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1 Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2 Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
3 Department of Industrial Chemistry, Faculty of Engineering, Shibaura Institute of Technology, 3-9-14 Shibaura, Minato-ku, Tokyo 108-8548, Japan
Correspondence
Hiroshi Habe
hiroshi.habe{at}aist.go.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence of the arh genes and their flanking regions reported in this paper is AB240454.
Present address: National Institute of Advanced Industrial Science and Technology (AIST), Central 5-2, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-4 subgroup of the Proteobacteria (Takeuchi et al., 2001
). Sphingomonads are ubiquitous in the environment, occurring in soil, water and sediments (White et al., 1996). They are attracting interest as a result of their high potential for bioremediation. In recent years, many Sphingomonas strains, which have been isolated from a variety of contaminated environments, have been described with respect to their ability to degrade xenobiotics and aromatic compounds, such as biphenyl (Fredrickson et al., 1995), dibenzo-p-dioxin (Wittich et al., 1992), carbazole (Habe et al., 2002), tetralin (Hernáez et al., 1999), pentachlorophenol (Saber & Crawford, 1985), 4-nonylphenol (Gabriel et al., 2005), 
-hexachlorocyclohexane (Imai et al., 1989) and herbicides (Adkins, 1999; Kohler, 1999). In addition, Sphingomonas strains are often isolated from contaminated soils because of their ability to degrade polycyclic aromatic hydrocarbons (PAHs), which are ubiquitous environmental pollutants with toxic, mutagenic and carcinogenic properties (Bastiaens et al., 2000; Khan et al., 1996; Mueller et al., 1990; Pinyakong et al., 2000).
Recent genetic analyses of the aromatic degradation pathways in PAH-degrading sphingomonads have increasingly revealed that most of the genes necessary for degrading an aromatic compound are scattered in several clusters and not organized in coordinately regulated operons (recently reviewed by Pinyakong et al., 2003a). This raises the question of how these complexly arranged genes are efficiently regulated and transcribed. The system regulating PAH-degradative genes in sphingomonads has not been investigated fully, probably owing to their complexity. However, it was recently reported that the LysR-type transcriptional regulators (LTTRs) ThnR, PcpR and LinR, which activate the expression of target degradative genes, were isolated from Sphingomonas strains that degraded tetralin, pentachlorophenol and -hexachlorocyclohexane, respectively (Martínez-Pérez et al., 2004; Cai & Xun, 2002; Miyauchi et al., 2002).
To investigate the diverse and complex aromatic-degradation systems of sphingomonads, including their regulatory mechanisms, it is necessary first to isolate the gene(s) responsible for each degradation step and then to examine the regulatory mechanisms related to their functions. For this purpose, we investigated acenaphthene degradation by Sphingomonas sp. strain A4 at the molecular level (Pinyakong et al., 2004). Strain A4 was isolated based on its ability to utilize acenaphthene as a sole source of carbon and energy (Komatsu et al., 1993). Acenaphthene, a PAH that possesses a single alicyclic five-membered ring sharing three carbon atoms with two aromatic rings, is an abundant constituent of coal tar and creosote (Wise et al., 1988). Commercial creosotes contain 0.95–6.1 % (w/w) acenaphthene (Kohler et al., 2000). Nevertheless, the biodegradation of the compound has been poorly studied, especially in terms of bacterial catabolic genes. To our knowledge, there are only a few acenaphthene-utilizing bacterial strains, almost all of which are Sphingomonas spp. isolated by our group (Komatsu et al., 1993) or by Shi et al. (2001), except for Alcaligenes spp. reported by Selifonov et al. (1993). Previously, we demonstrated that the degradation of acenaphthene by strain A4 proceeds via 1-acenaphthenol and 1-acenaphthenone (Komatsu et al., 1993). Recently, we isolated the terminal oxygenase genes, arhA1 and arhA2, required for the initial oxygenation of acenaphthene into 1-acenaphthenol, using the shotgun cloning method (Pinyakong et al., 2004). That was the first report to identify the genes responsible for PAH oxygenation in sphingomonads, along with a report on phnA genes published almost simultaneously by Demanèche et al. (2004). We also demonstrated that several PAHs other than acenaphthene, i.e. acenaphthylene, naphthalene, phenanthrene, anthracene and fluoranthene, could be dioxygenated to their corresponding cis-dihydrodiol products in resting Escherichia coli cells coexpressing arhA1A2 with the genes encoding ferredoxin (ahdA3) and ferredoxin reductase (ahdA4) derived from another sphingomonad (Pinyakong et al., 2004). However, the genes for the intrinsic electron-transport protein for ArhA1A2, the genes for the degradation pathway of their metabolic products, and their regulatory genes, have not been isolated.
In this study, we performed transposon mutagenesis on strain A4 to isolate the genes, other than arhA1A2, responsible for acenaphthene degradation. We also investigated the regulatory mechanism of a newly isolated gene cluster, which includes the arhA genes, as the first step for elucidating the entire acenaphthene degradation system in strain A4.
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Bacterial strains, plasmids and culture conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. Sphingomonas sp. strain A4 was cultivated at 30 °C in Luria–Bertani (LB) medium (Sambrook et al., 1989) or carbon-free mineral medium (CFMM) as described previously (Pinyakong et al., 2004) supplemented with 0.1 % (w/v) acenaphthene, 0.1 % (w/v) acenaphthylene, 0.1 % (w/v) 1,8-naphthalic anhydride (1,8-NA), or 0.1 % or 0.2 % (w/v) fructose. Acenaphthene, acenaphthylene and 1,8-NA solutions were prepared by dissolving the appropriate compounds in DMSO (acenaphthene and acenaphthylene, 100 mg ml–1; 1,8-NA, 25 mg ml–1). The E. coli strains were grown at 37 °C in LB medium. When necessary, ampicillin (Ap), chloramphenicol (Cm), kanamycin (Km) and tetracycline (Tc) were used at 100, 34, 50 and 10 µg ml–1, respectively. Gentamicin (Gm) was used at 10 µg ml–1 for E. coli strains and at 30 µg ml–1 for mutants of strain A4. IPTG was used at a final concentration of 100 µM.
