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Applied and Environmental Microbiology, August 2002, p. 3716-3723, Vol. 68, No. 8
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.8.3716-3723.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Plasmid-Borne Genes Code for an Angular Dioxygenase Involved in Dibenzofuran Degradation by Terrabacter sp. Strain YK3

Toshiya Iida,1* Yuki Mukouzaka,1,2 Kaoru Nakamura,1 and Toshiaki Kudo1,3,4

Microbiology Laboratory, RIKEN (The Institute of Physical and Chemical Research),1 Bio-Recycle Project, Japan Science and Technology Corporation (JST), Wako, Saitama 351-0198,3 Graduate School of Science and Engineering, Saitama University, Saitama 338-0825,2 Science of Biological Supramolecular Systems, Graduate School of Integrated Science, Yokohama City University, Suehiro, Tsurumi-ku, Yokohama 230-0045, Japan4

Received 23 January 2002/ Accepted 30 April 2002


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The genes responsible for angular dioxygenation of dibenzofuran in actinomycetes were cloned by using a degenerate set of PCR primers designed by using conserved sequences of the dioxygenase alpha subunit genes. One sequence of alpha subunit genes was commonly amplified from four dibenzofuran-utilizing actinomycetes: Terrabacter sp. strains YK1 and YK3, Rhodococcus sp. strain YK2, and Microbacterium sp. strain YK18. A 5.2-kb PstI fragment encoding the alpha and beta subunits of the terminal dioxygenase, ferredoxin, and ferredoxin reductase (designated dfdA1 to dfdA4, respectively) was cloned from the large circular plasmid pYK3 isolated from Terrabacter sp. strain YK3. We confirmed that transcription of the dfdA1 gene was induced by dibenzofuran in Terrabacter sp. strain YK3. Southern blot hybridization analysis revealed that this type of dioxygenase gene is distributed among diverse dibenzofuran-utilizing actinomycetes. However, genes homologous to dfdA1 were not detected in dibenzofuran utilization-deficient mutants of Terrabacter, Rhodococcus, and Microbacterium species. When the dfdA1 to dfdA4 genes were introduced into a non-dibenzofuran-degrading mutant of Rhodococcus sp. strain YK2, strain YK2-RD2, which had spontaneously lost the gene homologous to dfdA1, the ability to degrade dibenzofuran was restored. Analysis of the breakdown products indicated that DfdA has angular dioxygenase activity. This dfdA transformant degraded several aromatic compounds, indicating that the novel angular dioxygenase possesses broad substrate specificity.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Environmental pollution caused by dioxins, which is a general term for polychlorinated dibenzofuran (DF), dibenzo-p-dioxin (DD), and biphenyl (specifically the coplanar polychlorinated biphenyl), is of increasing concern. These chemicals are produced as by-products from processes such as incineration, pulp bleaching, and chemical synthesis and are stable in the environment, especially they are when adsorbed to particles such as those in soils and fly ash.

Microbial degradation of dioxins was studied by using DF and DD as model compounds. Several bacteria capable of assimilating DF and DD have been described (10, 13, 15, 18, 22, 30, 33). The first step in the biodegradation of DF and DD is characteristic of the substrate specificity. The naphthalene and biphenyl dioxygenases that catalyze dioxygenation of DF mediate oxygenation at DF and DD lateral positions, such as the [1,2] position, and produce relatively stable cis-dihydrodiol derivatives (6, 11, 24). In contrast, the activity found in DF- and DD-utilizing bacteria is specific for the [4,4a] or so-called angular position (reviewed in references 7 and 32). The resulting hemiacetal intermediates of DF and DD produced by the angular dioxygenase are unstable and spontaneously convert to the trihydroxy derivatives of biphenyl and diphenylether, respectively. Although several DF- and DD-utilizing bacteria have been isolated, there are only a few examples in which the gene encoding the dioxygenase responsible for the initial oxygenation of these substrates has been cloned. Currently, the only examples are DxnA and the cognate electron transport proteins from Sphingomonas wittichii RW1 (2-5, 8) and DbfA from Terrabacter sp. strain DBF63 (16). The carbazole dioxygenase (CarA) from the carbazole-utilizing organism Pseudomonas sp. strain CA10 was reported to possess angular dioxygenase activity against DF and DD (23, 28).

In the field of dioxin bioremediation, there is enormous interest in isolating angular dioxygenases with different substrate specificities. For this reason, we isolated several DF-utilizing actinomycetes, including Rhodococcus, Terrabacter, and Microbacterium strains, from a number of soil samples (14). Five extradiol dioxygenase genes were identified from Rhodococcus sp. strain YK2, and one of these (dfdB) was determined to be important for DF degradation both by transcriptional analysis and by studying mutants of the DF degradation pathway which arose spontaneously. We also investigated whether genes homologous to dfdB could be detected in a diverse range of DF-utilizing actinomycete strains.

