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Appl Environ Microbiol. 2006 May; 72(5): 3198–3205. doi: 10.1128/AEM.72.5.3198-3205.2006. | PMCID: PMC1472349 |
Copyright © 2006, American Society for Microbiology Plasmid pCAR3 Contains Multiple Gene Sets Involved in the Conversion of Carbazole to Anthranilate † Masaaki Urata,1 Hiromasa Uchimura,1 Haruko Noguchi,1,2 Tomoya Sakaguchi,3 Tetsuo Takemura,3 Kaori Eto,1 Hiroshi Habe,1 Toshio Omori,1‡ Hisakazu Yamane,1 and Hideaki Nojiri1,2* Biotechnology Research Center,1 Professional Programme for Agricultural Bioinformatics, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657,2 Department of Chemistry, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan3 Received October 31, 2005; Accepted February 8, 2006. The carbazole degradative car-I gene cluster (carAaIBaIBbICIAcI) of Sphingomonas sp. strain KA1 is located on the 254-kb circular plasmid pCAR3. Carbazole conversion to anthranilate is catalyzed by carbazole 1,9a-dioxygenase (CARDO; CarAaIAcI), meta-cleavage enzyme (CarBaIBbI), and hydrolase (CarCI). CARDO is a three-component dioxygenase, and CarAaI and CarAcI are its terminal oxygenase and ferredoxin components. The car-I gene cluster lacks the gene encoding the ferredoxin reductase component of CARDO. In the present study, based on the draft sequence of pCAR3, we found multiple carbazole degradation genes dispersed in four loci on pCAR3, including a second copy of the car gene cluster (carAaIIBaIIBbIICIIAcII) and the ferredoxin/reductase genes fdxI-fdrI and fdrII. Biotransformation experiments showed that FdrI (or FdrII) could drive the electron transfer chain from NAD(P)H to CarAaI (or CarAaII) with the aid of ferredoxin (CarAcI, CarAcII, or FdxI). Because this electron transfer chain showed phylogenetic relatedness to that consisting of putidaredoxin and putidaredoxin reductase of the P450cam monooxygenase system of Pseudomonas putida, CARDO systems of KA1 can be classified in the class IIA Rieske non-heme iron oxygenase system. Reverse transcription-PCR (RT-PCR) and quantitative RT-PCR analyses revealed that two car gene clusters constituted operons, and their expression was induced when KA1 was exposed to carbazole, although the fdxI-fdrI and fdrII genes were expressed constitutively. Both terminal oxygenases of KA1 showed roughly the same substrate specificity as that from the well-characterized carbazole degrader Pseudomonas resinovorans CA10, although slight differences were observed. Carbazole is an N-heterocyclic aromatic compound derived from coal tar and shale oil ( 26) and is known to possess mutagenic and toxic activities ( 2, 16). To remediate carbazole-contaminated environments using biotechnological approaches, a wide variety of carbazole-degrading bacteria have been isolated and characterized ( 13, 14, 18, 28). Among them, the carbazole-catabolic car genes of Pseudomonas resinovorans CA10 ( carCA10 genes) have been studied most extensively. Carbazole is first dioxygenated at the angular (C-9a) and adjacent (C-1) positions to yield an unstable cis-hydrodiol (Fig. ) ( 28). Such initial dioxygenation is called an angular dioxygenation. Unstable cis-hydrodiol is spontaneously converted to 2′-aminobiphenyl-2,3-diol, which is further converted to anthranilate via meta-cleavage and hydrolysis (Fig. ). Genes encoding carbazole 1,9a-dioxygenase (CARDO) were first cloned from CA10, and CARDO CA10 was found to be a three-component system, in which NAD(P)H-dependent ferredoxin reductase (CARDO-R CA10; CarAd CA10 monomer) and ferredoxin (CARDO-F CA10; CarAc CA10 monomer) transfer electrons from NAD(P)H to oxygenase (CARDO-O CA10; CarAa CA10 trimer) (Fig. ) ( 25, 36). This multicomponent enzyme is a member of the Rieske non-heme iron oxygenase system (ROS). ROS members are classified into three classes and several subclasses based on the features of the electron transport chain ( 7). CARDO-R CA10 contains both a flavin adenine dinucleotide (FAD) and a plant-type [2Fe-2S] cluster, and CARDO-F CA10 is a ferredoxin having a Rieske-type [2Fe-2S] cluster (Rieske ferredoxin) ( 25). Thus, the CARDO CA10 is classified in class III. Sphingomonas sp. strain KA1 was isolated as a versatile carbazole-degrading bacterium ( 9) whose