1071, were found near the TnpA-encoding sequences, indicating that this tad gene cluster is surrounded by two IS1071 sequences (nt 11853 and nt 2440324681 in AY940090).
Expression of AD and C23O genes
Aniline oxygenase activity was measured using cell suspensions of E. coli harbouring pDA1 to examine whether the cloned AD genes (tadQTA1A2B) were functional. The E. coli cells harbouring pDA1 showed apparent aniline oxygenase activity [32±3 mg O2 (g dry wt)1 h1]. The endogenous respiration (the oxygen uptake without the addition of aniline) subtracted was 16±2 mg O2 (g dry wt)1 h1. Catechol was detected in the cell suspension as the oxidation product by GC/MS analysis (data not shown). These results indicate that the recombinant E. coli oxidized aniline to catechol. The 9·3 kb HindIII fragment of pDA1 was then subcloned into the broad-host-range plasmid pVK100 to make the recombinant plasmid pVD1. This plasmid was introduced by triparental mating (Winstanley et al., 1989) into the phenol-degrading bacterium A. calcoaceticus PHEA-2 (Xu et al., 2003
), which can assimilate both catechol and phenol, but not aniline. The resultant strain was able to grow on aniline as a sole carbon source, indicating that the AD genes in pVD1 allowed strain PHEA-2 to convert aniline into catechol (data not shown). Furthermore, when pVD1 was introduced into the parent strain AD9, the resultant strain accumulated a large amount of a dark brown compound, probably auto-oxidized catechol from aniline, in liquid cultures, because of the multi-copy dose effect of the AD genes. These results indicate that the cloned AD genes were functional in AD9 and also in E. coli and A. calcoaceticus.
In the catechol spray selection, the recombinant E. coli strain harbouring pDB2 was selected based on yellow colour formation as an index of C23O activity. To evaluate the C23O activity, the cell extract of the recombinant strain was prepared and used to measure C23O activity. The activity measured was 3·2 units (mg crude protein)1.
Copy number and location of the tad gene cluster in AD9
Southern hybridization was carried out using a 526 bp gene probe including a part of the AD genes (tadQ region, see Fig. 1) in order to determine the copy number of the tad gene cluster in strain AD9. The total DNA extracted from AD9 was digested independently by three restriction enzymes (HindIII, EcoRI or PstI) for this study. As shown in Fig. 2
(a, b), the gene probe hybridized to one band in each lane of the Southern blot (Fig. 2b
) corresponding to the digested DNA sample lanes of the agarose gel electrophoresis (Fig. 2a
), suggesting that AD9 has the tad gene cluster in a single copy, if it resides on the chromosome. In order to clarify the location of the tad gene cluster in AD9, PFGE was carried out using the AD9 cells. Fig. 2(c)
clearly demonstrates that there are no detectable large plasmids in AD9, although several plasmids were detected in Bacillus thuringiensis A-01 (a plasmid-positive control strain), and the chromosome of AD9 could be seen together with that of E. coli (negative control and size marker). Southern hybridization with the same gene probe revealed that the tad gene cluster is located on the AD9 chromosome (Fig. 2d
).
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Comparison of the chromosome-encoded tad gene cluster with the plasmid-encoded tdn gene cluster
As shown in Table 3, the putative products of the tad genes showed striking identity to those of the plasmid-encoded tdn genes of P. putida UCC22. Hence, the gene organization of the tad gene cluster was compared with that of the tdn gene cluster in detail (Fig. 3). It was found that the gene organization of both gene clusters is quite similar and both are surrounded by two IS1071 sequences including TnpA-encoding regions. However, there are some differences between them. First, the tad gene cluster lacks the genes corresponding to orf3 and tdnH of the tdn gene cluster. The function of the orf3 gene product has not been characterized yet, and the tdnH gene product, which showed similarity to short-chain dehydrogenases (Fukumori & Saint, 2001
), seems to be unnecessary for catechol metabolism, because many meta-cleavage pathways do not contain this enzyme. Secondly, in the region between tadC1/tdnC and tadD2/tdnD2 (region I in Fig. 3
), the tdn gene cluster lacks a 270 bp DNA segment of the tad gene cluster (nt 1126611535 in AY940090). The loss of this small segment in the tad gene cluster caused the disruption of orfU and orfS and resulted in the formation of longer orf1 and orf2 in the tdn gene cluster. Thirdly, in the region downstream of tadL/tdnL (region II in Fig. 3
), there is a substitution of a 2·3 kb DNA segment. In region II of the tad gene cluster, there are three ORFs, orfXYZ, as mentioned above. In contrast, in region II of the tdn gene cluster, there are four ORFs, which have not been described so far (Fukumori & Saint, 2001
). Herein, we tentatively call them orf5678. The putative gene products of orf5678 showed considerable similarity to MarR-type regulators (e.g. AAL02068, identity 58 %), the C terminal half of
-ketoadipate enol-lactone hydrolases (e.g. CAD13826, identity 39 %), KfrA-like proteins (e.g. AAP22622, identity 27 %) and integrases/recombinases (e.g. CAI47894, identity 71 %), respectively. As shown in Fig. 3
, a 2·3 kb DNA segment containing half of orfY and intact orfZ in the tad gene cluster (nt 2212424402 in AY940090) is substituted by a smaller segment containing orf7 and orf8 in the tdn gene cluster.