In this study we cloned and characterized a novel plasmid-encoded angular dioxygenase from the DF-utilizing actinomycete Terrabacter sp. In this paper we also discuss the evolution of DF-degradative genes in actinomycetes.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacteria, media, and culture conditions.
Most chemicals used in this study were obtained from Nacalai Tesque and Wako Pure Chemical Industries and were the highest purity available. DF and DD were purchased from Tokyo Chemical Industry. The DF used for the bioconversion assay, phenoxathiin, and 9-hydroxyfluorene were purchased from Aldrich. Escherichia coli JM109 was used for routine cloning experiments and was cultured at 37°C on Luria-Bertani (LB) medium (27). Ampicillin, isopropyl-ß-D-thiogalactopyranoside (IPTG), and 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-Gal) were added at final concentrations of 100 µg ml-1, 1 mM, and 40 µg ml-1, respectively, for plasmid selection. For solid media, 1.5 to 2.5% agar powder or agar purified powder (Nacalai Tesque) was added. The compositions of minimal medium supplements of yeast extract (MM3Y) and appropriate carbon sources (DF, glucose, and salicylate) have been reported previously (14). The DF-utilizing bacteria Terrabacter sp. strainsYK1 and YK3, Rhodococcus sp. strain YK2, and Microbacterium sp. strain YK18 were isolated from soil samples in Japan by selecting the organisms able to grow on DF as a sole carbon and energy source (14) at 30°C. Derivatives of Terrabacter sp. stain YK3 cured of plasmid pYK3 (strains YK3-TD1 and YK3-TD2) were isolated as follows. A mid-log-phase culture of YK3 on LB medium incubated at 30°C was shifted to 37°C and cultured for an additional 2 days. Diluted cultures were spread on LB agar to obtain single colonies. Three hundred heat-shocked cells were analyzed for the ability to assimilate DF by replication onto MM3Y-DF and MM3Y-glucose. The presence of a plasmid in the strains that did not grow on DF was determined. Mutants of Microbacterium sp. strain YK18 (strains YK18-MD1 and YK18-MD2) which had spontaneously lost the ability to degrade DF were isolated from an overnight culture of YK18 on LB medium with glucose by replicating single colonies on MM3Y-DF and MM3Y-glucose. Spontaneous DF degradation mutants of Rhodococcus sp. strain YK2 (strains YK2-RD1 and YK2-RD2) were isolated as described previously (14).

DNA and RNA manipulation and nucleotide sequence analysis.
Plasmid DNA was isolated from Terrabacter, Rhodococcus, and Microbacterium strains by using a modified version of the method described by Sambrook et al. (27). To assist cell lysis, cell pellets were treated with lysozyme (4 mg ml-1 for Terrabacter and Microbacterium spp. and 20 mg ml-1 for Rhodococcus sp.) for up to 3 h at 37°C. For purification of plasmid pYK3, cesium chloride-ethidium bromide gradient ultracentrifugation was carried out with a Beckman ultracentrifuge (Optima TL) equipped with a TLN-100 rotor at 100,000 rpm for 4 h. Ethidium bromide was subsequently removed by butanol extraction, and the plasmid DNA was recovered by ethanol precipitation. Total DNA from actinomycetes was prepared by the method of Marmur (20), modified by adding the lysozyme treatment procedure used in the plasmid isolation protocol. Total RNA was isolated from DF-utilizing actinomycetes by the modified method of Sherman et al. (29) without cycloheximide treatment. Southern blotting and Northern blotting were performed by the methods described previously (14). Plasmid DNA from E. coli was prepared by a standard method (27) and was sequenced by using an ABI PRISM BigDye terminator cycle sequencing Ready Reaction kit (Applied Biosystems). Sequencing reactions were analyzed by using an automatic sequence analyzer (model 377ABI sequencer). GENETYX software (Software Development) was used for general analysis of the nucleotide and amino acid sequences. Alignment of sequences and construction of phylogenetic trees were performed as described previously (14).

PCR amplification.
PCRs were performed by using ExTaq DNA polymerase (Takara) according to the manufacturer's instructions. The following degenerate PCR primers were used for amplification of the alpha subunit of the multicomponent dioxygenase genes: DO{alpha}-2 (5'-TGYHSNTAYCAYGGNTGG-3'; amino acid sequence C[P/S]YHGW) and DO{alpha}-3 (5'-TCNRCNGCNARYTTCCARTT-3'; amino acid sequence NWK[L/F]A[V/A][E/D]) (in the oligonucleotide sequences N = A, T, G, or C; H = A, C, or T; Y = C or T; S = G or C; R = A or G). The reaction conditions were as follows: after 8 min of incubation at 94°C, 35 cycles consisting of 94°C for 20 s, 50°C for 30 s, and 72°C for 40 s, followed by incubation at 72°C for 5 min. The PCR primers used for amplification of the dfdA1 gene were dfdA1-5' (5'-GCAACAATGCTGACTGTGA-3') and dfdA1-3' (5'-AAGAACGCTCATCAGGATTCT-3'). The reaction conditions were as follows: after 8 min of incubation at 94°C, 25 cycles consisting of 94°C for 20 s, 56°C for 20 s, and 72°C for 60 s, followed by incubation at 72°C for 5 min. PCR products were purified by agarose gel electrophoresis followed by gel extraction with a QIAEX II DNA extraction kit (QIAGEN). Purified PCR products were cloned directly into the pGEM-T vector (Promega) by following the manufacturer's guidelines.