degradation pathway of carbazole is similar to that by CA10 ( 14). Previous study of the carbazole degradation ( carKA1) gene cluster of KA1 ( 14) revealed that, unlike the carCA10 gene cluster, the carKA1 gene cluster does not contain the CARDO-R gene but does contain the genes for CARDO-O ( carAa) and CARDO-F ( carAc), meta-cleavage enzyme ( carBaBb), and meta-cleavage compound hydrolase ( carC). Interestingly, although CarAa KA1 shows high homology with CarAa CA10 (60% identity), CarAc KA1 had no relatedness with the Rieske ferredoxin, including CarAc CA10, and showed similarity to the putidaredoxin-type ferredoxins. Because CARDO-O KA1 can receive electrons from CARDO-F KA1 and catalyze angular dioxygenation of carbazole ( 14), CARDO KA1 consisting of CarAa KA1 and CarAc KA1 is likely to be assigned to class IIA. To confirm this consideration, we should definitely clarify all CARDO components, especially ferredoxin reductase CARDO-R, that are involved in carbazole metabolism by KA1. In the genus Sphingomonas, it has been reported that catabolic genes are sometimes dispersed on the genome ( 4, 23). Considering that the carKA1 gene cluster is located on the 254-kb circular plasmid pCAR3 ( 9), it is possible that the ferredoxin reductase gene is also located on this plasmid. In the present study, using the draft sequence covering >99.9% of pCAR3, we found the multiple gene sets involved in carbazole degradation. In addition, based on the reverse transcription-PCR (RT-PCR) analysis of each CARDO KA1 component gene and reconstitution assays of CARDO KA1 components in Escherichia coli cells, we discuss the multiplicity of the carbazole degradation function of pCAR3. Bacterial strains, plasmids, and media. The bacterial strains and plasmids used in this study are listed in Table 1. LB or 2×YT medium ( 35) was used for bacterial growth. To prepare the KA1 RNA, nitrogen-containing mineral medium NMM1 supplemented with carbazole or succinate was used. NMM1 has the same composition as CNFMM ( 29) except for the addition of 3.0 g of NH 4NO 3 per liter. Carbazole was added to NMM1 as described previously ( 29). E. coli JM109 ( 35) and DH5α ( 35) were used as hosts for pUC119 and its derivatives. Ampicillin (Ap) was added to selective media at a final concentration of 50 μg/ml. For plate cultures, the media mentioned above, solidified with 1.6% (wt/vol) agar, were used. | TABLE 1.Bacterial strains and plasmids used in this study |
DNA manipulations. Total DNA of KA1 was prepared as described previously ( 29). Plasmids were prepared from E. coli by the alkaline lysis method ( 35) or with a Quantum Prep plasmid miniprep kit (Bio-Rad Laboratories, Hercules, CA). DNA fragments were extracted from agarose gels with an EZNA gel extraction kit (Omega Bio-tek, Inc., Doraville, GA). Other DNA manipulations were performed according to standard protocols ( 35). Sequencing of pCAR3 and annotation. Shotgun sequencing of pCAR3 was performed by Dragon Genomics Co. Ltd. (Shiga, Japan). Open reading frames (ORFs) were found by DNASIS-Mac, version 3.7 (Hitachi Software Engineering Co. Ltd., Yokohama, Japan). Homologous sequences were searched from the DDBJ/EMBL/GenBank DNA databases using the BLAST program (version 2.2.10) ( 1). The deduced amino acid sequences of ORFs were aligned using CLUSTAL W (version 1.83) ( 15). RNA preparation and RT-PCR. After the precultivation of KA1 in 5 ml of NMM1 supplemented with 10 mM succinate at 30°C, cells were gathered by centrifugation at 5,000 × g and then washed twice using CFMM ( 17). The washed cells were suspended in 500 μl of CFMM. Fifty microliters of the resultant cell suspension was added to 5 ml of NMM1 supplemented with 10 mM succinate, 10 mM each succinate and carbazole, or 10 mM carbazole (all media contained 1% [vol/vol] dimethyl sulfoxide [DMSO]). After a 2-h incubation with reciprocal shaking (300 strokes/min) at 30°C, the cells were harvested and used for extraction of total RNA by a NucleoSpin RNA II (Macherey-Nagel & Co., Düren, Germany) combined with RQ1 RNase-free DNase (Promega, Madison, WI). For RT-PCR, a One Step RNA PCR kit (AMV) (Takara Bio Inc.) was used. In RT-PCR, 100 ng of total RNA was used as a template. Detailed information on the RT-PCR