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DISCUSSION |
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We found some differences between the tad gene cluster and the tdn gene cluster. In region I of Fig. 3, the tdn gene cluster lacks a 270 bp segment of the tad gene cluster, resulting in the disruption of orfS and the formation of a longer orf2. orfS is predicted to encode a LysR-type regulator with a normal size (301 aa) compared to those of other LysR-type regulators (around 300 aa), whereas orf2 is expected to encode a larger protein with an unusual size (532 aa) for a member of this family. The additional sequence of orf2 compared to orfS encodes no known proteins. Thus, it is natural to think that the 270 bp segment was deleted from the tad-type gene cluster to form the tdn-type gene cluster. In region II, a putative transcriptional unit, orfYZ, is disrupted by the substitution of the 2·3 kb DNA segment. The reasons why orfY and orfZ were presumed to form a transcriptional unit are that (i) both ORFs are thought to be a part of a catechol ortho-cleavage pathway operon, judging from the sequence similarity of their gene products (Table 2
), and (ii) the 5' terminus of orfY overlaps by 3 bp the 3' terminus of orfZ. Therefore, this substitution also might have happened after the tad gene cluster had been established. These sequence analyses suggest that the tad gene cluster is more ancestral than the tdn gene cluster.
Our isolate, AD9, grew on and degraded aniline at concentrations as high as those used by the most aniline-tolerant aniline degrader Delftia sp. AN3. In the aniline degradation pathway of AD9, the activities of the key enzymes, AD and C23O, were experimentally confirmed. The AD genes of strain AD9 were expressed in E. coli and more efficiently in A. calcoaceticus PHEA1 and the parent strain AD9. Although the function of each subunit of ADs remains unknown, catechol was detected as a metabolite of aniline after the expression of the AD genes. Moreover, cell extracts of recombinant E. coli harbouring pDB11 showed C23O activity. However, pDB11 contains two C23O genes, tadC1 and tadC2. The phylogenetic analysis of the gene products TadC1 and TadC2 (Fig. 4) illustrates that these two C23Os belong to different phylogenetic branches. This may mean that they have come from different meta-cleavage pathways. The phylogenetic tree also revealed that TadC1 and TadC2 are most closely related to TdnC and TdnC2 of P. putida strain UCC22, respectively. Fukumori & Saint (2001)
reported that TdnC and TdnC2 have distinct substrate specificity: the E. coli cell extract containing TdnC showed relatively high activity on substituted catechols (catechol, 100 %; 3-methylcatechol, 93 %; 4-methylcatechol, 43 %), while that containing TdnC2 showed less activity on substituted catechols (catechol, 100 %; 3-methylcatechol, 4·6 %; 4-methylcatechol, 19 %). Strain UCC22 can assimlate m-toluidine (3-methylaniline) and p-toluidine (4-methylaniline) via 3-methylcatechol and 4-methylcatechol, respectively. Therefore, it might be necessary for cells to acquire another C23O, TdnC, for these methylcatechols, in addition to TdnC2 for unsubstituted catechol, to expand the assimilation range for toluidines. Strain AD9 can also assimilate m-toluidine and p-toluidine. In our preliminary study using recombinant E. coli cell extracts containing TadC1 or TadC2, C23O activity was seen on catechol (100 % and 100 %, respectively), 3-methylcatechol (56 % and 18 %) and 4-methylcatechol (28 % and <1 %). Therefore, we confirmed that both tadC1 and tadC2 can produce an active C23O. The activities of TadC2 towards methylcatechols were lower than those of TadC1, as reported for TdnC2 and TdnC by Fukumori & Saint (2001)
. However, detailed analysis should be carried out using the purified enzymes.
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ACKNOWLEDGEMENTS |
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Received 20 April 2005;
revised 18 July 2005;
accepted 18 July 2005.
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