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Dioxin catabolic genes are dispersed on the Terrabacter sp. DBF63 genome
BBRC Biochemical and Biophysical Research Communications 296 (2002) 233–240 www.academicpress.com
Dioxin catabolic genes are dispersed on the Terrabacter sp. DBF63 genome Hideaki Nojiri,a Mayuko Kamakura,b Masaaki Urata,a Takahiro Tanaka,b Jin-Sung Chung,a Tetsuo Takemura,b Takako Yoshida,a Hiroshi Habe,a and Toshio Omoria,* a
b
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Department of Chemistry, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan Received 1 July 2002
Abstract Reverse transcription-PCR of the dbfA1A2, dbfBC, and pht genes, encoding oxygenase component of multicomponent dioxygenase, meta cleavage enzyme and hydrolase, and phthalate-degrading enzymes, respectively, revealed their role in the aromatic compound degradation by Terrabacter sp. strain DBF63. The specific expression in strain DBF63 cells grown on dibenzofuran (the model compound of dioxin; DF) and/or fluorene (FN) indicated that the DbfA1A2 and DbfBC catalyze the conversion of DF to salicylate, and that the DbfA1A2 and Pht enzymes are involved in FN degradation. Pulsed-field gel electrophoresis analyses revealed that the dbfA1A2 cistron and pht operon were located on the two linear plasmids, pDBF1 (160 kb) and pDBF2 (190 kb), while dbfBC genes were located on the chromosome. Because the pht operon is located immediately upstream of the dbfA1A2 cistron, the dioxin-catabolic genes were dispersed on the genome of strain DBF63, while FN-catabolic genes were gathered on the plasmids. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Biodegradation; dbf Gene cluster; Expression; Degradative plasmid; pDBF1; Dibenzofuran; Dioxin; Fluorene; Terrabacter sp. strain DBF63
Polychlorinated dibenzo-p-dioxins and dibenzofurans, called dioxins, are produced by incineration processes and, unintentionally, as by-products during the synthesis of pesticides and herbicides. Many of their congeners are highly toxic compounds with undesirable effects on the environment and living beings [1,2], but have been widely distributed in the environment [3–5]. The dioxins are recalcitrant molecules and show extremely high persistence in soil sediments [6,7]. Because of the relatively low cost and minimal impact on the environment, dioxin-degrading bacteria have been isolated and investigated by using the dibenzofuran (DF) as a model substrate in enrichment culture as reviewed previously [8–12]. In the microbial degradation pathway of DF, the angular dioxygenation at 4 and 4a positions is the initial *
Corresponding author. Fax: +81-3-5841-8030. E-mail address: [email protected] (T. Omori).
degradation reaction (Fig. 1). This novel type of dioxygenation reaction is shown to be catalyzed by DF 4,4adioxygenase, and leads DF to a chemically unstable intermediate that is spontaneously rearomatized to 2,20 , 3-trihydroxybiphenyl (THB). Subsequent ring cleavage of THB is catalyzed by meta cleavage enzyme yielding 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-dienoic acid and salicylic acid (Fig. 1). Salicylic acid is then metabolized via catechol or gentisic acid. Among these metabolic enzymes, initial angular dioxygenase is a key determinant of the substrate range in the degradation of heterocyclic aromatic compounds including dioxin [13–15]. Terrabacter sp. strain DBF63 was isolated from soil as a microorganism having the ability to utilize DF or fluorene (FN) as a sole source of carbon and energy [16]. Based on the identification of the metabolic intermediates, Monna et al. [16] proposed the degradation pathways for DF and FN by strain DBF63 (Fig. 1). By the shotgun cloning using the meta cleavage activity as a
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 8 7 3 - 2
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Fig. 1. Comparison of the degradation pathways for DF and FN by Terrabacter sp. strain DBF63. The structures shown in brackets have not been characterized directly. The arrows with solid and broken lines indicate the enzymatic and spontaneous conversions, respectively. The bold letters indicate that the conversions are catalyzed by the corresponding enzymes. The conversion of 1,1a-dihydroxy-1-hydrofluorene-9-one to phthalic acid is catalyzed by the specific enzymes [H. Habe, H. Kato, K. Kasuga, T. Yoshida, H. Nojiri, T. Omori, unpublished results]. Compound designations: I, DF; II, cis-4,4a-dihydroxy-4-hydrodibenzofuran; III, 2,20 ,3-trihydroxybiphenyl (THB); IV, 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid; V, salicylic acid; VI, FN; VII, 9-fluorenol; VIII, 9-fluorenone; IX, 1,1a-dihydroxy-1-hydrofluorene-9-one; X, 20 -carboxy-2,3-dihydroxybiphenyl; XI, 2-hydroxy-6-(2-carboxyphenyl)-6-oxo-2,4-hexadienoic acid; XII, phthalic acid; XIII, protocatechuic acid.
selection marker, we have cloned the DNA fragments containing dbfBC genes and ORFK0K1K2 genes encoding the meta cleavage enzyme (DbfB and ORFK1 products) and meta cleavage compound hydrolase (DbfC and ORFK2 products) (Fig. 2) [17]. The fact that DbfB has strict preference to the binuclear substrate, such as 2,3-dihydroxybiphenyl and 2,20 ,3-trihydroxybiphenyl, indicated the possibility that DbfB constitutes the DF degradation pathway by this strain. Also, we successfully isolated the dbfA1A2 cistrons encoding the a and b subunits of terminal oxygenase component of DF 4,4a-dioxygenase from strain DBF63 genome by using the PCR-based method (Fig. 2) [18]. Although the electron transport components co-working with this oxygenase component were not identified, we could detect the angular dioxygenase activity of DbfA1A2 for
Fig. 2. Genetic organization of pht-dbfA1A2 and dbfBC loci encompassing the genes involved in DF and phthalate degradation [17,18, H. Habe, M. Miyakoshi, K. Kasuga, T. Yoshida, H. Nojiri, T. Omori, submitted] and locus K (Accession No. AB084286) in Terrabacter sp. strain DBF63 genome. The pentagons and triangles in the physical map indicate the size, location, and the direction of transcription of the ORFs derived from the nucleotide sequence data. DF-degrading genes and insertion sequence are shown by shaded pentagons and black box, respectively.
DF and FN by using the unspecific electron transport system of the Escherichia coli host strain [18, H. Habe, H. Kato, K. Kasuga, T. Yoshida, H. Nojiri, T. Omori, unpublished results]. As we expected, the DF-degrading system of strain DBF63 was able to decompose chlorinated DF/dibenzo-p-dioxin [15,19]. Because the DFmetabolism-deficient (DF ) derivative strain DBF63W did not contain the dbfA1A2 and dbfBC genes [17,18], we speculated that the DbfA1A2 and DbfBC functions in the DF and FN degradation by strain DBF63. Very recently, we revealed the phthalate degradation gene cluster [phtA1A2(ORF11)BA3A4CR] responsible for the conversion of phthalate to protocatechuate [H. Habe, M. Miyakoshi, K. Kasuga, T. Yoshida, H. Nojiri, T. Omori, submitted] in the upstream region of dbfA1A2 cistron as shown in Fig. 2. Because phthalate is a metabolic intermediate of FN, the degradation pathway encoded by the phtA1A2(ORF11)BA3A4CR genes should constitute the lower part of the entire FN degradation pathway. In this study, to confirm the role of the DbfA1A2, DbfBC, and phthalate-degrading enzymes in DF and FN degradation by strain DBF63, we carried out transcriptional analysis of the corresponding genes in strain DBF63 grown on various carbon sources. In addition, we discussed the evolution of the DF- and FN-degradation pathway based on the localization of these genes in the genome of strain DBF63.