primer sets and the conditions employed for respective gene amplifications are provided in Table S1 in the supplemental material. Control experiments without the addition of reverse transcriptase were also performed. qRT-PCR. The primer sets used in quantitative RT-PCR (qRT-PCR) and RT conditions are provided in Table S1 in the supplemental material. To synthesize each cDNA, ThermoScript reverse transcriptase (Invitrogen Corp., Carlsbad, CA) and 10 ng of total RNA (as a template) were used. After the RT reaction, quantitative real-time PCR with SYBR GREEN PCR Master Mix (Applied Biosystems, Foster City, CA) was performed using the synthesized cDNA as a template on the ABI7700 sequence detection system (Applied Biosystems). The copy number of each mRNA was determined by a standard curve using a series of known concentrations of the target sequence according to the method of Habe et al. ( 10). For normalization, 16S rRNA of KA1 was used as an internal standard. For each sample, the mean value from triplicate real-time PCRs was used to calculate the transcript abundance. The mRNA levels of each gene in the control sample (NMM1 supplemented with succinate) were set at 1.0. Construction of expression plasmids for car genes. Each of the carAaI, carAaII, carAcI, carAcII, fdxI, fdrI, and fdrII genes was separately amplified by PCR using the respective primer sets shown in Table S2 in the supplemental material, which were designed to introduce appropriate restriction sites and the effective Shine-Dalgarno sequence for the E. coli transcription system. In PCR amplification, total DNA of KA1 was used as a template. The amplified products were digested at the introduced restriction sites and were ligated into the corresponding sites of pUC119 to produce plasmids for the expression of single CARDOKA1 components. After their nucleotide sequences were confirmed to be identical to those designed, their insert fragments were used for the construction of plasmids to direct the expression of each of the CARDOKA1 components. For example, pUKA248 was constructed by cloning the 0.3-kb XbaI-KpnI fragment from pUKAcarAcI into the corresponding site of pUKAcarAaI. For the construction of pUKA249 and pUKA253, the 1.2-kb KpnI-EcoRI fragment from pUKAfdrII was ligated into the corresponding sites of pUKAcarAaI and pUKA248, respectively. Other plasmids were constructed similarly. Biotransformation of carbazole by E. coli cells expressing putative CARDO components. E. coli JM109 harboring appropriate plasmids was cultivated in 5 ml of LB supplemented with Ap at 37°C with reciprocal shaking (300 strokes min −1), and then 100 μl of the culture was transferred to 100 ml of the same medium. After cultivation at 30°C with shaking at 120 rpm until the optical density at 600 nm (OD 600) reached 0.4 to 0.5, isopropyl-1-thio-β- d-galactopyranoside (IPTG) was added to 0.5 mM and the cells further cultivated at 30°C for 15 h. Then the cells were harvested by centrifugation (5,000 × g, 10 min, 4°C), washed twice with CNF buffer (2.2 g Na 2HPO 4, 0.8 g KH 2PO 4 per liter), and resuspended in the same buffer to an OD 600 of 12 to 13. Carbazole was dissolved at 100 mM in DMSO and 50 μl added to 5-ml cell suspensions in the reaction tube. After incubation on a reciprocal shaker (300 strokes min −1) at 30°C for 20 h, the mixtures were extracted with an equal volume of ethyl acetate. The extracts were analyzed by gas chromatography-mass spectrometry (GC-MS) after derivatization with N-methyl- N-trimethylsilyltrifluoroacetamide (MSTFA) as described previously ( 27, 39). To define the conversion ratio, we used the following formula: conversion ratio (%) = 100 × (peak area for the TIC of the product)/(peak area for the TIC of the carbazole + peak area for the TIC of the product), where TIC is the total ion current. All experiments were conducted independently at least three times. Biotransformation analysis to determine substrate specificity. E. coli JM109 harboring pUKA253 or pUKA260 was cultivated as described above, and then 5 ml of the culture was transferred to 1 liter of the same medium. A resting cell suspension (OD600, 17 to 18) was prepared as described above except for the incubation temperature (25°C) and induction time