Construction of shuttle vector and transformation of Rhodococcus strain.
An E. coli-Rhodococcus shuttle vector was constructed by modifying pK4 (12). The blunted SacI-SmaI fragment of pK4, containing the regions for plasmid replication in Rhodococcus species, was cloned into the blunted AflIII site of pHSG298, yielding pRK401. Rhodococcus sp. strain YK2-RD2 was made competent by the method of Desomer et al. (9). The electroporation conditions for introduction of DNA with a Gene Pulser (Bio-Rad) and a 2-mm gapped cuvette were as follows: capacitance, 25 µF; parallel register, 400 {Omega}; and initial voltage, 2.0 kV. LB medium supplemented with glucose (10 mM) and kanamycin (25 µg ml-1) was used for selection of transformants.

HPLC and GC-MS analysis of metabolic intermediates.
Resting cells and high-performance liquid chromatography (HPLC) samples were prepared as described previously (14). The metabolic intermediates of DF and DD were analyzed by gas chromatography-mass spectrometry (GC-MS) by using an instrument (Shimadzu GCMS-QP5050A) equipped with a capillary column (Hewlett-Packard HP-5MS; 30 m by 0.25 mm [inside diameter]; film thickness, 0.25 µm). Analytical samples for GC-MS were prepared by extracting resting culture supernatants with 2 volumes of ethyl acetate, followed by acidification with 6 N HCl to approximately pH 2.0; then the samples were dehydrated with sodium sulfate, evaporated, and redissolved in 10% of resting culture volume of ethyl acetate. One microliter of each extract was analyzed by GC-MS. The GC conditions were as follows: helium flow rate, 1.1 ml/min; splitless mode; initial temperature, 100°C for 5 min; temperature increased to 280°C at a rate of 10°C min-1 and kept at 280°C for 10 min; and then temperature increased to 300°C at a rate of 20°C min-1 and kept at 300°C for 2 min. The head pressure of the helium carrier gas was 80 kPa.

Nucleotide sequence accession numbers.
The sequence data reported in this paper have been deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession numbers AB075233 to AB075242.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PCR amplification of the large-subunit gene fragment of the initial ring-hydroxylating dioxygenase.
A degenerate set of PCR primers was used to amplify dioxygenase alpha subunit genes from total DNA isolated from four DF-utilizing actinomycetes: Terrabacter sp. strains YK1 and YK3, Rhodococcus sp. strain YK2, and Microbacterium sp. strain YK18. PCR products from each strain were cloned, and randomly selected clones (13 clones from YK1, 26 clones from YK2, 11 clones from YK3, and 4 clones from YK18) were then sequenced. As expected, some of the clones derived from a given strain were identical. In total, we identified two different sequences from strains YK1 and YK18, four different sequences from strain YK2, and one sequence from strain YK3. Some of the sequences amplified from different strains were very closely related to each other, allowing the sequences to be classified into four separate groups. Interestingly, one of the PCR products was almost identical (99.5% at the nucleotide level) to sequences amplified from all four strains (accession numbers of representative sequences from strains YK1, YK2, YK3, and YK18 are AB075233, AB075238, AB075239, and AB075241, respectively). The translation product of this sequence displayed 53% identity to the large subunit of 3-phenylpropionate dioxygenase from E. coli (accession number Z37966). Another group of sequences (accession numbers of representative sequences from strains YK1, YK2, and YK18 are AB075234, AB075237, and AB075240, respectively) were identical to dbfA1, which was isolated from Terrabacter sp. strain DBF63 as an angular dioxygenase component gene for DF dioxygenation (16). Two other dioxygenase genes were identified from strain YK2. The deduced amino acid sequence encoded by one of these genes (accession number of representative sequence is AB075235) displayed 53% identity to IpbA1 (an isopropylbenzene 2,3-dioxygenase) from Rhodococcus erythropolis strain BD2 (accession number U24277), and the deduced amino acid sequence encoded by the other gene (accession number of representative sequence is AB075236) showed 70% identity to a biphenyl dioxygenase large-subunit protein (BphA1) from R. erythropolis TA421 (accession number D88021). Recently, we demonstrated that the extradiol dioxygenase gene for DF degradation (dfdB) was highly conserved among DF-utilizing actinomycetes (14). These results suggest that the initial dioxygenase genes for DF dioxygenation are also highly conserved in DF-utilizing actinomycetes. From the PCR-cloned library of dioxygenase alpha subunit genes, we selected the gene amplified from Terrabacter sp. strain YK3 for further study as this gene was highly conserved in three genera of actinomycetes.