Materials and methods Bacterial strains, plasmid, and culture conditions. Strain DBF63 is a DF-degrading bacterium that was isolated from a soil sample in Japan on the basis of its ability to grow on DF as a sole source of carbon and energy [16]. Strain DBF63W obtained by the continuous culture of strain DBF63 on the nutrient broth can utilize salicylic acid but not DF as a sole source of carbon and energy [17]. Strains DBF63 and
H. Nojiri et al. / Biochemical and Biophysical Research Communications 296 (2002) 233–240 DBF63W were cultivated in carbon-free mineral medium (CFMM) supplemented with the DF and nutrient medium, commercially available from Eiken Chemical (Tokyo, Japan), respectively, at 30 °C with reciprocal shaking (300 strokes/min) as described by Kasuga et al. [17]. E. coli strain JM109 [recA1, D(lac-proAB), endA1, gyrA96, thi-1, hsdR17, relA1, supE44, F0 (traD36, proAB, lacI q ZDM15)] [20] was used as a host strain of plasmid pUC118/119 [20] and their derivatives. E. coli strains were grown on LB or 2YT media [20] at 37 °C. For plate cultures, the above media solidified with 1.6% (wt/vol) agar were used. DNA manipulations. Plasmid DNA for routine DNA manipulation was prepared from the E. coli cell by the alkaline lysis method and purified as described by Sambrook et al. [20]. Plasmid DNA used for PCR template was prepared by using the Quantum Prep Plasmid Miniprep Kit (Bio-Rad Laboratories, Richmond, CA, USA) as recommended by the manufacturer. Restriction endonucleases were used according to manufacturer’s instructions. DNA fragments were fractionated by electrophoresis using 0.9% agarose in TAE buffer [20], and were extracted from the agarose gel by using Concert Rapid Gel Extraction Systems (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. Other DNA manipulation procedures were performed according to the standard methods [20]. Transcriptional analyses of dbfA1A2 and dbfBC genes by reverse transcription (RT)-PCR. After a 4-day cultivation of strain DBF63 in 5 ml CFMM supplemented with DF, cells were gathered from 2 ml of culture by centrifugation at 7200g, then washed twice using CFMM. After the washed cells were suspended in 2 ml CFMM, the cells were incubated at 30 °C with reciprocal shaking at 300 strokes/min for 12 h as starvation culture. The starved cells were washed twice as described above, and finally suspended in 2 ml CFMM. Five-milliliter CFMM supplemented with 0.1% (wt/vol; in final concentration) of CAR, FN, salicylic acid, or gentisic acid was inoculated with 2 ml of the resultant cell suspension of strain DBF63. After 12-h incubation with reciprocal shaking (300 strokes/min) at 30 °C, the cells were harvested and used for extraction of total RNA. Total RNA from harvested cell was extracted using an RNeasy Mini Kit (Qiagen, Santa Clarita, CA, USA) according to manufacturer’s instruction. By RT-PCR analyses, the transcriptions of dbfA1, dbfC, and ORFK2 were monitored. RT-PCR was performed using a Takara One Step RNA PCR Kit (AMV) (Takara Shuzo, Kyoto, Japan) as recommended by the manufacturer. In RT-PCR, 1 lg total RNA prepared from the strain DBF63 cells was used as a template RNA. Table 1 shows the names of primer sets and their nucleotide sequences. After the RT at 60 °C for 30 min followed by the initial denaturation step at 94 °C for 2 min, denaturation, annealing, and polymerization were performed for 25 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 2 min, respectively. Pulsed-field gel electrophoresis (PFGE). Strain DBF63 was grown on DF for 4 days, and then the cells were harvested from 5 ml of culture as described above, and washed with TE buffer [20]. After the
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resultant cells were suspended in 50 ll of the Cell Suspension Buffer in the CHEF Bacterial Genomic DNA Plug Kit (Bio-Rad Laboratories), the plug for PFGE analysis was prepared as recommended by the manufacturer with some modification. For effective lysis of strain DBF63 cells grown on DF, we used lysozyme and proteinase K at the final concentrations of 1 and 8 mg/ml, respectively. Strain DBF63W was grown on nutrient medium for 4 days, and the cells corresponding to 0.5 ml culture were used for the suspension with 50 ll of Cell Suspension Buffer. For strain DBF63W cell, we used lysozyme and proteinase K at the final concentrations of 0.5 and 4 mg/ml, respectively. PFGE was performed by using a CHEF DRIII PFGE System (BioRad Laboratories) and 0.5 Tris–Borate–EDTA buffer [20]. Electrophoresis was performed with electric field of 6 V/cm at 14 °C. The sets of pulse time and running time were from 0.2 to 22 s and 15 h, from 20 to 30 s and 15 h, or from 30 to 60 s and 22 h. The field angle was 120°. Southern hybridization analysis. After PFGE, DNA was transferred to a Biodyne B nylon membrane (Poll BioSupport, East Hills, NY, USA) by VacuGene XL Vacuum Blotting System (Amersham Pharmacia Biotech UK, Little Chalfont, Buckinghamshire, England) as recommended by the manufacturer. The preparation of probes and Southern blots were performed by using a nonradioactive DIG DNA labeling and detection kit (Roche Diagnostics GmbH) as described previously [21]. To prepare the dbfA1A2-, dbfBC-, and ORFK1K2specific probes, we used as templates a 1.2-kb PstI fragment of pDF32 [18], a 1.3-kb SalI fragment of pLM105R [17], and a 2.2-kb SphI–PstI fragment of pKN1 [17], respectively. The 16S rRNA gene was amplified as described previously [21], and used as a template for probe preparation. Chemicals. Chemicals used in this study were of the highest purity commercially available (Merck, Darmstadt, Germany; Sigma–Aldrich, Steinheim, Germany; Kanto Chemical, Tokyo, Japan; Wako Pure Chemical, Osaka, Japan; Nacalai Tesque, Kyoto, Japan).