after the addition of IPTG (12 h). Anthracene was dissolved in N,N-dimethylformamide at 10 mg/ml (wt/vol), and the other putative substrates shown in Table S3 in the supplemental material were dissolved in DMSO at the same concentration. Addition of substrate, subsequent incubation, and GC-MS analysis were performed as described above. Nucleotide sequence accession numbers. The nucleotide sequences of the car-I, car-II, fdxIfdrI, and fdrII loci were deposited in the DDBJ DNA database under accession numbers AB095953, AB220949, AB220950, and AB220951, respectively. pCAR3 loci encompassing the carbazole degradation genes. The draft sequence of pCAR3 having a single gap (estimated to be <80 bp) revealed the loci encompassing the genes that could be involved in the transformation of carbazole to anthranilate. In addition to the previously characterized car gene cluster ( 14), probable carbazole degradation genes were found in three other distinct loci of pCAR3 (Fig. and Table 2). | TABLE 2.ORFs found in four pCAR3 loci, which contain the genes probably involved in carbazole conversion to anthranilate |
The deduced amino acid sequences of six ORFs (ORFB1 to ORFB6) in locus B exhibited 51 to 75% identity with those of the previous car genes on pCAR3, and we designated this cluster the car-II gene cluster, carAaIIBaIIBbIICIIAcII-carRII. The previous car gene cluster was renamed the car-I gene cluster (Fig. , locus A). The amino acid sequence of CarAaII displayed 75 and 59% identity with those of CarAaI and CarAa CA10, respectively, and contained both the Rieske-type [2Fe-2S] cluster consensus and Fe(II)-binding motifs ( 31). The amino acid sequence of CarAcII showed similarity to that of several putidaredoxin-type ferredoxins and 55% identity with that of CarAcI. CarAcII contained four Cys residues (CX 5CX 2CX nC), typical of putidaredoxin-type [2Fe-2S] cluster ligands ( 3, 5, 38). ORFC2 (designated fdxI) at locus C (Fig. ) showed similarity to CarAcI and CarAcII. The deduced amino acid sequences of ORFC3 (designated fdrI), which may constitute an operon with the fdxI gene in locus C, and ORFD2 (designated fdrII) at locus D (Fig. ) displayed 59 and 76% identities with RedA2, the ferredoxin reductase component of the dioxin dioxygenase of Sphingomonas wittichii RW1 ( 6). FdrI and FdrII shared 38 to 41% identity with putidaredoxin reductase ( 20, 32) and rhodocoxin reductase ( 24), and also had a FAD-binding motif and two ADP-binding motifs (for FAD and NADH) ( 40). As well as CarBaI, CarBbI, and CarCI in the car-I gene cluster, CarBaII, CarBbII, and CarCII shared moderate homology with the CarBa, CarBb, and CarC from CA10 as summarized in Table 2. Their functions were predicted as follows: CarBaII and CarBbII, structural and catalytic subunits, respectively, of the meta-cleavage enzyme; CarCII, meta-cleavage compound hydrolase. Transcriptional analyses of the putative CARDO component genes. The primer set for the carAaIBaIBbICIAcI, carAaIIBaIIBbII, carBbIICII, carCIIAcII, fdxIfdrI, or fdrII genes could amplify DNA fragments with the expected sizes from the RNA of carbazole-grown KA1 cells (data not shown). Therefore, it was revealed that all seven genes (carAaI, carAaII, carAcI, carAcII, fdxI, fdrI, and fdrII) are expressed in carbazole-grown KA1 cells, suggesting that the gene products could be involved in CARDO system formation. Also, these results indicated that the carAaIBaIBbICIAcI and fdxIfdrI genes are transcribed as single transcriptional units. Furthermore, although we could not detect the RT-PCR product spanning the region from carAaII to carAcII (data not shown), the amplifications of three segmental fragments covering the entire car-II gene cluster (carAaIIBaIIBbII, carBbIICII, and carCIIAcII regions) were detected, and we concluded that the car-II gene cluster was also cotranscribed. To analyze the inducibilities of the putative CARDO component genes, the mRNA levels of each gene after 2 h in carbazole-exposed or nonexposed KA1 were investigated by qRT-PCR. The mRNAs of carAaI, carAcI, carAaII, and carAcII in carbazole-exposed cells were about 13-, 10-, 15-, and 11-fold more abundant than those in nonexposed cells. Together with the results of RT-PCR analyses, these findings