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). For Southern hybridization, DNA fragments within arhR and ORF16 were amplified using PCR. The PCR products were labelled using a DIG DNA labelling kit (Roche Diagnostics) and used as probes. The primers used in this study are listed in Table 2.
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pir harbouring suicide plasmid pBSL202 (Alexeyev et al., 1995). Both strains were grown on LB medium until the stationary phase and then suspended in 10 mM MgSO4 after being washed twice with the same solution. Equal numbers of cells from these suspensions were mixed and incubated on a filter membrane (0.45 µm pore size) on an LB agar plate at 30 °C for 6 h. The cells were resuspended in 10 mM MgSO4 and then spread on CFMM plates supplemented with 0.2 % (w/v) fructose and Gm. After incubation at 30 °C for 5 days, 3 % (w/v) acenaphthene solution dissolved in acetone was sprayed on the cells that had grown on the selective medium, and the cells were incubated overnight at 30 °C. The mutants that failed to produce a clear zone in the acenaphthene layer were isolated. For the indigo formation assay, the mutants were pre-incubated on an LB plate containing Gm at 30 °C for 4 days, and then a small amount of indole was put on the inside of the lid of the plate. After further incubation at 30 °C for 24 h, we observed whether the colour of the cells had changed to indigo blue.
The DNA regions flanking the mini-Tn5 insertion sites in mutants of strain A4 were cloned by digesting total DNA from the mutants with EcoRI, ligating them to EcoRI-digested pUC19, and transforming E. coli DH5. The clone libraries were selected on LB agar plates containing both Ap and Gm. Positive clones were subjected to nucleotide sequencing using primer GMR (Table 2
), which is specific for the Gmr cassette in the mini-Tn5, and an ABI PRISM 310 genetic analyser (Applied Biosystems) according to the manufacturer's instructions.
Nucleotide sequence analysis and annotation.
Previously, we obtained cosmid pSC288 containing arhA1A2 and its flanking region in a 40 kb insert from a genomic library of strain A4 (Pinyakong, 2003). In this study, the 16.4 kb KpnI fragment of pSC288 containing the arhA1A2 genes was subcloned into pUC19Kmr to yield pUK17 (Table 1), and the nucleotide sequence of this fragment was determined by the Dragon Genomics Center of Takara Bio. To construct pUC19Kmr, the Kmr cassette from pTKm (Yoshida et al., 2003) was excised by EcoRV digestion, and was ligated to the ScaI site of pUC19. The nucleotide sequences were analysed using DNASIS-Mac software (version 3.7; Hitachi Software). We searched for homology using the BLAST programs available at the web site of the National Center for Biotechnology Information (
http://www.ncbi.nlm.nih.gov/blast/blast_references.shtml). The deduced amino acid sequences of the observed ORFs were aligned using ClustalW (Thompson et al., 1994), available at the NPSA web site (http://npsa-pbil.ibcp.fr/NPSA/).
Gene disruption and complementation.
To disrupt arhR and ORF15, we first constructed plasmids pKGR16 and pKG15, respectively (Table 1, Fig. 1). The Gmr cassette used for gene disruption was prepared as described previously (Pinyakong et al., 2004). To construct pKGR16, the Gmr cassette was ligated to the blunt-ended BamHI site of pBSCN2 (Table 1, Fig. 1), as the arhR gene and the Gmr cassette are transcribed in the same direction. The 2.0 kb HindIII–SalI fragment of the resultant plasmid containing arhR : : Gmr was ligated to HindIII/SalI-digested pK19mobsacB to yield pKGR16. To construct pKG15, the 1.0 kb SphI fragment of pU351E1 (Table 1, Fig. 1) containing part of ORF15 was ligated to SphI-digested pK19mobsacB, in which the SmaI site had been eliminated by SalI/EcoRI digestion, blunting and self-ligation. The resultant plasmid was digested with SmaI and ligated to the Gmr cassette, as ORF15 and the Gmr cassette are transcribed in the same direction, to yield pKG15. Plasmids pKGR16 and pKG15 were introduced into strain A4 by filter-mating, and then the double-crossover recombinants were screened using a method described previously (Pinyakong et al., 2004). The resultant arhR and ORF15 disruptants were designated strains A4DR and A4D15, respectively. The insertion of the Gmr cassette into arhR and ORF15 was confirmed by Southern hybridization. To complement strain A4DR with arhR in trans, the 2.0 kb SmaI–HindIII fragment of pBSCN2 (Table 1, Fig. 1) containing arhR was ligated into SmaI/HindIII-digested pBBad22T (Table 1), as the arhR gene was located downstream from the arabinose-inducible ParaBAD promoter. The resultant plasmid, pBBadR (Table 1, Fig. 1), was introduced into strain A4DR by filter-mating to generate strain A4DRC.
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) to yield pBBadA12, pBBadA13 and pBBadA14, respectively (Table 1, Fig. 1). To obtain pBBadA12, the 2.9 kb NcoI fragment of pU288E1 (Table 1, Fig. 1) containing arhA1A2 was ligated into NcoI-digested pBBad22T. To obtain pBBadA13, the 0.7 kb SmaI–NheI fragment of pBSCN2 (Table 1, Fig. 1) containing arhA3 was ligated into SmaI/XbaI-digested pBBadA12. To obtain pBBadA14, the 1.5 kb SalI fragment of pBSCN1 (Table 1, Fig. 1) containing arhA4 was ligated into SalI-digested pBBadA13. Plasmids pBBadA12, pBBadA13, pBBadA14 and pBBad22T were introduced into strain A4-PCM1 (Pinyakong et al., 2004), which is a spontaneous mutant defective in the ability to utilize acenaphthene. The strains harbouring the different plasmids were pre-incubated at 30 °C for 4 days on an LB agar plate containing Tc and 0.2 % arabinose. After a small amount of indole was added on the lid of the plate, the cells were incubated at 30 °C for 5 more days and then checked for the ability to produce indigo.