Identification of a circular plasmid carrying the dioxygenase alpha subunit gene from Terrabacter sp. strain YK3.
We attempted to isolate a DF degradation mutant of the YK3 strain. A total of 30 of 300 colonies had spontaneously lost the ability to grow on DF following heat shock treatment at 37°C. These mutants were designated the YK3-TD strains. The mutants were still able to utilize salicylate, which is the late metabolic intermediate of DF degradation in strain YK3. These results suggest that the initial degradation of DF in YK3 is dependent on an unstable DNA element, such as a plasmid. We attempted to detect circular plasmid DNA in strain YK3 using an alkaline denaturation method. Following CsCl-ethidium bromide ultracentrifugation a layer corresponding to covalently closed circular DNA was observed. We estimated the size of the plasmid (designated pYK3) to be around 100 kb based on fragments generated by ClaI digestion (data not shown). Plasmid pYK3 was undetectable in the four YK3-TD strains examined (YK3-TD1 to YK3-TD4), and PCR analysis in which total DNA was used as the template failed to give a product (data not shown). However, a PCR product of the expected size was obtained when purified plasmid pYK3 was used as the template. The nucleotide sequences of 10 randomly selected clones containing the 0.3-kb fragment amplified from pYK3 were almost identical to the sequences amplified from total DNA of YK3. These results strongly suggest that the gene for initial dioxygenation of DF is carried on plasmid pYK3. We designated the dioxygenase gene dfdA.

Cloning and nucleotide sequence analysis of dfdA from Terrabacter sp. strain YK3.
The PCR product amplified from strain YK3 was used as a hybridization probe to clone a 5.2-kb PstI fragment of pYK3. Nucleotide sequence analysis of this fragment (accession number AB075242) revealed four complete open reading frames (ORFs) and one 5'-truncated ORF. Figure 1A shows the genetic arrangement of the dfdA gene cluster. The results of a phylogenetic analysis of the deduced amino acid sequences are shown in Fig. 2.



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  		FIG. 1. Genetic structures and relevant restriction sites of the dfdA gene cluster (A) and DF conversion activities of the YK2-RD2 dfdA gene transformant and deletion derivatives (B). Deduced ORFs found in the nucleotide sequence of the 5,211-bp PstI fragment are indicated by arrows. A truncated ORF located upstream of dfdA1 is indicated by dotted lines. A PstI fragment (dfdA1 to dfdA4), a PstI-SphI fragment (dfdA1 to dfdA3), a SmaI fragment (dfdA1 to dfdA3), and a PstI-ApaLI fragment (dfdA1 and dfdA2) were subcloned into the shuttle vector pRK401 and introduced into Rhodococcus sp. strain YK2-RD2. The DF conversion activity of the transformants is indicated for each clone. Abbreviations for restriction endonuclease sites: A, ApaLI; N, NspV; P, PstI; Sm, SmaI; Sp, SphI.

 


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  		FIG. 2. Phylogenetic analysis of amino acid sequences of DfdA gene products. Neighbor-joining trees inferred from amino acid sequences of the alpha (A) and beta (B) subunits of the terminal dioxygenase, ferredoxin (C), and ferredoxin reductase (D) are shown. The gene and strain designations for the sequences used for phylogenetic analysis are indicated, and the sequences isolated in this study are shaded. Bootstrap values greater than 50 based on 100 resamplings are shown at the nodes. Scale bar = 0.1 amino acid substitution per position. The following bacteria were used: for BphA, BphE, BphF, and BphG, Burkholderia sp. strain LB400 (accession number M86348); for BphA1-1, BphA2-1, BphA3-1, and BphA4-1, R. erythropolis TA421 (accession number D88020); for BphA1-2, BphA2-2, BphA3-2, and BphA4-2, R. erythropolis TA421 (accession number D88021); for BphA1 and BphA2, Rhodococcus sp. strain RHA1 (accession number D32142); for BphA1b and BphA2b, Sphingomonas aromaticivorans F199 (accession number AF079317); for CarAa, CarAb, CarAc, and CarAd, Sphingomonas sp. strain CB3 (accession number AF060489); for CumA1 and CumA2, Pseudomonas fluorescens IP01 (accession number D37828); for DbfA1 and DbfA2, Terrabacter sp. strain DBF63 (accession number AB054975); for DfdA1, DfdA2, DfdA3, and DfdA4, Terrabacter sp. strain YK3 (accession number AB075242); for DxnA1 and DxnA2, Sphingomonas sp. strain RW1 (accession number X72850); for HcaE, HcaA2, HcaA3, and HcaD, E. coli K-12 (accession numbers Z37966 and Y11070); for IpbA3 and IpbA4, R. erythropolis BD2 (accession number U24277); for NahAc and NahAb, Pseudomonas putida G7 (accession number M83949); for NidA and NidB, Rhodococcus sp. strain I24 (accession number AF121905); for ORF8, Terrabacter sp. strain DBF63 (accession number AB054975); for ORFG5, ORFG6, and ORFG3, Sphingomonas sp. strain RW1 (accession number AJ223219); for PhdA, PhdB, and PhdD, Nocardioides sp. strain KP7 (accession number AB031319); for PhnAc, Alcaligenes faecalis AFK2 (accession number AB024945); for RedA2, Sphingomonas sp. strain RW1 (accession number AJ002606); for SC7A8.08c, Streptomyces coelicolor A3(2) (accession number AL137187); and for ThcD, R. erythropolis NI86/21 (accession number U17130).