clearly indicated that transcription of the car-I and car-II gene clusters was induced when KA1 cells were exposed to carbazole. In contrast, the expression levels of ferredoxin and ferredoxin reductase genes were not elevated in response to carbazole exposure (1.0- to 1.2-fold induction). Functional analyses of putative CARDO. Biotransformation experiments were performed with carbazole using E. coli cells expressing putative CARDO components in various combinations. Although we could not detect their expression by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (data not shown), considering that the same promoter and Shine-Dalgarno sequence were used, the expression level of each gene was assumed to be similar. The production ratios of 2′-aminobiphenyl-2,3-diol from carbazole detected for cells harboring pUKA254 ( carAaI, carAcI, and fdrI) and pUKA253 ( carAaI, carAcI, and fdrII) were much higher than those seen with cells harboring any other recombinant plasmids containing carAaI (Table 3). These results clearly indicated that CarAaI can catalyze angular dioxygenation of carbazole efficiently in conjunction with both CarAcI and FdrI (or FdrII). Similarly, as shown in Table 3, E. coli cells expressing CarAaII exhibited a much higher conversion ratio when both CarAcII and FdrI (or FdrII) were coexpressed, indicating that CarAaII could function as a terminal oxygenase of CARDO with the aid of both CarAcII and FdrI (or FdrII). In addition, the expression of FdxI with CarAaI/FdrI or CarAaII/FdrI also increased the conversion ratio (see pUKA263 versus pUKA250 and pUKA265 versus pUKA257 in Table 3), although it was markedly lower than the expression observed when CarAcI or CarAcII was supplied as ferredoxin. These results suggested that FdxI could also transfer electrons from FdrI to CarAaI/CarAaII, but the efficiency of electron transfer was markedly lower than those of CarAcI and CarAcII. | TABLE 3.Carbazole conversion by E. coli expressing a putative CARDO component(s) |
We further examined whether CarAaI and CarAaII could receive electrons from ferredoxins not encoded on the same car gene clusters. When CarAcII was expressed with CarAaI/FdrI (pUKA268) or CarAaI/FdrII (pUKA267), 77% ± 3.3% and 87% ± 5.2% conversion occurred, respectively. Both of these figures were only a little lower than those seen with CarAcI (pUKA254 and pUKA253) (Table 3). These results suggested that CarAaI could receive electrons from CarAcII and CarAcI with similar efficiencies. However, the conversion ratio of carbazole by E. coli expressing CarAaII/CarAcI/FdrI (or FdrII) was markedly lower than that detected when CarAcII was replaced with CarAcI (8.3% ± 2.0% and 6.2% ± 1.2% conversion were observed for E. coli harboring pUKA270 [CarAaII/CarAcI/FdrI] and pUKA269 [CarAaII/CarAcI/FdrII], respectively). Therefore, we concluded that CarAcI could transfer electrons to CarAaII but that the electron transferability to CarAaII is substantially lower than that to CarAcII. Based on these results, although all CARDO components found on pCAR3 can theoretically function in various combinations, we concluded that two CARDO systems, composed of CarAaI/CarAcI/(FdrI or FdrII) (designated CARDOKA1-1) and CarAaII/CarAcII/(FdrI or FdrII) (designated CARDOKA1-2) primarily function as initial oxygenases for carbazole in KA1. Substrate ranges of two CARDOs of KA1. The substrate ranges of CARDO KA1-1 and CARDO KA1-2 were determined by biotransformation analyses using recombinant E. coli. (Detailed data are provided in Table S3 in the supplemental material). Both CARDOs catalyzed angular dioxygenation ( 28) of carbazole, dibenzofuran, dibenzo- p-dioxin (DD), and phenoxathiin. The ratio of angular dioxygenation of 9-fluorenone was markedly poorer in comparison with carbazole, dibenzofuran, DD, or phenoxathiin. Neither CARDO KA1-1 nor CARDO KA1-2 could catalyze any oxygenation reactions for dibenzothiophene sulfone. With fluorene, both CARDOs catalyzed the monooxygenation of the methylene carbon and probable lateral dioxygenation ( 28) at unidentified positions. For biphenyl, naphthalene, and anthracene, both CARDOs catalyzed lateral dioxygenations as was the case with CARDO CA10 ( 28). In conclusion, CARDOKA1-1 and CARDOKA1-2 have