Construction of E. coli clones expressing the arhA genes and an assay for acenaphthene oxygenase activity.
For the purpose of expression of the arhA3 or arhA3A4 genes in E. coli, the 0.7 or 2.3 kb KpnI–HindIII fragment containing the respective genes from pBBadA13 or pBBadA14 was cloned into the corresponding site of pSTV28, resulting in pSArhA3 or pSArhA3A4 (Table 1). E. coli JM109 was then cotransformed with plasmids pSArhA3 or pSArhA3A4 and pUArhA1A2 (Pinyakong et al., 2004; Table 1) containing arhA1A2. The resultant E. coli clones were grown at 37 °C in 100 ml LB medium containing Ap and Cm to an OD600 of 0.7–0.8. IPTG was added at a final concentration of 100 µM followed by further cultivation at 30 °C for 4 h. The cells obtained were then washed twice with CFMM and resuspended in the same medium to yield a cell suspension of OD600 9.8. A 5 ml sample of each cell suspension was placed in the reaction tube and supplemented with 0.01 % (w/v) acenaphthene. After incubation at 30 °C for 2 or 16 h, 0.01 % (w/v) fluoranthene, which served as an internal standard, was added to the reaction tube and the reaction mixture was extracted with an equal volume of ethyl acetate. The extracts were dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. The metabolite was derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) at 70 °C for 20 min, and analysed by GC-MS as described previously (Pinyakong et al., 2004). Control experiments using E. coli JM109 carrying pUArhA1A2 and pSTV28 (Table 1), or pUC18 (Table 1) and pSTV28, were performed in parallel. To detect acenaphthene-cis-1,2-diol, the metabolite was derivatized with methaneboronic acid dissolved in dehydrated pyridine (2 mg ml–1) at 70 °C for 30 min.
RNA preparation.
Total RNA from strain A4 and its derivatives was extracted using a NucleoSpin RNA II (Macherey-Nagel), according to the manufacturer's instructions. The culture conditions used for total RNA extraction from the cells were as follows. For RT-PCR and primer extension, strain A4 was inoculated into CFMM supplemented with 0.1 % (w/v) acenaphthene, and the cells were harvested at approximately exponential phase. For quantitative RT-PCR, strains A4, A4DR and A4DRC were cultured on CFMM supplemented with 0.1 % (w/v) fructose to an OD600 of 0.5 and were then supplemented with 2 % (w/v) acenaphthene solution dissolved in DMSO at a final concentration of 0.02 % (w/v) or the same volume of DMSO. After further cultivation for 2 h, the cells were harvested. The RNA was treated with RQ1 RNase-free DNase (Promega) before being used in further experiments.
RT-PCR.
RT-PCR was performed with a One Step RNA PCR kit (AMV) (Takara). The DNA regions between arhA3 and ORF6 (Fig. 1) were amplified using the primer sets shown in Table 2. The reaction mixture (25 µl) contained 2.5 µl 10-fold One Step RNA PCR buffer, 5 mM MgCl2, 1 mM dNTP, 20 U RNase inhibitor, 2.5 U AMX reverse transcriptase XL, 2.5 U AMV-optimized Taq, 0.4 µM each primer, and 0.1 µg total RNA, prepared as described above. After the RT reaction at 50 °C for 30 min, PCR was performed using the following conditions: 94 °C for 2 min, and 30 cycles of 94 °C for 30 s, 65 °C for 30 s and 72 °C for 2.5 min. Negative control reactions were performed similarly, except the reverse transcriptase was omitted from the reaction mixture.
Quantitative RT-PCR.
First-strand cDNA was synthesized in four separate RT reactions using the reverse primers for arhA1, arhA3, arhR and the 16S rRNA gene (rrn) (arhA1qR, arhA3qR, arhRqR and 16SqR, respectively; Table 2). The RT reaction was performed using SuperScript III reverse transcriptase (Invitrogen). The RT reaction mixture (20 µl) contained 0.3 µg total RNA from each cell culture, 0.1 µM each primer (Table 2), 4 µl fivefold First Strand buffer, 1 µl 0.1 M DTT, 1 µl RNaseOUT recombinant RNase inhibitor and 200 U SuperScript III reverse transcriptase. The reaction mixture was heated to 65 °C for 5 min and then chilled on ice. The RT enzymes were added, and the reaction mixture was incubated at 50 °C for 1 h. The reaction was terminated by incubation at 70 °C for 15 min. The cDNA solution was diluted 10-fold in deionized water before PCR amplification.
Real-time PCR was carried out with the ABI PRISM 7700 Sequence Detection System (Applied Biosystems) according to the manufacturer's instructions. The PCR mixture (22 µl) contained 2 µl of the cDNA solution, 0.4 µM each primer set (Table 2) and 11 µl SYBR Green PCR Master Mix (Applied Biosystems). Real-time PCR was performed using the following conditions: 50 °C for 2 min, 94 °C for 10 min, and 40 cycles of 94 °C for 5 s, 65 °C for 5 s and 72 °C for 30 s. For the standard curves, the DNA fragments of arhA1, arhA3, arhR and rrn were amplified by PCR with total DNA from strain A4 and the primer sets for the respective genes (Table 2), and dilution series were subjected to real-time PCR analyses as templates. The specificity of the real-time PCR was verified from the dissociation curve analysis and agarose gel electrophoresis of the PCR products. Negative control reactions were performed similarly, except the reverse transcriptase was omitted from the RT reaction mixture. The mRNA levels of the target genes (arhA1, arhA3 and arhR) were normalized to that of the reference gene (rrn) to correct for sample-to-sample variation in the amount of total RNA.