Results and discussion The role of the dbf and pht genes in the cell of Terrabacter sp. strain DBF63 We have revealed the genes encoding the terminal oxygenase of angular dioxygenase (DbfA1A2), meta cleavage enzyme (DbfB), and meta cleavage compound hydrolase (DbfC) (pht-dbfA1A2 and dbfBC loci in Fig. 2). The catabolic activities of these enzymes suggest that they are involved in the DF degradation by this strain. In the upstream region of the dbfA1A2 cistron, there exist the pht genes proposed to be involved in the conversion of
Table 1 Primers used in the RT-PCR analyses Target gene
Name of primer
Nucleotide sequence
dbfA1
dbfA1A2-F dbfA1A2-R
50 -CGGCGTCTTCCACGTCTTCG-30 50 -CCCGGGGTGGTCATGAGTTC-30
dbfC
dbfC-F dbfC-R
50 -CTGGAAGATCCACTACAACG-30 50 -GTGATCAGGTGCGAGATCCG-30
ORFK2
ORFK2-750F ORFK2-1120R
50 -CGCCTTCGACGACTACGACA-30 50 -TTCTGCGAGAAGTTGCTCCA-30
ORFK0
ORFK0-F ORFK0-R
50 -TTCGGCAACATCATCAAGCC-30 50 -TCGGACATGAAGGAGACGAA-30
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phthalate to protocatechuate (pht-dbfA1A2 and dbfBC loci in Fig. 2) [Habe et al., submitted for publication]. In addition, in another locus (designated as locus K as shown in Fig. 1) (DDBJ/EMBL/GenBank Accession No. AB084286) there are genes encoding putative hydrolase (ORFK0), meta cleavage enzyme (ORFK1), and hydrolase (ORFK2) which is possibly involved in the degradation of monocyclic aromatic compounds based on the substrate specificity of meta cleavage enzyme [17] and homology search results (data not shown). To obtain the direct evidence for the role of these enzymes in the degradation of aromatic compounds by this strain, we examined the presence of the transcripts of these genes in strain DBF63 cells grown on DF and/or FN. As shown in Fig. 3A, the clear amplifications of the portions of the dbfA1 and the dbfC genes were detected in the RT-PCR analyses using the total RNA isolated from the DF-grown strain DBF63 cells, while the PCRamplicons of other genes were not detected. In case of total RNA extracted from FN-grown strain DBF63 cells, only the dbfA1 and phtA3A4 genes were amplified as shown in Fig. 3B. On the other hand, none of the genes were amplified from mRNAs prepared from cells grown on salicylate or gentisate (data not shown). Together with the fact that no amplified products were detected for any genes in the negative control analyses lacking the RT (data not shown), these findings demonstrate that the dbfA1A2 genes were specifically transcribed in the strain DBF63 cells grown on DF or FN, and suggest that DbfA1A2 functions as the initial angular dioxygenase in the DF degradation pathway, and as the initial 9-monooxygenase for FN and the angular dioxygenase for 9-fluorenone in FN degradation pathways. Specific expression of dbfC gene in DF-grown strain DBF63 cells indicated that DbfB and DbfC are involved in DF metabolism as second and third conversion steps, but not in FN metabolism. These results indicate that the DF degradation system of strain DBF63 consists of the enzymes encoded in the distinct loci in the genome. The dbfA1A2 cistron is a part of the
well-clustered operon involved in the degradation of other aromatic compound [H. Habe, H. Kato, K. Kasuga, T. Yoshida, H. Nojiri, T. Omori, unpublished results]. Iida et al. [22] reported that the DF-degrading Rhodococcus sp. strain YK2 has different DF degradation enzymes. It was also revealed that, in addition to its own DF-degrading enzymes, strain YK2 had the dbfA1A2 cistron on the genome, but that the dbfA1A2 cistron of strain YK2 was not expressed in DF-grown strain YK2 cells [22]. These results indicate the possibilities that, in strain DBF63 cells, the expression mechanism of dbfA1A2 cistron has been changed for induced expression in the presence of DF, and that the employment of the DbfA1A2 as an initial degradation enzyme for DF made this strain a DF-degrader. Similarly, the dioxin (DF) degradation pathway by Gramnegative dioxin-degrading bacterium Sphingomonas wittichii strain RW1 was shown to consist of the elements separately encoded on the genome [23]. Because of the rareness of the enzymes that can participate in the degradation pathway in nature, the recruitment of these enzymes might be indispensable for the formation of the entire metabolic pathway of dioxin. Accordingly, a longer period of evolution might be necessary to constitute the well-clustered genetic structure. Similar dispersion of the catabolic elements on the genome has been mainly reported in the genera Sphingomonas and Rhodococcus. In addition to the dioxin-degradation pathway of S. wittichii strain RW1, the degradation pathway for c-hexachlorocyclohexane by S. paucimobilis strain UT26 was encoded by five structural genes and one regulatory gene encoded on distinct loci on the chromosome [24]. The biphenyl/ polychlorinated biphenyl degradation genes of Rhodococcus sp. strain RHA1 were shown to be located on the chromosome and two linear plasmids (pRHL1 and pRHL2) [25–28]. From the information we now have, it is unclear whether this tendency originates from the characteristics of the genera or not. dbfA1A2 genes was isolated from not only Terrabacter strains but also
Fig. 3. Transcriptional analysis of dbfA1A2, dbfBC, pht gene, ORFK0, and ORFK2 in the strain DBF63 cells grown on DF (A) and FN (B) by RTPCR. Lanes: 1 and 7, DNA size standards (1057, 770, 612, 495, 392, 340, 297, 210, 162, and 79 bp; top to bottom); lane 2, dbfA1; lane 3, dbfC; lane 4, phtA3A4; lane 5, ORFK0; lane 6, ORFK2.
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Rhodococcus strains [T. Noumura, H. Habe, J. Widada, T. Yoshida, H. Nojiri, T. Omori, unpublished results]. Under these situations, it seems to be interesting to compare the frequency of the change of the genetic structure in response to the environmental condition among the bacteria classified to the various genera, including Sphingomonas, Rhodococcus, and Terrabacter. While the meta cleavage and subsequent hydrolysis are involved in the FN degradation pathway, the results obtained in this study clearly indicated that strain DBF63 should have another set of meta cleavage enzyme and hydrolase specific for the FN degradation pathway. In fact, there exist another meta cleavage enzyme gene and hydrolase gene in the downstream region of dbfA1A2 cistron [18, H. Habe, H. Kato, K. Kasuga, T. Yoshida, H. Nojiri, T. Omori, unpublished results]. Although phthalate is a key intermediate of the FN degradation pathway [29], strain DBF63 was unable to grow on this compound, probably because of its low permeability through the cell membrane. In the RT-PCR experiment, phtA3A4 genes were shown to be induced in strain DBF63 cells grown on FN (Fig. 3B), indicating that the phthalate degradation pathway encoded by pht gene cluster physiologically functions in FN degradation by strain DBF63. As shown in Fig. 2, dbfA1A2 genes were located on an operon distinct from the pht operon. The results obtained indicated that two gene clusters involved in the FN degradation are encoded tandemly in this locus on the DBF63 genome. Considering the fact that only the dbfA1A2 cistron is transcribed in the strain DBF63 cells grown on DF, it is quite interesting to reveal how to control the expression of these two operons depending on the carbon source. As reported by Iida et al. [22], the dbfA1A2 cistron of strain YK2 was not expressed in the strain YK2 cells grown on DF. Accordingly, it is considered that the induction mechanism of dbfA1A2 genes of strain DBF63 is different from that of YK2. Previous data on the substrate specificity of meta cleavage enzyme encoded by ORFK1 suggested that this enzyme is related to monocyclic aromatic compound degradation [17]. A homology search of the three ORFs observed in locus K revealed that the products of ORFK0 and ORFK1 showed the highest homologies to the hydroxylase and meta cleavage enzyme in 3-(3-hydroxyphenyl)propionate degradation pathway in E. coli and Comamonas testosteroni (data not shown). However, the amplification products of ORFK0 and ORFK2 were not observed in RT-PCR analyses using mRNAs prepared from the strain DBF63 cells grown on DF, FN (Fig. 3), salicylate, or gentisate (data not shown). In addition, because the expression of these genes were not observed in the cells grown on FN, it is quite likely that these genes are not involved in phthalate degradation. Thus, it was revealed that the enzymes encoded by ORFK0, ORFK1, and ORFK2 are not related to the degradation of DF or FN by this strain.