 
A portion of ORF1, designated dfdA1, was identical to the PCR-amplified product generated with the degenerate primer set. The dfdA1 gene encodes a 53.1-kDa protein consisting of 474 amino acids. The protein sequence displays significant similarity to the sequences of a number of alpha subunits of aromatic hydrocarbon dioxygenases (Fig. 2A). A consensus motif of the Rieske type 2Fe-2S cluster (21) was identified within the translated sequence of dfdA1. ORF2 (designated dfdA2) encodes a 21.1-kDa protein consisting of 179 amino acids, which shows similarity to the small subunit of several aromatic hydroxylating dioxygenases (Fig. 2B). ORF3 (designated dfdA3) encodes a 13.2-kDa protein consisting of 123 amino acids. A Rieske type 2Fe-2S cluster consensus motif was found in the deduced amino acid sequence (21). Comparison with the protein database indicated that the translation product is a type 2Fe-2S ferredoxin (Fig. 2C). ORF4 (designated dfdA4) encodes a 43.2-kDa protein consisting of 407 amino acids and contains a consensus motif for a flavin adenine dinucleotide-binding (ADP-binding) site (GXGX2GX3AX6G) (21). The deduced amino acid sequence showed similarity to the sequences of several ferredoxin reductases (Fig. 2D). A truncated ORF upstream of the dfdA1 gene encodes a protein similar to the N-terminal portion of an integrase/recombinase protein found in Mesorhizobium loti (accession number AP003007).

Distribution of genes homologous to dfdA1 among DF-utilizing actinomycetes.
The PCR-amplified portion of the dfdA1 gene from Terrabacter sp. strain YK3 was used as a probe in Southern blot experiments to determine its distribution in DF-utilizing actinomycetes. PstI-digested total DNAs from diverse actinomycetes, including Terrabacter sp. strain YK1, Rhodococcus sp. strain YK2, Microbacterium sp. strain YK18, spontaneously lost DF mineralization mutants of Rhodococcus sp. strain YK2 (YK2-RD1 and YK2-RD2), Terrabacter sp. strain YK3 (YK3-TD1 and YK3-TD2), and Microbacterium sp. strain YK18 (YK18-MD1 and YK18-MD2), and plasmid pYK3 were used as targets (Fig. 3A). The total DNAs of several Terrabacter strains isolated in this study (YK6, YK7, YK14, YK17, and YK21) and strain DBF63 isolated by Monna et al. (22) were also used as targets (Fig. 3B). The hybridization signals were apparent in all of the DF-utilizing actinomycetes (Fig. 3A, lanes 1 to 4, and B). The sizes of the cross-reacting bands from pYK3 (Fig. 3A, lane 11) and total DNA of strain YK3 (Fig. 3A, lane 3) were identical. The dfdA1 gene was not detectable in the DF degradation-negative mutants, including members of the genera Rhodococcus, Terrabacter, and Microbacterium (Fig. 3A, lanes 5 to 10). We examined the nucleotide sequences of PCR-amplified genes homologous to dfdA1 from Terrabacter sp. strains YK1 and DBF63, Rhodococcus sp. strain YK2, and Microbacterium sp. strain YK18 using specific primers for the dfdA1 gene of YK3. The specific amplified DNA fragments (about 1.45 kb) observed in all PCR samples were cloned. Nucleotide sequence analysis of a 600-bp portion of the 5' region of the clones showed that they were almost identical. These sequences differed from the YK3 sequence as follows: one nucleotide in the YK1 sequence, two nucleotides in the YK18 sequence, and three nucleotides in the YK2 and DBF63 sequences. These results demonstrate that the dfdA1 gene is highly conserved in diverse DF-utilizing actinomycetes.