Primer extension analysis.
The total RNA from strain A4 cells grown on acenaphthene was subjected to an RT reaction with SuperScript III reverse transcriptase (Invitrogen) and an IRD800-labelled primer (Aloka), arhA3-PE1 (Table 2), using the method described above. The primer extension products were purified using phenol/chloroform extraction and ethanol precipitation. The products were subjected to electrophoresis together with a sequence reaction using the same primer and a Li-Cor model 4200I-2 Auto-DNA sequencer running Base ImaglR data collection software 4.0 (LI-COR), according to the manufacturer's instructions.
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), whereas strain AG3-69 had also lost the ability to grown on 1,8-NA. As strain A4 possesses the ability to produce indigo from indole, which involves the ring-hydroxylating oxygenase ArhA1A2 (Pinyakong et al., 2004), we examined the ability of these four mutants to produce indigo in order to estimate their ring-hydroxylating oxygenase activities. Strains AG2-45, AG2-48 and AG3-15 had also lost the ability to produce indigo (data not shown). These observations suggested that the genes involved in the initial oxygenation of acenaphthene did not function in strains AG2-45, AG2-48 and AG3-15.
To determine the location of the mini-Tn5 insertion in these four mutants, the region flanking the mini-Tn5 was cloned and partially sequenced. In strain AG2-45, the mini-Tn5 was inserted into the DNA region homologous (40 % amino acid identity) to part of the gene encoding an LTTR, DntR, from Burkholderia sp. strain DNT (a 2,4-dinitrotoluene degrader) (Lessner et al., 2003). In strains AG2-48 and AG3-15, the mini-Tn5 was inserted into ORF1 and arhA1, respectively, both of which we have sequenced previously (Pinyakong et al., 2004). In strain AG3-69, the mini-Tn5 was inserted into the DNA region homologous (59 % amino acid identity) to part of the gene encoding a ferredoxin reductase, RedA2, from Sphingomonas wittichii strain RW1 (a dibenzo-p-dioxin degrader) (Armengaud & Timmis, 1998) (data not shown).
Nucleotide sequence analysis and annotation of the 16.4 kb DNA region
Previously, we partially sequenced the 40 kb insert in cosmid pSC288 (Pinyakong, 2003), which contains a 5.0 kb EcoRI fragment carrying arhA1A2 and its flanking region. Consistent with part of the preliminary sequence of pSC288, the DNA region flanking the mini-Tn5 in AG2-45 appeared to be located in the same locus as the 5.0 kb EcoRI fragment, within approximately 16 kb (Fig. 1). To determine the complete nucleotide sequence of this locus, the corresponding 16.4 kb KpnI fragment of pSC288 was subcloned and sequenced, revealing 16 ORFs and a partial ORF at this locus (Fig. 1, Table 3).
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) (Table 3). The double-crossover disruptant of arhR (strain A4DR, Table 1) showed deficient growth on acenaphthene, and this deficiency was complemented in strain A4DR transformed with plasmid pBBadR (Table 1, Fig. 1) expressing the arhR gene (strain A4DRC, Table 1). These observations revealed that ArhR is clearly involved in acenaphthene metabolism in strain A4.
The deduced amino acid sequences of ORF10 and ORF12 shared the highest homology (50 % and 46 % amino acid identity) with a ferredoxin and a ferredoxin reductase (ThnA3 and ThnA4), respectively, from Sphingopyxis macrogoltabida strain TFA (a tetralin degrader) (Moreno-Ruiz et al., 2003). We postulated that ORF10 and ORF12 encoded an intrinsic ferredoxin and ferredoxin reductase for ArhA1A2 and tentatively designated them arhA3 and arhA4, respectively.
The deduced amino acid sequence of ORF11 shared the highest identity with a putative 1-hydroxy-2-naphthoaldehyde dehydrogenase (PhnF) from Alcaligenes faecalis strain AFK2 (aphenanthrene degrader) (Kiyohara et al., 1982). However, the product of ORF11 was approximately 280 aa shorter than other aldehyde dehydrogenases, including PhnF, and lacked both the substrate- and NADH-binding domains (Gruez et al., 2004), which are considered necessary for its enzymic activity. Therefore, ORF11 seems to be a pseudogene generated by deletion of the corresponding DNA region.
The ORF13 product showed homology with the large subunit of the terminal oxygenase component of salicylate 1-hydroxylase (AhdA1d) from Sphingobium sp. P2 (Pinyakong et al., 2003b). However, ORF13 also seems to be a pseudogene because its products lacked part of the Rieske [2Fe–2S] cluster domain which is required for oxygenase activity (Ferraro et al., 2005).
The ORF15 product displayed high homology with known 2,3-dihydroxybiphenyl 1,2-dioxygenases. However, the double-crossover disruptant of ORF15 (strain A4D15) did not lose its ability to grow on acenaphthene, revealing that ORF15 is not essential for acenaphthene utilization.
The ORF6 product showed the highest homology with ferredoxin reductase (NahAa) from Pseudomonas putida NCIB 9816-4 (a naphthalene degrader) (Dennis & Zylstra, 2004), but this homology was extremely low (18.1 % amino acid identity). Especially, the C-terminal amino acid sequence of ORF6 showed no significant homology with that of NahAa, which contains the NAD-binding domain. In a BLAST analysis of the C-terminal region of ORF6, where the NAD-binding domain should be, we failed to detect any significant homologous proteins or peptides. It is still unclear whether the ORF6 product acts as a ferredoxin reductase. This ORF may have a function quite different from that of a ferredoxin reductase gene.
