Cloning and Characterization of Pseudomonas sp. StrainDNT Genes for 2,4-Dinitrotoluene Degradation
WEN-CHEN SUEN AND JIM C. SPAIN*Air Force Civil Engineering Support Agency, Tyndall Air Force Base, Florida 32403-6001
Received 6 November 1992/Accepted 18 December 1992
The degradation of 2,4-dinitrotoluene (DNT) by Pseudomonas sp. strain DNT is initiated by a dioxygenaseattack to yield 4-methyl-5-nitrocatechol (MNC) and nitrite. Subsequent oxidation ofMNC by a monooxygenaseresults in the removal of the second molecule of nitrite, and further enzymatic reactions lead to ring fission.Initial studies on the molecular basis ofDNT degradation in this strain revealed the presence of three plasmids.Mitomycin-derived mutants deficient in either DNT dioxygenase only or DNT dioxygenase and MNCmonooxygenase were isolated. Plasmid profiles of mutant strains suggested that the mutations resulted fromdeletions in the largest plasmid. Total plasmid DNA partially digested by EcoRI was cloned into a broad-host-range cosmid vector, pCP13. Recombinant clones containing genes encoding DNT dioxygenase, MNC monoox-ygenase, and 2,4,5-trihydroxytoluene oxygenase were characterized by identification of reaction products and theability to complement mutants. Subcloning analysis suggests that the DNT dioxygenase is a multicomponentenzyme system and that the genes for the DNT pathway pre organized in at least three different operons.
2,4-Dinitrotoluene (DNT), a major by-product from man-ufacture of 2,4,6-trinitrotoluene (TNT), is regulated as ahazardous waste at concentrations of >0.13 ppm by theEnvironmental Protection Agency (19). It is widely used inindustry for production of polyurethane foams. Its commonpresence in industrial plant effluents and toxicity to experi-mental animals have raised environmental concerns (26, 31).DNT is reduced to amino and azoxy or nitroso compounds
by microorganisms, including both fungi and bacteria (23,25, 26). However, the reduction products are resistant tofurther degradation. Recently, it was reported that a lignin-degrading fungus, Phanerochaete chrysosporium, degradesDNT to carbon dioxide and nitrite. The first nitro group ofDNT is reduced to an aromatic amine, and subsequentreactions by a lignin peroxidase and/or a manganese perox-idase result in the removal of both nitro groups before ringcleavage (40). We recently reported the isolation of Pseudo-monas sp. strain DNT, which is able to completely miner-alize DNT by an oxidative pathway. The initial reaction is anattack on DNT by a dioxygenase which forms a brightyellow product, 4-methyl-5-nitrocatechol (MNC), and re-leases the first nitrite (Fig. 1) (37). A monooxygenase thencatalyzes the oxidation of MNC to 2-hydroxy-5-methylqui-none (HMQ) and nitrite. Further reactions catalyzed by aquinone reductase and 2,4,5-trihydroxytoluene (THT) oxy-genase result in colorless ring fission products (16).The structure, organization, and regulation of genes in-
volved in the pathway must be elucidated to facilitateunderstanding of the molecular basis of DNT degradation bythis strain. For this purpose, we have cloned and character-ized the genes for DNT degradation from Pseudomonas sp.strain DNT. Although there are many reports on the bio-chemical studies of nitroaromatic degradation (2, 6, 7, 17, 26,32, 36, 37, 40), to our knowledge, this is the first report onthe genetic analysis of pathways involved in the removal ofaromatic nitro groups.
* Corresponding author.
MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. Pseudo-
monas sp. strain DNT was grown as described previously(37). For plasmid isolation, it was grown on tryptic soy agar(TSA) (Difco Laboratories, Detroit, Mich.) Escherichia coliHB101 (hsdS20 recA13 proA2 leuB6 thi-1) (1) and JM109[recA1 endAl gyrA96 thi hsdR17supE44 reLU1 A(lac-proAB)(F' traD36proAB lacIZAM15)] (44) were used as the hostsfor gene cloning. Plasmid pCP13 (5), used as the vector forcosmid cloning, was a gift from I. S. You, California StateUniversity, Fresno. Plasmid pGEM7Z(+) (Promega, Madi-son, Wis.) was used as the vector for subcloning. E. colistrains were grown on TSA at 30 or 37°C. Tetracycline (12 to50 ,ug/ml) or ampicillin (100 ,ug/ml) was added to the growthmedium when needed to select for the presence of recombi-nant plasmids. IPTG (isopropyl-j3-D-thiogalactopyranoside)(0.5 mM) and X-Gal (5-bromo-4-chloro-3-indolyl-13-D-galac-topyranoside) (40 ,ug/ml) were also added where appropriate.
Screening methods and identification of Pseudomnonas sp.DNT mutants and recombinant E. coli clones. DNT degrada-tion by Pseudomonas sp. strain DNT produced severalcolored intermediates, and all the enzymes in this pathwayhave some constitutive activity (16). These properties al-lowed the development of a rapid screening method (mi-croplate assay) for detection of DNT dioxygenase, MNCmonooxygenase, and THT oxygenase. This method involveswhole-cell transformation of substrate to metabolic interme-diates with concomitant color changes in the medium.Toothpicks were used to transfer cells from colonies
grown on TSA overnight to 100 ,ul of buffer in the wells of96-well microplates. Suspensions were incubated at roomtemperature overnight. For detecting the presence of DNTdioxygenase activity, cells were suspended in the wellcontaining SMSB (37) or phosphate buffer (pH 7.0) with100-ppm DNT. The accumulation of MNC by the action ofDNT dioxygenase resulted in conversion of the mediumfrom colorless to bright yellow. Nitrite was detected directlyin the microplate wells by means of a small-scale nitriteassay (35).MNC monooxygenase activity was detected by suspen-
sion of cells in the same buffer containing 50 ,M MNC. The
FIG. 1. Proposed pathway for the degradation of DNT byPseudomonas sp. strain DNT. Also shown are the gene designationsfor different enzymes in the pathway. dntA, DNT dioxygenasegene(s); dntB, MNC monooxygenase gene; dntC, HMQ reductasegene; dntD, THT oxygenase gene.
conversion of the yellow color to a light pink due to HMQaccumulation was taken to indicate MNC monooxygenaseactivity. The nitrite assay could not be used for detection ofnitrite released from MNC by the action of MNC monoox-ygenase because HMQ and THT inhibited the nitrite assayreaction (15).
Conversion of HMQ to THT requires NADH and iscatalyzed by a quinone reductase in intact cells, but the THTrapidly decomposes to HMQ in neutral buffer (16). E. colicells have quinone reductases that reduce HMQ to THT,which eventually converts back to HMQ if the cells have noTHT oxygenase activity (this study). For detection of THToxygenase activity, cells were suspended in microplate wellscontaining 50 mM phosphate buffer (pH 6.8) with 100 ,uMTHT. The reaction mixtures with cells expressing THToxygenase activity remained colorless after overnight incu-bation at room temperature. Controls were pink because ofthe decomposition of THT to HMQ. Alternatively, THToxygenase activity was detected in a similar manner with 100,uM 4-methylcatechol as the substrate. THT oxygenasecatalyzed the conversion of 4-methylcatechol to a yellowring fission product in a reaction analogous to that describedby Cain and Farr (3).
Strains expressing enzyme activity initially detected bythe microplate assay were further characterized by analysisof substrate disappearance and reaction product accumula-tion in large-scale experiments. Cells were grown overnightin tryptic soy broth with appropriate antibiotics at 30°C,harvested by centrifugation, washed, and suspended inphosphate buffer to a final optical density of 3.0 at 600 nmand incubated at 30°C. At appropriate intervals, cells wereremoved by centrifugation and supernatant fluids were ana-lyzed by high-performance liquid chromatography (HPLC)as described previously (37).