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  		FIG. 3. Southern hybridization analysis of several DF-utilizing actinomycetes and their mutant strains performed with the dfdA1 gene as a probe. (A) PstI-digested total DNA was loaded in each lane. Lane 1, Terrabacter sp. strain YK1; lane 2, Rhodococcus sp. strain YK2; lane 3, Terrabacter sp. strain YK3; lane 4, Microbacterium sp. strain YK18; lane 5, Rhodococcus sp. strain YK2-RD1; lane 6, Rhodococcus sp. strain YK2-RD2; lane 7, Terrabacter sp. strain YK3-TD1; lane 8, Terrabacter sp. strain YK3-TD2; lane 9, Microbacterium sp. strain YK18-MD1; lane 10, Microbacterium sp. strain YK18-MD2. Lane 11 contained plasmid pYK3 digested with PstI. (B) PstI-digested total DNA of DF-utilizing Terrabacter strains was loaded in each lane. Lane 1, YK1; lane 2, YK3; lane 3, YK6; lane 4, YK7; lane 5, YK14; lane 6, YK17; lane 7, YK21; lane 8, DBF63. The positions of size markers are indicated on the left.

 
Transcriptional expression of the dfdA1 gene in Terrabacter sp. strain YK3.
Transcriptional expression of the dfdA1 gene in Terrabacter sp. strainYK3 was studied by Northern hybridization analysis. Washed LB medium-grown cells were resuspended in MM3Y supplemented with either 10 mM glucose or 0.2% DF. Following growth at 30°C for either 2 or 6 h, total RNA was isolated. Transcripts were detected by using the dfdA1 gene as a hybridization probe to analyze total RNA isolated from DF-induced cells (Fig. 4, lanes 4 and 5). The signal intensity after 6 h of induction (lane 5) was stronger than that after 2 h of induction (lane 4). No hybridization signals were observed in the RNAs isolated from induction cultures grown with glucose (lanes 2 and 3) or from LB medium-cultured cells (lane 1). These results suggest that transcription of the dfdA gene cluster is induced by DF in Terrabacter sp. strain YK3. The strongest signals appeared between the 23S and 16S rRNA genes (lanes 4 and 5), suggesting that transcription termination of the dfdA gene cluster occurs downstream of dfdA3 (transcriptional mRNA length, about 2.5 kb) or dfdA2 (about 2.0 kb), although we could not find a plausible stem-loop structure in either of the intervening regions. This implies that the dfdA4 gene, encoding the ferredoxin reductase, is not part of the transcription unit.



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  		FIG. 4. Northern hybridization analysis of Terrabacter sp. strain YK3 performed with the coding region of the dfdA1 gene as a probe. Total RNA was extracted from either LB medium cultures (lane 1) or cultures induced with glucose (lanes 2 and 3) or DF (lanes 4 and 5) at 2 h (lanes 2 and 4) or 6 h (lanes 3 and 5). Two micrograms of total RNA was loaded in each lane. (A) Denatured agarose gel stained by ethidium bromide; (B) hybridization signals. The arrowheads in panel B indicate the positions of the small-subunit rRNA genes.

 
Heterologous expression of dfdA.
Initially, we attempted to express the dfdA gene in the heterologous host E. coli. An NspV-PstI fragment containing the complete dfdA1 to dfdA4 genes was cloned into the ClaI and PstI sites of pBluescript II KS+. The expression construct was introduced into E. coli, and following IPTG induction, the ability to transform DF was assayed. Analysis of a culture broth by HPLC failed to detect any hydroxylated products.

Therefore, we attempted to express the dfdA gene in a Rhodococcus strain. For this we chose a derivative of Rhodococcus sp. strain YK2-RD2 which had spontaneously lost the ability to degrade DF found in Rhodococcus sp. strain YK2. This mutant still harbored three dioxygenase alpha subunit genes, including dbfA1YK2, whose gene product displayed some angular dioxygenation activity towards DF when it was expressed in E. coli (data not shown). However, no hybridization signals were obtained for induction cultures of the mutant grown on LB medium, MM3Y-DF, and MM3Y-glucose by Northern hybridization analysis when the coding region of the dbfA1YK2 gene was used as a probe (data not shown). These results suggest that the YK2-RD2 strain is a suitable host for heterologous expression of the dfdA gene from YK3 when the aim is to study the enzyme's substrate specificity profile.