Assay of ArhA oxygenase activity
To examine whether the tentatively designated ArhA3 (ORF10) and ArhA4 (ORF12) really act as ferredoxin and ferredoxin reductase for ArhA1A2, respectively, we constructed three plasmids, pBBadA12, pBBadA13 and pBBadA14 (Table 1, Fig. 1), which coexpressed the arhA1A2, arhA1A2A3 and arhA1A2A3A4 genes, respectively, from the arabinose-inducible ParaBAD promoter. Plasmids pBBadA12 and pBBad22T (Table 1) were used as negative controls. These four plasmids were introduced into strain A4-PCM1, which is a spontaneous mutant defective in the ability to utilize acenaphthene (Pinyakong et al., 2004). In strain A4-PCM1, the loss of the DNA region from arhR to arhA1 (Fig. 1) was confirmed by Southern hybridization (data not shown). After pre-incubation on an LB agar plate containing Tc and 0.2 % arabinose, the ability of strain A4-PCM1 cells harbouring these four plasmids to produce indigo from indole was investigated by adding indole on the lid of the plate. After further incubation for 5 days, slight indigo formation was observed for strain A4-PCM1(pBBadA13), compared with the negative controls (data not shown). By contrast, significant indigo formation was observed only for strain A4-PCM1(pBBadA14) (data not shown). These observations showed that ArhA3 and ArhA4 function as the electron-transport system for ArhA1A2. To investigate acenaphthene oxygenase activity in the cells with or without ArhA3 and ArhA4 more quantitatively, we constructed E. coli strains harbouring plasmids pUArhA1A2 (Pinyakong et al., 2004) and pSArhA3 or pSArhA3A4 (Table 1). E. coli strains harbouring pUC18 and pSTV28 or pUArhA1A2 and pSTV28 were used in control experiments. After resting-cell reactions with these E. coli strains and acenaphthene as a substrate, the metabolites were derivatized with MSTFA and subjected to GC-MS analysis. Two major products, 1-acenaphthenol and acenaphthene-1,2-diol, were detected and their relative quantities were determined (Table 4). The amounts of 1-acenaphthenol and acenaphthene-1,2-diol after 16 h of incubation were increased 2.5-fold and 10-fold, respectively, in the cells expressing arhA1A2A3 as compared with the cells expressing only arhA1A2, and further increased in the cells expressing arhA1A2A3A4. These results clearly showed that ArhA3 and ArhA4 were necessary for maximal acenaphthene oxygenase activity.
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, Fig. 1). DNA fragments of the expected size were amplified with all the primer sets used. No amplification was observed when the reverse transcriptase was omitted from the reaction mixture (date not shown). These results suggested that the DNA region from arhA3 to ORF6 is transcribed within the same transcription unit, although some promoters might be included within this region.
Involvement of ArhR in arh gene expression
To investigate how the disruption of arhR affects the expression of the arh genes, quantitative RT-PCR analyses of the arhR, arhA3 and arhA1 genes were performed. The differences in their mRNA levels in strains A4, A4DR (the arhR disruptant) and A4DRC (A4DR carrying arhR in trans) were compared under two conditions, i.e. with and without induction by acenaphthene (Fig. 2). In the wild-type cells, acenaphthene induced transcription of arhA3 and arhA1 (Fig. 2a, b). By contrast, in strain A4DR cells, the mRNA levels of arhA3 and arhA1 were much lower than those of the wild-type cells, even when the cells were not induced by acenaphthene. In strain A4DRC, the mRNA levels of arhA3 and arhA1 were restored to almost the same as those in the wild-type cells. These results strongly suggest that ArhR acts as an activator for the transcription of arhA genes and that transcription was induced more in the presence of acenaphthene (or its metabolites). By contrast, the transcription of arhR was not induced by acenaphthene under the experimental conditions, and it was not affected by the disruption of arhR (Fig. 2c). Therefore, the arhR gene appears to be regulated in a different manner from the arhA genes, perhaps constitutively.
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) results, we predicted the existence of at least one acenaphthene-inducible promoter, which is activated by ArhR, in the region upstream from the arhA3 gene. Therefore, we performed a primer extension analysis to determine the transcription start point of the arhA3 gene. One transcription start point was found 44 bp upstream from the translation start point of arhA3 (Fig. 3a, b). The possible promoter sequence was assigned to TTGACG (–35 region) and TATCCG (–10 region) (Fig. 3b). This promoter sequence is similar to the consensus sequence of the E. coli 
70-dependent promoter (Record et al., 1996). Upstream from this promoter, between bases –56 and –70, there is a consensus sequence for LTTR binding (ATCACTGTCCGTGAT), which is a palindromic T-N11-A motif (beginning and end underlined) (Schell, 1993) (Fig. 3b). This suggests that ArhR binds to this motif and regulates transcription from this promoter region. We did not find any other sequence similar to this motif in the sequenced 16.4 kb DNA region.
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DISCUSSION |
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). The gene organization in this locus was interesting: the ORFs located in the region from arhA3 to ORF6, all of which are oriented in the same transcription direction (Fig. 1), include a gene not essential for acenaphthene degradation (ORF15) and ORFs apparently not involved in aromatic degradation, such as two putative pseudogenes (ORF11 and ORF13) and a putative transposase gene or its remnant (ORF16). RT-PCR analysis revealed that the entire region between arhA3 and ORF6 is actually transcribed in the same direction when strain A4 is grown on acenaphthene (Fig. 1), suggesting that the region from arhA3 to ORF6 forms an operon. This loose operon structure, including the long regions unnecessary for acenaphthene degradation, seems inefficient in terms of the energy required for transcription, in contrast to the well-studied operons for the aromatic degradation of pseudomonads (e.g. the nah and sal operons for naphthalene degradation; Dennis & Zylstra, 2004). Apparently, such loose genetic structures have been found in many PAH-degrading sphingomonads (Pinyakong et al., 2003a). However, their actual transcriptional units have not been extensively investigated. Therefore, further study is necessary to prove that such an operon structure is typical for other PAH-degrading sphingomonads.