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Localization of pht-dbfA1A2 and dbfBC loci in the genome of Terrabacter sp. strain DBF63 The results of PFGE analyses clearly showed the presence of three replicons in strain DBF63 cells, although a large amount of DNA remained in the well as shown in Fig. 4A (lane 2). Among the three replicons observed, the 16S rRNA gene probe was specifically hybridized to the band with the smallest mobility (Fig. 4B, lane 2), suggesting that it is the chromosome of strain DBF63. Strong hybridization was also observed in the region corresponding to the well, suggesting that the efficiency of the cell lysis was not high. Thus, it was revealed that strain DBF63 has at least two plasmids, besides the chromosome. On the other hand, only one DNA band with a mobility on the gel similar to that of the chromosome, was detected in the DF mutant strain DBF63W (Fig. 4A, lane 3). The mobility of the band observed in strain DBF63W varied depending on the cell lysis and embedding conditions (data not shown). Because of the hybridization to the 16S rRNA gene probe (Fig. 4B, lane 3), the band observed in strain DBF63W was a chromosome. These results clearly indicated that strain DBF63 had lost both plasmids to give the DF mutant strain DBF63W. The two plasmids observed in strain DBF63 cell appeared to be linear, because they showed migration patterns similar to the marker DNA (k DNA concatemer with k DNA HindIII fragments; Fig. 4A, lane 1) under the different pulse times (data not shown) like other linear plasmids isolated from the Gram-positive bacteria [25,30,31]. The plasmids were estimated to be about 160 and 190 kb. The probe prepared from dbfA1 specifically hybridized to two plasmids (Fig. 4C, lane 2), suggesting that the dbfA1A2 cistron was located on these plasmids. Therefore, we designated them pDBF1 (about 160 kb) and pDBF2 (about 190 kb). The relative intensity of the two bands observed in the PFGE gel (Fig. 4A, lane 2) was quite similar to that of the hybridization observed after the probing with the dbfA1 probe (Fig. 4C, lane 2). This suggests the possibility that the two plasmids each contain the same number of dbfA1 gene(s). Considering that a larger amount of pDBF1 was detected by PFGE analysis (Fig. 4A, lane 2), it is possible that pDBF2 was derived from pDBF1. Because we used a single colony of strain DBF63 as an inoculum to prepare the cells for the DNA plug in the PFGE analysis, the gene rearrangement might have occurred on pDBF1 during the cultivation. There might be a hot spot for the gene rearrangement on the plasmid pDBF1. Now, we are carrying out the determination of the entire nucleotide sequence of pDBF1. The sequence data will be a useful tool to reveal the difference of these two plasmids. Since Hayakawa et al. [32] discovered the first bacterial linear plasmid in Streptomyces rochei, many linear
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Fig. 4. Localization of pht-dbfA1A2 locus, dbfBC locus, and locus K in Terrabacter sp. strain DBF63 genome. (A) The results of PFGE analysis of strains DBF63 (lane 2) and DBF63W (lane 3) is shown. Lane 1 is the Low Range PFG Marker (New England Biolabs, Beverly, MA, USA). (B–E), Southern hybridization analyses of total DNAs extracted from strains DBF63 (lane 2) or DBF63W (lane 3) were performed using the 16S rRNA gene (B), dbfA1A2 (C), dbfC (D), and ORFK1K2 as probes. The pulse time was increased from 0.2 to 22 s in PFGE analyses shown in (A)–(E).
double-stranded DNA plasmids of various sizes (from 12 kb to 1 Mb) have been reported in actinomycete genera such as Streptomyces, Rhodococcus, Mycobacterium, and Planbispora (for a review, see [33]). Up to now, several linear plasmids encoding the enzymes involved in the aromatic compound degradation have been reported in genus Rodococcus [25,27,34–38]. The plasmids pDBF1 and pDBF2 are the first to be demonstrated on the linear plasmid isolated from the Terrabacter strains and are involved in the bacterial dioxin degradation. The dbfA1A2 genes have been isolated from not only Terrabacter strains but also Rhodococcus strains [T. Noumura, H. Habe, J. Widada, T. Yoshida, H. Nojiri, T. Omori, unpublished results]. In addition, Rhodococcus sp. strain YK2 was shown to have the dbfA1A2 cistron on the genome [22]. From these results, it is quite likely that plasmid pDBF1 and/or pDBF2 are selftransmissible plasmids, and that these plasmids play an important role in the distribution of the dioxin-degrading dbfA1A2 genes in nature (probably in Gram-positive bacteria). Interspecific transfers of several linear plasmids have been reported in Streptomyces and Rhodococcus strains [27,34,39–43]. Together with the fact that the DF 4,4a-dioxygenase system encoded on the dbfA1A2 cistron can decompose dioxin, it is quite interesting to reveal the conjugative host range and the host range for replication of these plasmids. The entire structure of pDBF1 (and/or pDBF2) will provide us useful information on the mechanisms of maintenance
and lateral transfer of this plasmid between the bacterial cells. As reported by Kasuga et al. [17], it was confirmed that the dbfC gene was lost in strain DBF63W, while the locus K was retained in both strains DBF63 and DBF63W (Figs. 4D and E). Clear and specific hybridization of dbfC probe to the chromosome of strain DBF63 was observed (Fig. 4D). Thus, it was revealed that dbfA1A2 cistron and dbfBC genes were encoded on different replicons in strain DBF63. In the PFGE analyses under the conditions suitable for a larger size of DNA fragments (0.2–2.2 Mb), no additional bands hybridized to the dbfC probe different from the chromosome were detected (data not shown). The above results suggested that dbfBC genes were encoded on the chromosome, although the possibility that the dbfC gene is encoded on the megaplasmid whose molecular size is similar to that of the chromosome cannot be excluded. In addition to the curing of the pDBF1 (pDBF2), an additional deletion event had occurred to give strain DBF63W. As shown in Fig. 2, the insertion sequence, ISTesp1, was found in the vicinity of the dbfBC cistron [17]. Based on the results of Southern hybridization analysis, strain DBF63 was shown to have at least four copies of ISTesp1 on its genome [17]. This ISTesp1 might be involved in the deletion of dbfBC genes from the strain DBF63 genome. Genetic analysis of the flanking region of dbfBC gene and comparison of the DNA region with the corresponding region of strain DBF63W will be necessary.