Mutants. Mitomycin curing was performed as describedby Chakrabarty (4). Survivors from treatment with 10 ,ug ofmitomycin were grown on TSA plates at 30°C overnight.Colonies were transferred to fresh TSA plates and incubatedovernight. Mutants were screened by the microplate assay.The phenotypes were confirmed by identification of trans-formation products by HPLC and by auxanography (30).
DNA isolation and manipulation. Plasmids from Pseudo-monas sp. strain DNT and its derivatives were isolated on asmall scale by the method of Kado and Liu (22). Large-scaleisolation of plasmids was done by the procedure describedby Hansen and Olsen (18) with subsequent purification bycentrifugation in cesium chloride-ethidium bromide densitygradients (33). Recombinant plasmids derived from pCP13were packaged into lambda phages in vitro by using aGigapack II plus packaging extracts from Stratagene, LaJolla, Calif. Phages containing recombinant plasmids weretransfected into E. coli HB101 as recommended by Strata-gene. Recombinant plasmids were isolated from E. coli asdescribed by Ish-Horowitz and Burke (21) and purified bycesium chloride-ethidium bromide ultracentrifugation (33).Plasmid DNA was digested by restriction enzymes under theconditions recommended by the suppliers (GIBCO/BRL,Gaithersburg, Md., and Promega Corp.). DNA fragmentswere analyzed by agarose gel electrophoresis (33). DNAfragments of interest were isolated from agarose gels with anElu-quick DNA purification kit by the protocols recom-mended by the supplier (Schleicher & Schuell, Keene,N.H.). DNA fragments were ligated to vectors with T4 DNAligase and were transformed into JM109 by the calciumchloride-thymidine-glycerol procedures (33). E. coli plas-mids (24), TOL plasmid pTN2 (28), a supercoiled-DNAladder, high-molecular-weight DNA markers, and a 1-kbDNA ladder (GIBCO/BRL) were used as DNA molecularweight markers.
Conjugation. Recombinant cosmids derived from pCP13were transferred into the mutants ofPseudomonas sp. strainDNT by triparental mating using the mobilizing plasmidpRK2013 (14). The transconjugants were selected and grownon SMSB (37) supplemented with 20 mM sodium succinateand 12 pg of tetracycline per ml.
Chemicals. IPTG, X-Gal, and cesium chloride were fromGIBCO/BRL. SeaKem GTG agarose was from FMC Bio-Products, Rockland, Maine. DNT was from Aldrich Chem-ical Company, Milwaukee, Wis. MNC and THT were kindlyprovided by R. J. Spanggord, SRI International, MenloPark, Calif. Ethidium bromide, ampicillin, and tetracyclinewere from Sigma Chemical Company, St. Louis, Mo.
RESULTSPlasmids and mutants of Pseudomonas sp. strain DNT.
Three plasmids of approximately 180 (pJS1), 50 (pJS2), and2.1 (pJS3) kb were isolated from strain DNT. One sponta-neous mutant, strain S1, was isolated from screening 100colonies after three passages on TSA plates. Two hundredcolonies were screened for mutants after mitomycin treat-ment. Ninety-five percent of these colonies were deficient inDNT dioxygenase activity. One colony isolate had lost bothDNT dioxygenase and MNC monooxygenase activities. Theremaining nine colonies still maintain Dnt+ phenotypes.Two mutants were chosen for further study; mutant S5 lacksonly DNT dioxygenase, and mutant S52 lacks both DNTdioxygenase and MNC monooxygenase. The plasmid pro-files and phenotypes of these mutants (Fig. 2 and Table 1)indicate that phenotypic changes of mutant strains S5 andS52 correlated with a large deletion in pJS1. These prelimi-nary results suggested that the genes encoding DNT dioxy-genase and MNC monooxygenase were located on pJS1.Cosmid cloning of genes for DNT degradation. Attempts toisolate and purify pJS1 in the quantities and of the purityneeded for cloning (33, 42) were unsuccessful. Therefore,total plasmid DNA from strain DNT was partially digested
FIG. 2. Plasmid profile of Pseudomonas' sp. strain DNT and itsmutants. Agarose gel (0.4%) electrophoresis was performed at 41 Vfor 15 h at room temperature. The far left lane shows the super-coiled-DNA ladder molecular weight marker.