In this study, ArhR, which is an LTTR that activates the gene cluster from arhA3, was also isolated. LTTRs are one of the most common transcriptional regulators in prokaryotes. To date, numerous LTTRs have been reported, and their transcriptional regulation mechanisms have been investigated in detail (Schell, 1993; Tropel & van der Meer, 2004). Usually, LTTRs act as transcriptional activators and increase transcription from target promoters between 6- and 200-fold, only in the presence of an inducer (Schell, 1993). We demonstrated that ArhR also activates transcription of the arhA genes in the presence of acenaphthene (or its metabolites) (Fig. 2a, b). Nevertheless, the mRNA levels of arhA3 and arhA1 in strain A4 increased only 2.4- and 6.2-fold, respectively, in the presence of acenaphthene. Moreover, in strain A4DR (the arhR disruptant), the mRNA levels of arhA3 and arhA1 were much lower (less than 10 %) than those in strain A4 in the absence of acenaphthene. These facts suggest that ArhR increases some transcription of its target genes even in the absence of an inducer. Although this is unusual for LTTRs, a similar tendency has been observed for the activation of clcABD (3-chlorocatechol degradative operon) by CatR, which is the LTTR that activates both catBCA (catechol degradative operon) and clcABD (McFall et al., 1998). In that study, an in vitro transcription assay was used to demonstrate that CatR could activate the clcABD promoter in a concentration-dependent manner in the absence of an inducer and that the activation was increased two- to threefold in the presence of inducer (McFall et al., 1998, 1997). This was thought to be due to the binding of CatR to the clcABD promoter in its active conformation in the absence of an inducer (McFall et al., 1998). In strain A4, ArhR is thought to activate its target promoter in a manner similar to that of CatR activation of the clc promoter. The difference in the fold-increase of the mRNA level in the presence of acenaphthene between arhA3 (2.4-fold) and arhA1 (6.2-fold) remains to be explained, but it is possible that another, more inducible promoter(s) exists between these genes. Also, there may be a difference in stability between the mRNA fragments transcribed from arhA3 and arhA1.
In general, the genes for LTTRs are located just upstream from their target metabolic operons and are transcribed in the opposite direction; LTTRs repress their own expression, probably because the binding sites responsible for activating the target operon overlap the promoter of the genes for LTTRs (Schell, 1993). In strain A4, arhR is located upstream from arhA3 in the gene cluster; however, the intergenic region between arhR and arhA3 is about 1 kb in length and includes putative transposase genes or their remnants (ORF8 and ORF9). Therefore, autorepression as with typical LTTRs does not seem likely to occur with the expression of arhR. In fact, the transcription of arhR was not affected by the disruption of arhR under the conditions examined in this study (Fig. 2c). Therefore, the existence of ORF8 and ORF9 may affect the expression of the arhR gene.
For acenaphthene degradation in strain A4, ArhR first activates the expression of the arhA genes, and then the ArhA oxygenase converts acenaphthene into 1-acenaphthenol and acenaphthene-1,2-diol by two sequential monooxygenations. In our previous work, it was shown that 1-acenaphthenol can be dehydrogenated into 1-acenaphthenone (Komatsu et al., 1993; Pinyakong et al., 2004). Although the degradation pathway downstream of 1-acenaphthenone and acenaphthene-1,2-diol was not clearly identified in strain A4, Selifonov et al. (1996) reported that 1,2-acenaphthoquinone was produced by two sequential monooxygenations and subsequent nonspecific dehydrogenations in resting cells of Pseudomonas aeruginosa strain PAO1 that expressed naphthalene dioxygenase genes (nahA). Furthermore, they reported that 1,2-acenaphthoquinone was oxidized spontaneously to 1,8-naphthalenedicarboxylic acid (1,8-NDCA), which was identified as its anhydride (1,8-NA). This degradation pathway may also be present in strain A4, because one mini-Tn5-inserted mutant, strain AG3-69, lost the ability to utilize 1,8-NA in addition to acenaphthene, suggesting that acenaphthene is degraded via 1,8-NDCA (or 1,8-NA) in strain A4. There have been no reports on the degradation pathway of 1,8-NDCA, although strain AG3-69 should provide an important clue for clarifying this. In strain AG3-69, the mini-Tn5 was inserted in the DNA region homologous to the redA2 gene, which encodes the ferredoxin reductase component of the dioxin dioxygenase in Sphingomonas wittichii strain RW1 (Armengaud et al., 1998). Therefore, this redA2 homologue is likely involved in the oxygenation of 1,8-NDCA or its metabolites in combination with other oxygenase components, which have not been identified. We performed a preliminary nucleotide sequence analysis of the flanking region of this redA2 homologue, but no other genes homologous to known aromatic degradative genes were found (data not shown). This suggests that the genes responsible for the further degradation of acenaphthene metabolites are also dispersed throughout the strain A4 genome, as are the dioxin dioxygenase genes of strain RW1 (Armengaud et al., 1998). Apparently, ArhR is not involved in regulating these genes, because the arhR disruptant (strain A4DR) did not lose the ability to utilize 1,8-NA (data not shown). Although some difficulty will arise because of the scattered gene organization that is common among sphingomonads, further isolation of the degradative genes and investigation of their regulatory mechanisms are needed to clarify the entire acenaphthene degradation system in strain A4.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Alexeyev, M. F., Shokolenko, I. N. & Croughan, T. P. (1995). New mini-Tn5 derivatives for insertion mutagenesis and genetic engineering in gram-negative bacteria. Can J Microbiol 41, 1053–1055.[Medline]
Armengaud, J. & Timmis, K. N. (1998). The reductase RedA2 of the multi-component dioxin dioxygenase system of Sphingomonas sp. RW1 is related to class-I cytochrome P450-type reductases. Eur J Biochem 253, 437–444.[Medline]
Armengaud, J., Happe, B. & Timmis, K. N. (1998). Genetic analysis of dioxin dioxygenase of Sphingomonas sp. strain RW1: catabolic genes dispersed on the genome. J Bacteriol 180, 3954–3966.