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Acknowledgments Part of this work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) in Japan.
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Dioxin catabolic genes are dispersed on the Terrabacter sp. DBF63 genome Hideaki Nojiri,a Mayuko Kamakura,b Masaaki Urata,a Takahiro Tanaka,b Jin-Sung Chung,a Tetsuo Takemura,b Takako Yoshida,a Hiroshi Habe,a and Toshio Omoria,* a
b
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Department of Chemistry, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan Received 1 July 2002
Abstract Reverse transcription-PCR of the dbfA1A2, dbfBC, and pht genes, encoding oxygenase component of multicomponent dioxygenase, meta cleavage enzyme and hydrolase, and phthalate-degrading enzymes, respectively, revealed their role in the aromatic compound degradation by Terrabacter sp. strain DBF63. The specific expression in strain DBF63 cells grown on dibenzofuran (the model compound of dioxin; DF) and/or fluorene (FN) indicated that the DbfA1A2 and DbfBC catalyze the conversion of DF to salicylate, and that the DbfA1A2 and Pht enzymes are involved in FN degradation. Pulsed-field gel electrophoresis analyses revealed that the dbfA1A2 cistron and pht operon were located on the two linear plasmids, pDBF1 (160 kb) and pDBF2 (190 kb), while dbfBC genes were located on the chromosome. Because the pht operon is located immediately upstream of the dbfA1A2 cistron, the dioxin-catabolic genes were dispersed on the genome of strain DBF63, while FN-catabolic genes were gathered on the plasmids. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Biodegradation; dbf Gene cluster; Expression; Degradative plasmid; pDBF1; Dibenzofuran; Dioxin; Fluorene; Terrabacter sp. strain DBF63
Polychlorinated dibenzo-p-dioxins and dibenzofurans, called dioxins, are produced by incineration processes and, unintentionally, as by-products during the synthesis of pesticides and herbicides. Many of their congeners are highly toxic compounds with undesirable effects on the environment and living beings [1,2], but have been widely distributed in the environment [3–5]. The dioxins are recalcitrant molecules and show extremely high persistence in soil sediments [6,7]. Because of the relatively low cost and minimal impact on the environment, dioxin-degrading bacteria have been isolated and investigated by using the dibenzofuran (DF) as a model substrate in enrichment culture as reviewed previously [8–12]. In the microbial degradation pathway of DF, the angular dioxygenation at 4 and 4a positions is the initial *
Corresponding author. Fax: +81-3-5841-8030. E-mail address: [email protected] (T. Omori).
degradation reaction (Fig. 1). This novel type of dioxygenation reaction is shown to be catalyzed by DF 4,4adioxygenase, and leads DF to a chemically unstable intermediate that is spontaneously rearomatized to 2,20 , 3-trihydroxybiphenyl (THB). Subsequent ring cleavage of THB is catalyzed by meta cleavage enzyme yielding 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-dienoic acid and salicylic acid (Fig. 1). Salicylic acid is then metabolized via catechol or gentisic acid. Among these metabolic enzymes, initial angular dioxygenase is a key determinant of the substrate range in the degradation of heterocyclic aromatic compounds including dioxin [13–15]. Terrabacter sp. strain DBF63 was isolated from soil as a microorganism having the ability to utilize DF or fluorene (FN) as a sole source of carbon and energy [16]. Based on the identification of the metabolic intermediates, Monna et al. [16] proposed the degradation pathways for DF and FN by strain DBF63 (Fig. 1). By the shotgun cloning using the meta cleavage activity as a
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 8 7 3 - 2
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Fig. 1. Comparison of the degradation pathways for DF and FN by Terrabacter sp. strain DBF63. The structures shown in brackets have not been characterized directly. The arrows with solid and broken lines indicate the enzymatic and spontaneous conversions, respectively. The bold letters indicate that the conversions are catalyzed by the corresponding enzymes. The conversion of 1,1a-dihydroxy-1-hydrofluorene-9-one to phthalic acid is catalyzed by the specific enzymes [H. Habe, H. Kato, K. Kasuga, T. Yoshida, H. Nojiri, T. Omori, unpublished results]. Compound designations: I, DF; II, cis-4,4a-dihydroxy-4-hydrodibenzofuran; III, 2,20 ,3-trihydroxybiphenyl (THB); IV, 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid; V, salicylic acid; VI, FN; VII, 9-fluorenol; VIII, 9-fluorenone; IX, 1,1a-dihydroxy-1-hydrofluorene-9-one; X, 20 -carboxy-2,3-dihydroxybiphenyl; XI, 2-hydroxy-6-(2-carboxyphenyl)-6-oxo-2,4-hexadienoic acid; XII, phthalic acid; XIII, protocatechuic acid.
selection marker, we have cloned the DNA fragments containing dbfBC genes and ORFK0K1K2 genes encoding the meta cleavage enzyme (DbfB and ORFK1 products) and meta cleavage compound hydrolase (DbfC and ORFK2 products) (Fig. 2) [17]. The fact that DbfB has strict preference to the binuclear substrate, such as 2,3-dihydroxybiphenyl and 2,20 ,3-trihydroxybiphenyl, indicated the possibility that DbfB constitutes the DF degradation pathway by this strain. Also, we successfully isolated the dbfA1A2 cistrons encoding the a and b subunits of terminal oxygenase component of DF 4,4a-dioxygenase from strain DBF63 genome by using the PCR-based method (Fig. 2) [18]. Although the electron transport components co-working with this oxygenase component were not identified, we could detect the angular dioxygenase activity of DbfA1A2 for
Fig. 2. Genetic organization of pht-dbfA1A2 and dbfBC loci encompassing the genes involved in DF and phthalate degradation [17,18, H. Habe, M. Miyakoshi, K. Kasuga, T. Yoshida, H. Nojiri, T. Omori, submitted] and locus K (Accession No. AB084286) in Terrabacter sp. strain DBF63 genome. The pentagons and triangles in the physical map indicate the size, location, and the direction of transcription of the ORFs derived from the nucleotide sequence data. DF-degrading genes and insertion sequence are shown by shaded pentagons and black box, respectively.
DF and FN by using the unspecific electron transport system of the Escherichia coli host strain [18, H. Habe, H. Kato, K. Kasuga, T. Yoshida, H. Nojiri, T. Omori, unpublished results]. As we expected, the DF-degrading system of strain DBF63 was able to decompose chlorinated DF/dibenzo-p-dioxin [15,19]. Because the DFmetabolism-deficient (DF ) derivative strain DBF63W did not contain the dbfA1A2 and dbfBC genes [17,18], we speculated that the DbfA1A2 and DbfBC functions in the DF and FN degradation by strain DBF63. Very recently, we revealed the phthalate degradation gene cluster [phtA1A2(ORF11)BA3A4CR] responsible for the conversion of phthalate to protocatechuate [H. Habe, M. Miyakoshi, K. Kasuga, T. Yoshida, H. Nojiri, T. Omori, submitted] in the upstream region of dbfA1A2 cistron as shown in Fig. 2. Because phthalate is a metabolic intermediate of FN, the degradation pathway encoded by the phtA1A2(ORF11)BA3A4CR genes should constitute the lower part of the entire FN degradation pathway. In this study, to confirm the role of the DbfA1A2, DbfBC, and phthalate-degrading enzymes in DF and FN degradation by strain DBF63, we carried out transcriptional analysis of the corresponding genes in strain DBF63 grown on various carbon sources. In addition, we discussed the evolution of the DF- and FN-degradation pathway based on the localization of these genes in the genome of strain DBF63.