The 5.2-kb PstI fragment encoding the dfdA1 to dfdA4 genes was introduced into the E. coli-Rhodococcus shuttle vector pRK401, which was constructed in this study (see Materials and Methods). The construct was introduced into Rhodococcus sp. strain YK2-RD2 by electroporation. At the same time, we transformed the YK2-RD2 strain with the shuttle vector pRK401 to obtain a negative control. Resting culture supernatants were analyzed by HPLC. In the DF conversion assay, no specific peaks were obtained for the negative control culture. In contrast, a specific peak at a retention time of 4.4 min was observed for the dfdA transformant cultures (Table 1). The UV spectrum between 200 and 360 nm of the peak resembles that of 2,2',3-trihydroxybiphenyl, with spectrum maxima at 208, 245, and 283 nm (3). This was confirmed by analyzing ethyl acetate extracts of the resting cell cultures by GC-MS. The molecular ion peak (m/z 202) and the mass fragmentation pattern of the major peak detected in the dfdA transformant (15.4 min) closely resembled those of 2,2',3-trihydroxybiphenyl (10), confirming that DfdA-catalyzed angular dioxygenation of DF occurs.


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  		TABLE 1. Retention times of major metabolites of several polycyclic and heterocyclic aromatic hydrocarbons by a dfdA gene transformant as detected by HPLC analysis

 
The resting cells of the dfdA transformant also converted several aromatic compounds into compounds detectable by HPLC. The metabolite of DD displayed UV spectrum maxima at 201 and 275 nm and was identified as 2,2',3-trihydroxydiphenyl ether (3). This was confirmed by GC-MS analysis (33). We observed unidentified metabolites in the resting culture supernatant along with carbazole, naphthalene, phenanthrene, anthracene, and biphenyl, which were not present in negative control cultures analyzed by HPLC (Table 1). We also detected products in dfdA transformant cultures along with dibenzothiophene, xanthene, phenoxathiine, and fluorene, although minor peaks at the same retention times were also observed in the negative control cultures (Table 1) (at levels about 1.2 to 1.7% of the dfdA transformant levels as determined by mean peak areas of 220-nm chromatograms [data not shown]). Fluoren-9-one was effectively converted to 9-hydroxyfluorene (retention time, 6.4 min; identified by retention time and UV spectrum of the standard) by the negative control strain, and metabolites of fluorene from the dfdA transformant were identified as 9-hydroxyfluorene along with a trace amount of fluoren-9-one. These results suggest that the conversion of fluorene to 9-hydroxyfluorene by the dfdA transformant is catalyzed by the DfdA enzyme, as reported previously for other angular dioxygenases (16, 23, 31). The latter compound may then be converted to fluoren-9-one by an unidentified enzyme, which is constitutively expressed in strain YK2-RD2.

Three truncated subclones of the PstI fragment (Fig. 1B) were constructed in the vector pRK401. These subclones were introduced into Rhodococcus sp. strain YK2-RD2, and the abilities of the transformants to degrade DF were assayed in kanamycin-supplemented LB broth. The presence of 2,2',3-trihydroxybiphenyl in culture supernatants was determined by HPLC. Recombinant strains harboring either the PstI-SphI- or SmaI-truncated forms of the PstI fragment (Fig. 1B), in which the ferredoxin reductase gene was deleted, were able to mediate the conversion of DF to 2,2',3-trihydroxybiphenyl. This suggests that the ferredoxin reductase component, DfdA4, might be replaceable with other homologous activities that are present in the Rhodococcus strain. However, a recombinant strain harboring the PstI-ApaLI fragment (i.e., lacking both ferredoxin and ferredoxin reductase) was unable to perform the biotransformation, suggesting that the ferredoxin component, DfdA3, is essential for dioxygenation of DF.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
An angular dioxygenase gene cluster (dfdA1 to dfdA4) was cloned from circular plasmid pYK3 in DF-utilizing Terrabacter species. The phylogenetic positions of the terminal dioxygenase components (DfdA1 and DfdA2) were deeply branched within the portions of trees containing several dioxygenase sequences (Fig. 2A and B), suggesting that they are a novel type. In contrast, the electron transport enzymes, DfdA3 and DfdA4 (Fig. 2C and D), were clustered with counterparts in one of the biphenyl dioxygenase systems found in R. erythropolis TA421 with support by bootstrap analysis. The TA421 strain harbored at least two dioxygenase genes (1): a bphA1A2A3A4BCSTD gene cluster found on clone pTB1 (accession number D88020) and a bphA1A2A3BA4CD gene cluster found on clone pTT45 (accession number D88021). The amino acid sequences of DfdA3 and DfdA4 were similar to those of BphA3 and BphA4, respectively, encoded on pTT45. Interestingly, we found a partial gene fragment immediately downstream of the dfdA3 coding region, whose translation product displays homology to the N-terminal portion of BphB that is encoded in the clone pTT45 (21 amino acids plus 21 amino acids, divided by a frameshift mutation in the DNA segment). In addition, the stop codon of dfdA1 overlaps the start codon of dfdA2 by one nucleotide, whereas there are noncoding segments of DNA between dfdA2 and dfdA3 (82 bp) and between dfdA3 and dfdA4 (366 bp). Evidence both from the arrangement of the cluster and from the remnant of a bphB-like gene strongly suggests that the genes encoding the electron transfer partner of DfdA (dfdA3 and dfdA4) were derived from the pTT45 type of bph gene cluster found in R. erythropolis TA421 and that the terminal dioxygenase genes (dfdA1 and dfdA2) came from an ancestor different from the ancestor that provided the electron transporters. The bphB-like gene probably existed as an ancestor of the dfdA gene cluster and may have been selectively lost during evolution of the DF metabolic pathway since the cis-dihydrodiol dehydrogenase (BphB) activity is not needed in DF degradation.