a wide substrate range, which is similar to that of CARDOCA10. However, there are two noteworthy differences (see Table S3 in the supplemental material). (i) CARDOKA1-1 preferably oxygenates anthracene, probably at C-2 and C-3, whereas CARDOKA1-2 and CARDOCA10 preferably oxygenate at C-1 and C-2 to yield cis-1,2-dihydroxy-1,2-dihydroanthracene. (ii) It is likely that the angular dioxygenation activities of CARDOKA1-2 for DD or phenoxathiin are relatively lower than those in the other two CARDO systems. Two car gene clusters were present on plasmid pCAR3, and two CARDOs, composed of CarAaI/CarAcI/(FdrI or FdrII) (CARDO KA1-1) and CarAaII/CarAcII/(FdrI or FdrII) (CARDO KA1-2), function in KA1. Plural isofunctional degradation genes on a single bacterial genome have been reported. For example, multiple gene sets for biphenyl/polychlorinated biphenyl degradation are known to be dispersed on the chromosome and large linear plasmids in Rhodococcus sp. strain RHA1 ( 11, 19, 22, 42). Duplication of degradation genes has also been reported in several degradation plasmids, such as the 2,4-dichlorophenoxyacetic acid-degrading plasmids pJP4 ( 21) and pEST4011 ( 41) and the toluene/xylene-degrading plasmid pWW53 ( 30). It would be of great interest to determine the physiological roles of the two copies of the car gene cluster in carbazole metabolism by KA1. As described above, the substrate specificities of the two CARDOs are nearly identical, although slight differences were observed. These facts suggest that duplication does not broaden the degradation (or growth) substrate range of KA1 but raises its degradation rate of carbazole. Estimation of the in vivo concentration of each CARDO component and generation of knockouts of either car-I or car-II (or either fdrI or fdrII) followed by quantitative determination of the carbazole catabolic capacities will yield information to reveal the importance of multiple degradation genes. The draft sequence of pCAR3 showed that the fdxIfdrI cistron is located 50 and 85 kb downstream of the car-I and car-II gene clusters, respectively (data not shown). The fdrII gene is located about 80 and 115 kb downstream of the car-I and car-II gene clusters, respectively (data not shown). The dispersion of the ROS component genes in pCAR3 contrasts with the well-organized degradation operon in pseudomonads, where the four or three genes are assembled, or at least cotranscribed, as is observed with the carCA10 gene cluster (Fig. ) ( 36). Because similar dispersions of ROS component genes have been reported many times in sphingomonads ( 4, 23, 33, 34), it is possible that gene dispersion is one of the characteristics of sphingomonads. It is possible that constitutive expression of the fdrI and fdrII genes provides enough reductase to transfer electrons from NAD(P)H to ferredoxin. If this is the case, a well-organized operon is not necessary to achieve the appropriate carbazole degradation capacity. Another possibility is that the organization of car gene clusters of pCAR3 has not yet fully evolved and been optimized and that there is room for further evolution of the carbazole catabolic operon when KA1 is exposed to some selective pressure. On the other hand, the ferredoxin reductases, FdrI and FdrII, may be shared with other redox systems, possibly to maximize the catabolic potential while limiting its genetic burden. In this case, carbazole-dependent control of their expression would be disadvantageous for KA1. According to the classification of Batie et al. ( 7), both CARDOs of KA1 are classified in class IIA. Class IIA ROS is a three-component oxygenase, in which the electron transfer components comprise a simple flavoprotein and a putidaredoxin-type ferredoxin. Until now, almost all ROSs have been assigned to class IIB or III. As for class IIA ROSs, only a few examples have been reported, such as the pyrazon dioxygenase of an unidentified bacterium (37), the dioxin dioxygenase of S. wittichii RW1 ( 6), and the dicamba O-demethylase of Pseudomonas maltophilia DI-6 ( 8, 12). Whereas CARDO KA1-1/2 belongs to class IIA, according to the properties of ferredoxin and ferredoxin reductase, CARDO CA10 is a class III ROS. 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