Materials and methods Bacterial strains, plasmid, and culture conditions. Strain DBF63 is a DF-degrading bacterium that was isolated from a soil sample in Japan on the basis of its ability to grow on DF as a sole source of carbon and energy [16]. Strain DBF63W obtained by the continuous culture of strain DBF63 on the nutrient broth can utilize salicylic acid but not DF as a sole source of carbon and energy [17]. Strains DBF63 and
H. Nojiri et al. / Biochemical and Biophysical Research Communications 296 (2002) 233–240 DBF63W were cultivated in carbon-free mineral medium (CFMM) supplemented with the DF and nutrient medium, commercially available from Eiken Chemical (Tokyo, Japan), respectively, at 30 °C with reciprocal shaking (300 strokes/min) as described by Kasuga et al. [17]. E. coli strain JM109 [recA1, D(lac-proAB), endA1, gyrA96, thi-1, hsdR17, relA1, supE44, F0 (traD36, proAB, lacI q ZDM15)] [20] was used as a host strain of plasmid pUC118/119 [20] and their derivatives. E. coli strains were grown on LB or 2YT media [20] at 37 °C. For plate cultures, the above media solidified with 1.6% (wt/vol) agar were used. DNA manipulations. Plasmid DNA for routine DNA manipulation was prepared from the E. coli cell by the alkaline lysis method and purified as described by Sambrook et al. [20]. Plasmid DNA used for PCR template was prepared by using the Quantum Prep Plasmid Miniprep Kit (Bio-Rad Laboratories, Richmond, CA, USA) as recommended by the manufacturer. Restriction endonucleases were used according to manufacturer’s instructions. DNA fragments were fractionated by electrophoresis using 0.9% agarose in TAE buffer [20], and were extracted from the agarose gel by using Concert Rapid Gel Extraction Systems (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. Other DNA manipulation procedures were performed according to the standard methods [20]. Transcriptional analyses of dbfA1A2 and dbfBC genes by reverse transcription (RT)-PCR. After a 4-day cultivation of strain DBF63 in 5 ml CFMM supplemented with DF, cells were gathered from 2 ml of culture by centrifugation at 7200g, then washed twice using CFMM. After the washed cells were suspended in 2 ml CFMM, the cells were incubated at 30 °C with reciprocal shaking at 300 strokes/min for 12 h as starvation culture. The starved cells were washed twice as described above, and finally suspended in 2 ml CFMM. Five-milliliter CFMM supplemented with 0.1% (wt/vol; in final concentration) of CAR, FN, salicylic acid, or gentisic acid was inoculated with 2 ml of the resultant cell suspension of strain DBF63. After 12-h incubation with reciprocal shaking (300 strokes/min) at 30 °C, the cells were harvested and used for extraction of total RNA. Total RNA from harvested cell was extracted using an RNeasy Mini Kit (Qiagen, Santa Clarita, CA, USA) according to manufacturer’s instruction. By RT-PCR analyses, the transcriptions of dbfA1, dbfC, and ORFK2 were monitored. RT-PCR was performed using a Takara One Step RNA PCR Kit (AMV) (Takara Shuzo, Kyoto, Japan) as recommended by the manufacturer. In RT-PCR, 1 lg total RNA prepared from the strain DBF63 cells was used as a template RNA. Table 1 shows the names of primer sets and their nucleotide sequences. After the RT at 60 °C for 30 min followed by the initial denaturation step at 94 °C for 2 min, denaturation, annealing, and polymerization were performed for 25 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 2 min, respectively. Pulsed-field gel electrophoresis (PFGE). Strain DBF63 was grown on DF for 4 days, and then the cells were harvested from 5 ml of culture as described above, and washed with TE buffer [20]. After the
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resultant cells were suspended in 50 ll of the Cell Suspension Buffer in the CHEF Bacterial Genomic DNA Plug Kit (Bio-Rad Laboratories), the plug for PFGE analysis was prepared as recommended by the manufacturer with some modification. For effective lysis of strain DBF63 cells grown on DF, we used lysozyme and proteinase K at the final concentrations of 1 and 8 mg/ml, respectively. Strain DBF63W was grown on nutrient medium for 4 days, and the cells corresponding to 0.5 ml culture were used for the suspension with 50 ll of Cell Suspension Buffer. For strain DBF63W cell, we used lysozyme and proteinase K at the final concentrations of 0.5 and 4 mg/ml, respectively. PFGE was performed by using a CHEF DRIII PFGE System (BioRad Laboratories) and 0.5 Tris–Borate–EDTA buffer [20]. Electrophoresis was performed with electric field of 6 V/cm at 14 °C. The sets of pulse time and running time were from 0.2 to 22 s and 15 h, from 20 to 30 s and 15 h, or from 30 to 60 s and 22 h. The field angle was 120°. Southern hybridization analysis. After PFGE, DNA was transferred to a Biodyne B nylon membrane (Poll BioSupport, East Hills, NY, USA) by VacuGene XL Vacuum Blotting System (Amersham Pharmacia Biotech UK, Little Chalfont, Buckinghamshire, England) as recommended by the manufacturer. The preparation of probes and Southern blots were performed by using a nonradioactive DIG DNA labeling and detection kit (Roche Diagnostics GmbH) as described previously [21]. To prepare the dbfA1A2-, dbfBC-, and ORFK1K2specific probes, we used as templates a 1.2-kb PstI fragment of pDF32 [18], a 1.3-kb SalI fragment of pLM105R [17], and a 2.2-kb SphI–PstI fragment of pKN1 [17], respectively. The 16S rRNA gene was amplified as described previously [21], and used as a template for probe preparation. Chemicals. Chemicals used in this study were of the highest purity commercially available (Merck, Darmstadt, Germany; Sigma–Aldrich, Steinheim, Germany; Kanto Chemical, Tokyo, Japan; Wako Pure Chemical, Osaka, Japan; Nacalai Tesque, Kyoto, Japan).