Bastiaens, L., Springael, D., Wattiau, P., Harms, H., deWachter, R., Verachtert, H. & Diels, L. (2000). Isolation of adherent polycyclic aromatic hydrocarbon (PAH)-degrading bacteria using PAH-sorbing carriers. Appl Environ Microbiol 66, 1834–1843.
Brooks, C. J. W., Cole, W. J., Borthwick, J. H. & Brown, G. M. (1982). Characterization of dihydroarenediols and related compounds by gas chromatography-mass spectrometry: comparison of derivatives. J Chromatogr 239, 191–216.[CrossRef]
Cai, M. & Xun, L. (2002). Organization and regulation of pentachlorophenol-degrading genes in Sphingobium chlorophenolicum ATCC 39723. J Bacteriol 184, 4672–4680.
de Lorenzo, V. & Timmis, K. N. (1994). Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons. Methods Enzymol 235, 386–405.[Medline]
Demanèche, S., Meyer, C., Micoud, J., Louwagie, M., Willison, J. C. & Jouanneau, Y. (2004). Identification and functional analysis of two aromatic-ring-hydroxylating dioxygenases from a Sphingomonas strain that degrades various polycyclic aromatic hydrocarbons. Appl Environ Microbiol 70, 6714–6725.
Dennis, J. J. & Zylstra, G. J. (2004). Complete sequence and genetic organization of pDTG1, the 83 kilobase naphthalene degradation plasmid from Pseudomonas putida strain NCIB 9816-4. J Mol Biol 341, 753–768.[CrossRef][Medline]
Ferraro, D. J., Gakhar, L. & Ramaswamy, S. (2005). Rieske business: structure-function of Rieske non-heme oxygenases. Biochem Biophys Res Commun 338, 175–190.[CrossRef][Medline]
Fredrickson, J. K., Balkwill, D. L., Drake, G. R., Romine, M. F., Ringelberg, D. B. & White, D. C. (1995). Aromatic-degrading Sphingomonas isolates from the deep subsurface. Appl Environ Microbiol 61, 1917–1922.[Abstract]
Gabriel, F. L., Giger, W., Guenther, K. & Kohler, H. P. (2005). Differential degradation of nonylphenol isomers by Sphingomonas xenophaga Bayram. Appl Environ Microbiol 71, 1123–1129.
Gruez, A., Roig-Zamboni, V., Grisel, S., Salomoni, A., Valencia, C., Campanacci, V., Tegoni, M. & Cambillau, C. (2004). Crystal structure and kinetics identify Escherichia coli YdcW gene product as a medium-chain aldehyde dehydrogenase. J Mol Biol 343, 29–41.[CrossRef][Medline]
Habe, H., Ashikawa, Y., Saiki, Y., Yoshida, T., Nojiri, H. & Omori, T. (2002). Sphingomonas sp. strain KA1, carrying a carbazole dioxygenase gene homologue, degrades chlorinated dibenzo-p-dioxins in soil. FEMS Microbiol Lett 211, 43–49.[CrossRef][Medline]
Hernáez, M. J., Reineke, W. & Santero, E. (1999). Genetic analysis of biodegradation of tetralin by a Sphingomonas strain. Appl Environ Microbiol 65, 1806–1810.
Imai, R., Nagata, Y., Senoo, K., Wada, H., Fukuda, M., Takagi, M. & Yano, K. (1989). Dehydrochlorination of -hexachlorocyclohexane (-BHC) by -BHC-assimilating Pseudomonas paucimobilis. Agric Biol Chem 53, 2015–2017.
Khan, A. A., Wang, R. F., Cao, W. W., Franklin, W. & Cerniglia, C. E. (1996). Reclassification of a polycyclic aromatic hydrocarbon-metabolizing bacterium, Beijerinckia sp. strain B1, as Sphingomonas yanoikuyae by fatty acid analysis, protein pattern analysis, DNA-DNA hybridization, and 16S ribosomal DNA sequencing. Int J Syst Bacteriol 46, 466–469.
Kiyohara, H., Nagao, K., Kouno, K. & Yano, K. (1982). Phenanthrene-degrading phenotype of Alcaligenes faecalis AFK2. Appl Environ Microbiol 43, 458–461.
Kohler, H. P. (1999). Sphingomonas herbicidovorans MH: a versatile phenoxyalkanoic acid herbicide degrader. J Ind Microbiol Biotechnol 23, 336–340.[CrossRef][Medline]
Kohler, M., Künnifer, T., Schmid, P., Gujer, E., Crockett, R. & Wolfensberger, M. (2000). Inventory and emission factor of creosote, polycyclic aromatic hydrocabons (PAH), and phenols from railroad ties treated with creosote. Environ Sci Technol 34, 4766–4772.[CrossRef]
Komatsu, T., Omori, T. & Kodama, T. (1993). Microbial degradation of the polycyclic aromatic hydrocarbons acenaphthene and acenaphthylene by a pure bacterial culture. Biosci Biotechnol Biochem 57, 864–865.[Medline]
Lessner, D. J., Parales, R. E., Narayan, S. & Gibson, D. T. (2003). Expression of the nitroarene dioxygenase genes in Comamonas sp. strain JS765 and Acidovorax sp. strain JS42 is induced by multiple aromatic compounds. J Bacteriol 185, 3895–3904.
Martínez-Pérez, O., Moreno-Ruiz, E., Floriano, B. & Santero, E. (2004). Regulation of tetralin biodegradation and identification of genes essential for expression of thn operons. J Bacteriol 186, 6101–6109.