We also propose that the dfdA gene products are important for DF degradation not only in Terrabacter sp. strain YK3 but also in diverse DF-utilizing actinomycetes. This study, together with our previous results (14), showed that dfdA1 and dfdB, and possibly all of the genes involved in the degradation of DF to salicylic acid (dfdA1A2A3A4BC), are conserved in diverse actinomycetes. Furthermore, the gene cluster for DF degradation appears to have been transferred laterally within actinomycetes relatively recently in evolutionary terms. We have preliminary results based on Southern blot hybridization analysis that indicates that the dfdBC genes also are localized on pYK3 (data not shown). These results suggest that lateral transfer for spreading the DF degradation genes in actinomycetes is mediated by a plasmid such as pYK3.

Together with the DfdA dioxygenases isolated in this study, the DbfA dioxygenases isolated from Terrabacter sp. strain DBF63 (16) and Rhodococcus sp. strain YK2 (14) are also present in some DF-utilizing actinomycetes. The terminal dioxygenase subunits and electron transport enzymes of the dfdA gene cluster were tandemly arranged in the same orientation. In contrast, the terminal dioxygenase component genes (dbfA1A2) of DBF63 and YK2 were not clustered adjacent to the genes encoding the electron transport proteins. We found a 4Fe-4S type of ferredoxin gene 2.45 kb downstream of the dbfA1A2YK2 cluster of Rhodococcus sp. strain YK2 (14), but we have not yet established whether its gene product acts as the cognate redox partner for DbfA1 and DbfA2. Additionally, transcription of dfdA1 was induced by DF in Terrabacter sp. strain YK3 and Rhodococcus sp. strain YK2 (data not shown), while apparent induction of the dbfA1YK2 gene could not be observed in Rhodococcus sp. strain YK2. The true function of the dbfA gene cluster in Rhodococcus sp. strain YK2 has yet to be established. However, the fact that dbfA is highly conserved in a number of actinomycetes is intriguing and perhaps indicates a common natural function which presumably gives the organism a selective advantage.

DfdA was found to exhibit a substrate specificity different from that of other angular dioxygenases. For example, the DfdA enzyme could convert various aromatic compounds, such as polycyclic aromatic hydrocarbons, including naphthalene, phenanthrene, and anthracene, which were not oxidized by a recombinant E. coli strain expressing the dbfA gene (16). In addition, the heterocyclic nitrogen compound carbazole, which was not oxidized by either DbfA (16) or DxnA (8), was degraded to several unidentified metabolites by the dfdA transformants. The broad substrate specificity displayed by DfdA for aromatic hydrocarbons is comparable to that of the CarA system involved in the carbazole degradation pathway of Pseudomonas sp. strain CA10 (23), which was classified differently than DfdA.

Using a set of degenerate primers, we were able to amplify four dioxygenase genes from DF-utilizing actinomycetes, and three of these genes were novel gene types. These results suggest that the primer set designed in this study should be useful in screening for novel dioxygenase genes. This study also demonstrated that Rhodococcus sp. strain YK2 harbors at least four kinds of dioxygenase alpha subunit genes. The existence of multiple dioxygenase genes in a single bacterium has been reported previously. For example, between four and six dioxygenase alpha subunit genes were found in various species of Sphingomonas (3, 17, 25, 26, 34). Recently, it was reported that the gram-positive biphenyl-utilizing organism Rhodococcus sp. strain RHA1 encodes five dioxygenase alpha subunit genes (19). The multiple aromatic ring hydroxylases found in Sphingomonas and Rhodococcus species may, in part at least, explain how these organisms are able to efficiently degrade a wide range of aromatic hydrocarbons.


   ACKNOWLEDGMENTS
 
We are grateful to H. Nojiri for kindly providing the total DNA of Terrabacter sp. strain DBF63 and to W. Mizunashi for providing the E. coli-Rhodococcus shuttle vector pK4.

This work was supported in part by grants for the Ecomolecular Science Research Program and the Bioarchitect Research Program from RIKEN.


   FOOTNOTES
 
* Corresponding author. Mailing address: Microbiology Laboratory, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan. Phone: 81-(48)-462-1111, ext. 5725. Fax: 81-(48)-462-4672. E-mail: tiida{at}postman.riken.go.jp. Back


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Applied and Environmental Microbiology, August 2002, p. 3716-3723, Vol. 68, No. 8
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.8.3716-3723.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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