Results and discussion The role of the dbf and pht genes in the cell of Terrabacter sp. strain DBF63 We have revealed the genes encoding the terminal oxygenase of angular dioxygenase (DbfA1A2), meta cleavage enzyme (DbfB), and meta cleavage compound hydrolase (DbfC) (pht-dbfA1A2 and dbfBC loci in Fig. 2). The catabolic activities of these enzymes suggest that they are involved in the DF degradation by this strain. In the upstream region of the dbfA1A2 cistron, there exist the pht genes proposed to be involved in the conversion of
Table 1 Primers used in the RT-PCR analyses Target gene
Name of primer
Nucleotide sequence
dbfA1
dbfA1A2-F dbfA1A2-R
50 -CGGCGTCTTCCACGTCTTCG-30 50 -CCCGGGGTGGTCATGAGTTC-30
dbfC
dbfC-F dbfC-R
50 -CTGGAAGATCCACTACAACG-30 50 -GTGATCAGGTGCGAGATCCG-30
ORFK2
ORFK2-750F ORFK2-1120R
50 -CGCCTTCGACGACTACGACA-30 50 -TTCTGCGAGAAGTTGCTCCA-30
ORFK0
ORFK0-F ORFK0-R
50 -TTCGGCAACATCATCAAGCC-30 50 -TCGGACATGAAGGAGACGAA-30
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phthalate to protocatechuate (pht-dbfA1A2 and dbfBC loci in Fig. 2) [Habe et al., submitted for publication]. In addition, in another locus (designated as locus K as shown in Fig. 1) (DDBJ/EMBL/GenBank Accession No. AB084286) there are genes encoding putative hydrolase (ORFK0), meta cleavage enzyme (ORFK1), and hydrolase (ORFK2) which is possibly involved in the degradation of monocyclic aromatic compounds based on the substrate specificity of meta cleavage enzyme [17] and homology search results (data not shown). To obtain the direct evidence for the role of these enzymes in the degradation of aromatic compounds by this strain, we examined the presence of the transcripts of these genes in strain DBF63 cells grown on DF and/or FN. As shown in Fig. 3A, the clear amplifications of the portions of the dbfA1 and the dbfC genes were detected in the RT-PCR analyses using the total RNA isolated from the DF-grown strain DBF63 cells, while the PCRamplicons of other genes were not detected. In case of total RNA extracted from FN-grown strain DBF63 cells, only the dbfA1 and phtA3A4 genes were amplified as shown in Fig. 3B. On the other hand, none of the genes were amplified from mRNAs prepared from cells grown on salicylate or gentisate (data not shown). Together with the fact that no amplified products were detected for any genes in the negative control analyses lacking the RT (data not shown), these findings demonstrate that the dbfA1A2 genes were specifically transcribed in the strain DBF63 cells grown on DF or FN, and suggest that DbfA1A2 functions as the initial angular dioxygenase in the DF degradation pathway, and as the initial 9-monooxygenase for FN and the angular dioxygenase for 9-fluorenone in FN degradation pathways. Specific expression of dbfC gene in DF-grown strain DBF63 cells indicated that DbfB and DbfC are involved in DF metabolism as second and third conversion steps, but not in FN metabolism. These results indicate that the DF degradation system of strain DBF63 consists of the enzymes encoded in the distinct loci in the genome. The dbfA1A2 cistron is a part of the
well-clustered operon involved in the degradation of other aromatic compound [H. Habe, H. Kato, K. Kasuga, T. Yoshida, H. Nojiri, T. Omori, unpublished results]. Iida et al. [22] reported that the DF-degrading Rhodococcus sp. strain YK2 has different DF degradation enzymes. It was also revealed that, in addition to its own DF-degrading enzymes, strain YK2 had the dbfA1A2 cistron on the genome, but that the dbfA1A2 cistron of strain YK2 was not expressed in DF-grown strain YK2 cells [22]. These results indicate the possibilities that, in strain DBF63 cells, the expression mechanism of dbfA1A2 cistron has been changed for induced expression in the presence of DF, and that the employment of the DbfA1A2 as an initial degradation enzyme for DF made this strain a DF-degrader. Similarly, the dioxin (DF) degradation pathway by Gramnegative dioxin-degrading bacterium Sphingomonas wittichii strain RW1 was shown to consist of the elements separately encoded on the genome [23]. Because of the rareness of the enzymes that can participate in the degradation pathway in nature, the recruitment of these enzymes might be indispensable for the formation of the entire metabolic pathway of dioxin. Accordingly, a longer period of evolution might be necessary to constitute the well-clustered genetic structure. Similar dispersion of the catabolic elements on the genome has been mainly reported in the genera Sphingomonas and Rhodococcus. In addition to the dioxin-degradation pathway of S. wittichii strain RW1, the degradation pathway for c-hexachlorocyclohexane by S. paucimobilis strain UT26 was encoded by five structural genes and one regulatory gene encoded on distinct loci on the chromosome [24]. The biphenyl/ polychlorinated biphenyl degradation genes of Rhodococcus sp. strain RHA1 were shown to be located on the chromosome and two linear plasmids (pRHL1 and pRHL2) [25–28]. From the information we now have, it is unclear whether this tendency originates from the characteristics of the genera or not. dbfA1A2 genes was isolated from not only Terrabacter strains but also
Fig. 3. Transcriptional analysis of dbfA1A2, dbfBC, pht gene, ORFK0, and ORFK2 in the strain DBF63 cells grown on DF (A) and FN (B) by RTPCR. Lanes: 1 and 7, DNA size standards (1057, 770, 612, 495, 392, 340, 297, 210, 162, and 79 bp; top to bottom); lane 2, dbfA1; lane 3, dbfC; lane 4, phtA3A4; lane 5, ORFK0; lane 6, ORFK2.
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Rhodococcus strains [T. Noumura, H. Habe, J. Widada, T. Yoshida, H. Nojiri, T. Omori, unpublished results]. Under these situations, it seems to be interesting to compare the frequency of the change of the genetic structure in response to the environmental condition among the bacteria classified to the various genera, including Sphingomonas, Rhodococcus, and Terrabacter. While the meta cleavage and subsequent hydrolysis are involved in the FN degradation pathway, the results obtained in this study clearly indicated that strain DBF63 should have another set of meta cleavage enzyme and hydrolase specific for the FN degradation pathway. In fact, there exist another meta cleavage enzyme gene and hydrolase gene in the downstream region of dbfA1A2 cistron [18, H. Habe, H. Kato, K. Kasuga, T. Yoshida, H. Nojiri, T. Omori, unpublished results]. Although phthalate is a key intermediate of the FN degradation pathway [29], strain DBF63 was unable to grow on this compound, probably because of its low permeability through the cell membrane. In the RT-PCR experiment, phtA3A4 genes were shown to be induced in strain DBF63 cells grown on FN (Fig. 3B), indicating that the phthalate degradation pathway encoded by pht gene cluster physiologically functions in FN degradation by strain DBF63. As shown in Fig. 2, dbfA1A2 genes were located on an operon distinct from the pht operon. The results obtained indicated that two gene clusters involved in the FN degradation are encoded tandemly in this locus on the DBF63 genome. Considering the fact that only the dbfA1A2 cistron is transcribed in the strain DBF63 cells grown on DF, it is quite interesting to reveal how to control the expression of these two operons depending on the carbon source. As reported by Iida et al. [22], the dbfA1A2 cistron of strain YK2 was not expressed in the strain YK2 cells grown on DF. Accordingly, it is considered that the induction mechanism of dbfA1A2 genes of strain DBF63 is different from that of YK2. Previous data on the substrate specificity of meta cleavage enzyme encoded by ORFK1 suggested that this enzyme is related to monocyclic aromatic compound degradation [17]. A homology search of the three ORFs observed in locus K revealed that the products of ORFK0 and ORFK1 showed the highest homologies to the hydroxylase and meta cleavage enzyme in 3-(3-hydroxyphenyl)propionate degradation pathway in E. coli and Comamonas testosteroni (data not shown). However, the amplification products of ORFK0 and ORFK2 were not observed in RT-PCR analyses using mRNAs prepared from the strain DBF63 cells grown on DF, FN (Fig. 3), salicylate, or gentisate (data not shown). In addition, because the expression of these genes were not observed in the cells grown on FN, it is quite likely that these genes are not involved in phthalate degradation. Thus, it was revealed that the enzymes encoded by ORFK0, ORFK1, and ORFK2 are not related to the degradation of DF or FN by this strain.