McFall, S. M., Klem, T. J., Fujita, N., Ishihama, A. & Chakrabarty, A. M. (1997). DNase I footprinting, DNA bending and in vitro transcription analyses of ClcR and CatR interactions with the clcABD promoter: evidence of a conserved transcriptional activation mechanism. Mol Microbiol 24, 965–976.[CrossRef][Medline]
McFall, S. M., Chugani, S. A. & Chakrabarty, A. M. (1998). Transcriptional activation of the catechol and chlorocatechol operons: variations on a theme. Gene 223, 257–267.[CrossRef][Medline]
Miyauchi, K., Lee, H. S., Fukuda, M., Takagi, M. & Nagata, Y. (2002). Cloning and characterization of linR, involved in regulation of the downstream pathway for -hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26. Appl Environ Microbiol 68, 1803–1807.
Moreno-Ruiz, E., Hernáez, M. J., Martínez-Pérez, O. & Santero, E. (2003). Identification and functional characterization of Sphingomonas macrogolitabida strain TFA genes involved in the first two steps of the tetralin catabolic pathway. J Bacteriol 185, 2026–2030.
Mueller, J. G., Chapman, P. J., Blattmann, B. O. & Pritchard, P. H. (1990). Isolation and characterization of a fluoranthene-utilizing strain of Pseudomonas paucimobilis. Appl Environ Microbiol 56, 1079–1086.
Pinyakong, O. (2003). Biochemical and genetic analyses of polycyclic aromatic hydrocarbons degradation in sphingomonads. PhD thesis, University of Tokyo, Tokyo.
Pinyakong, O., Habe, H., Supaka, N. & 7 other authors (2000). Identification of novel metabolites in the degradation of phenanthrene by Sphingomonas sp. strain P2. FEMS Microbiol Lett 191, 115–121.[CrossRef][Medline]
Pinyakong, O., Habe, H. & Omori, T. (2003a). The unique aromatic catabolic genes in sphingomonads degrading polycyclic aromatic hydrocarbons (PAHs). J Gen Appl Microbiol 49, 1–19.
Pinyakong, O., Habe, H., Yoshida, T., Nojiri, H. & Omori, T. (2003b). Identification of three novel salicylate 1-hydroxylases involved in the phenanthrene degradation of Sphingobium sp. strain P2. Biochem Biophys Res Commun 301, 350–357.[CrossRef][Medline]
Pinyakong, O., Habe, H., Kouzuma, A., Nojiri, H., Yamane, H. & Omori, T. (2004). Isolation and characterization of genes encoding polycyclic aromatic hydrocarbon dioxygenase from acenaphthene and acenaphthylene degrading Sphingomonas sp. strain A4. FEMS Microbiol Lett 238, 297–305.
Record, M. T., Jr, Reznikoff, W. S., Craig, M. L., McQuade, K. L. & Schlax, P. J. (1996). Escherichia coli RNA polymerase (E70), promoters, and the kinetics of the steps of transcription initiation. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn, pp. 792–820. Edited by F. C. Neidhardt & others. Washington, DC: American Society for Microbiology.
Saber, D. L. & Crawford, R. L. (1985). Isolation and characterization of Flavobacterium strains that degrade pentachlorophenol. Appl Environ Microbiol 50, 1512–1518.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schäfer, A., Tauch, A., Jager, W., Kalinowski, J., Thierbach, G. & Pühler, A. (1994). Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145, 69–73.[CrossRef][Medline]
Schell, M. A. (1993). Molecular biology of the LysR family of transcriptional regulators. Annu Rev Microbiol 47, 597–626.[CrossRef][Medline]
Selifonov, S. A., Slepen'kin, A. V., Adanin, V. M., Grechkina, G. M. & Starovoitov, I. I. (1993). Acenaphthene catabolism by strains of Alcaligenes eutrophus and Alcaligenes paradoxus. Microbiology [English translation of Mikrobiologiia] 62, 85–92.
Selifonov, S. A., Grifoll, M., Eaton, R. W. & Chapman, P. J. (1996). Oxidation of naphthenoaromatic and methyl-substituted aromatic compounds by naphthalene 1,2-dioxygenase. Appl Envir Microbiol 62, 507–514.[Abstract]
Shi, T., Fredrickson, J. K. & Balkwill, D. L. (2001). Biodegradation of polycyclic aromatic hydrocarbons by Sphingomonas strains isolated from the terrestrial subsurface. J Ind Microbiol Biotechnol 26, 283–289.[CrossRef][Medline]
Sukchawalit, R., Vattanaviboon, P., Sallabhan, R. & Mongkolsuk, S. (1999). Construction and characterization of regulated L-arabinose-inducible broad host range expression vectors in Xanthomonas. FEMS Microbiol Lett 181, 217–223.[Medline]
Takeuchi, M., Hamana, K. & Hiraishi, A. (2001). Proposal of the genus Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analyses. Int J Syst Evol Microbiol 51, 1405–1417.[Abstract]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Tropel, D. & van der Meer, J. R. (2004). Bacterial transcriptional regulators for degradation pathways of aromatic compounds. Microbiol Mol Biol Rev 68, 474–500.
White, D. C., Sutton, S. D. & Ringelberg, D. B. (1996). The genus Sphingomonas: physiology and ecology. Curr Opin Biotechnol 7, 301–306.[CrossRef][Medline]
Wise, S. A., Benner, B. A., Burd, G. D., Chester, S. N., Rebbert, R. E. & Schantz, M. M. (1988). Determination of polycyclic aromatic hydrocarbons in a coal tar standard reference material. Anal Chem 60, 887–894.[CrossRef]
Wittich, R. M., Wilkes, H., Sinnwell, V., Francke, W. & Fortnagel, P. (1992). Metabolism of dibenzo-p-dioxin by Sphingomonas sp. strain RW1. Appl Environ Microbiol 58, 1005–1010.
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–119.[CrossRef][Medline]
Yoshida, T., Ayabe, Y., Yasunaga, M., Usami, Y., Habe, H., Nojiri, H. & Omori, T. (2003). Genes involved in the synthesis of the exopolysaccharide methanolan by the obligate methylotroph Methylobacillus sp. strain 12S. Microbiology 149, 431–444.
Received 6 January 2006;
revised 4 April 2006;
accepted 13 April 2006.
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