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Localization of pht-dbfA1A2 and dbfBC loci in the genome of Terrabacter sp. strain DBF63 The results of PFGE analyses clearly showed the presence of three replicons in strain DBF63 cells, although a large amount of DNA remained in the well as shown in Fig. 4A (lane 2). Among the three replicons observed, the 16S rRNA gene probe was specifically hybridized to the band with the smallest mobility (Fig. 4B, lane 2), suggesting that it is the chromosome of strain DBF63. Strong hybridization was also observed in the region corresponding to the well, suggesting that the efficiency of the cell lysis was not high. Thus, it was revealed that strain DBF63 has at least two plasmids, besides the chromosome. On the other hand, only one DNA band with a mobility on the gel similar to that of the chromosome, was detected in the DF mutant strain DBF63W (Fig. 4A, lane 3). The mobility of the band observed in strain DBF63W varied depending on the cell lysis and embedding conditions (data not shown). Because of the hybridization to the 16S rRNA gene probe (Fig. 4B, lane 3), the band observed in strain DBF63W was a chromosome. These results clearly indicated that strain DBF63 had lost both plasmids to give the DF mutant strain DBF63W. The two plasmids observed in strain DBF63 cell appeared to be linear, because they showed migration patterns similar to the marker DNA (k DNA concatemer with k DNA HindIII fragments; Fig. 4A, lane 1) under the different pulse times (data not shown) like other linear plasmids isolated from the Gram-positive bacteria [25,30,31]. The plasmids were estimated to be about 160 and 190 kb. The probe prepared from dbfA1 specifically hybridized to two plasmids (Fig. 4C, lane 2), suggesting that the dbfA1A2 cistron was located on these plasmids. Therefore, we designated them pDBF1 (about 160 kb) and pDBF2 (about 190 kb). The relative intensity of the two bands observed in the PFGE gel (Fig. 4A, lane 2) was quite similar to that of the hybridization observed after the probing with the dbfA1 probe (Fig. 4C, lane 2). This suggests the possibility that the two plasmids each contain the same number of dbfA1 gene(s). Considering that a larger amount of pDBF1 was detected by PFGE analysis (Fig. 4A, lane 2), it is possible that pDBF2 was derived from pDBF1. Because we used a single colony of strain DBF63 as an inoculum to prepare the cells for the DNA plug in the PFGE analysis, the gene rearrangement might have occurred on pDBF1 during the cultivation. There might be a hot spot for the gene rearrangement on the plasmid pDBF1. Now, we are carrying out the determination of the entire nucleotide sequence of pDBF1. The sequence data will be a useful tool to reveal the difference of these two plasmids. Since Hayakawa et al. [32] discovered the first bacterial linear plasmid in Streptomyces rochei, many linear
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Fig. 4. Localization of pht-dbfA1A2 locus, dbfBC locus, and locus K in Terrabacter sp. strain DBF63 genome. (A) The results of PFGE analysis of strains DBF63 (lane 2) and DBF63W (lane 3) is shown. Lane 1 is the Low Range PFG Marker (New England Biolabs, Beverly, MA, USA). (B–E), Southern hybridization analyses of total DNAs extracted from strains DBF63 (lane 2) or DBF63W (lane 3) were performed using the 16S rRNA gene (B), dbfA1A2 (C), dbfC (D), and ORFK1K2 as probes. The pulse time was increased from 0.2 to 22 s in PFGE analyses shown in (A)–(E).
double-stranded DNA plasmids of various sizes (from 12 kb to 1 Mb) have been reported in actinomycete genera such as Streptomyces, Rhodococcus, Mycobacterium, and Planbispora (for a review, see [33]). Up to now, several linear plasmids encoding the enzymes involved in the aromatic compound degradation have been reported in genus Rodococcus [25,27,34–38]. The plasmids pDBF1 and pDBF2 are the first to be demonstrated on the linear plasmid isolated from the Terrabacter strains and are involved in the bacterial dioxin degradation. The dbfA1A2 genes have been isolated from not only Terrabacter strains but also Rhodococcus strains [T. Noumura, H. Habe, J. Widada, T. Yoshida, H. Nojiri, T. Omori, unpublished results]. In addition, Rhodococcus sp. strain YK2 was shown to have the dbfA1A2 cistron on the genome [22]. From these results, it is quite likely that plasmid pDBF1 and/or pDBF2 are selftransmissible plasmids, and that these plasmids play an important role in the distribution of the dioxin-degrading dbfA1A2 genes in nature (probably in Gram-positive bacteria). Interspecific transfers of several linear plasmids have been reported in Streptomyces and Rhodococcus strains [27,34,39–43]. Together with the fact that the DF 4,4a-dioxygenase system encoded on the dbfA1A2 cistron can decompose dioxin, it is quite interesting to reveal the conjugative host range and the host range for replication of these plasmids. The entire structure of pDBF1 (and/or pDBF2) will provide us useful information on the mechanisms of maintenance
and lateral transfer of this plasmid between the bacterial cells. As reported by Kasuga et al. [17], it was confirmed that the dbfC gene was lost in strain DBF63W, while the locus K was retained in both strains DBF63 and DBF63W (Figs. 4D and E). Clear and specific hybridization of dbfC probe to the chromosome of strain DBF63 was observed (Fig. 4D). Thus, it was revealed that dbfA1A2 cistron and dbfBC genes were encoded on different replicons in strain DBF63. In the PFGE analyses under the conditions suitable for a larger size of DNA fragments (0.2–2.2 Mb), no additional bands hybridized to the dbfC probe different from the chromosome were detected (data not shown). The above results suggested that dbfBC genes were encoded on the chromosome, although the possibility that the dbfC gene is encoded on the megaplasmid whose molecular size is similar to that of the chromosome cannot be excluded. In addition to the curing of the pDBF1 (pDBF2), an additional deletion event had occurred to give strain DBF63W. As shown in Fig. 2, the insertion sequence, ISTesp1, was found in the vicinity of the dbfBC cistron [17]. Based on the results of Southern hybridization analysis, strain DBF63 was shown to have at least four copies of ISTesp1 on its genome [17]. This ISTesp1 might be involved in the deletion of dbfBC genes from the strain DBF63 genome. Genetic analysis of the flanking region of dbfBC gene and comparison of the DNA region with the corresponding region of strain DBF63W will be necessary.
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Acknowledgments Part of this work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) in Japan.
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