by EcoRI and ligated into the cosmid vector pCP13, whichhad been cleaved by EcoRI. The ligated DNA was packagedinto lambda phages and transfected into E. coli HB101. Of400 recombinants screened for genes in the DNT pathway, 3exhibiting only DNT dioxygenase activity were isolated.These clones produced pink to blue colonies when grown on
TSA plates, indicating the formation of indigo (12). Elevenclones with MNC monooxygenase activity only and fiveclones with both MNC monooxygenase and THT oxygenaseactivities were isolated. Examination of the plasmid profilesof these clones revealed the presence of multiple EcoRIfragments (data not shown). Recombinant clones with thesmallest plasmids (pJS6 and pJS7 for dnt4, pJS8 for dntB,and pJS19 for dntB and dntD) were chosen for furtheranalysis. Restriction digestion with EcoRI (Fig. 3) revealedthat both pJS7 and pJS6 have one 14.7-kb, one 1.8-kb, one0.6-kb, and two 2.1-kb EcoRI fragments. Both pJS8 andpJS19 have one 14.3-kb and two 2.1-kb EcoRI fragments.However, pJS7 has two more EcoRI fragments (3.0 and 8.2kb) than pJS6. Plasmid pJS19 has three more EcoRI frag-ments (0.7, 2.75, and 3.4 kb) than pJS8.
Characterization of cosmid clones. The enzyme activity ofeach clone was confirmed by measurement of substratedisappearance and/or product identification by HPLC. BothHB101(pJS6) and HB1O1(pJS7) transformed DNT to MNC.HB1O1(pJS8) oxidized MNC to HMQ. HB1O1(pJS19) oxi-dized MNC and THT to colorless products. HB1O1(pJS19)also transformed 4-methylcatechol to a yellow ring fissionproduct with a UV absorbance spectrum similar to thatobtained from transformation of 4-methylcatechol by cate-
TABLE 1. Phenotypes and some properties of Pseudomonas sp.strain DNT and its mutants
DNT Dnt+ Three plasmidsSia Dnt- Mnc+ Yes Indistinguishable from DNTS5b Dnt- Mnc+ No Deletion in pJS1S52" Dnt- Mnc- Tht' No Deletion in pJS1
a Spontaneous mutant.bMitomycin-derIved mutant.
FIG. 3. EcoRI fragments of different recombinant cosmids. Aga-rose gel (0.7%) electrophoresis was performed at 40 V for 16 h atroom temperature. The lane to the left of lane pJS6 is a 1-kb DNAladder as a molecular weight marker.
chol 2,3-dioxygenase (3, 16). The negative control, HB101(pCP13), did not transform MNC, THT, or 4-methylcate-chol. However, it slowly transformed DNT to unidentifiedcompounds but MNC and nitrite were not detected. Theseresults are consistent with the previous observation (23, 25,26) that some bacteria, including E. coli, catalyze the reduc-tion of TNT and DNT to amino and azoxy or nitrosocompounds. Therefore, MNC accumulation and nitrite re-lease but not the disappearance of DNT were reliableindicators of DNT dioxygenase activity.The results for complementation of mutants with recom-
binant plasmids are shown in Table 2. Both pJS8 and pJS19complemented mutant S52 to allow growth on MNC. Al-though pJS6 contains fully active DNT dioxygenase gene(s)expressed in E. coli, it failed to complement S1 and S5mutants. However, pJS7, which contains two more EcoRIfragments than pJS6, complemented both mutants.
Subcloning of genes for DNT degradation. Recombinantcosmids pJS6, pJS8, and pJS19 were used to subclone dntA,dntB, and dntD, respectively. Various EcoRI fragmentswere cloned into pGEM7Zf(+) and transformed into JM109.The recombinant clones were screened for enzyme expres-sion by the microplate assay. The results indicated thatdntA, dntB, and dntD were located in three different EcoRIfragments of approximately 14.7, 14.3, and 3.4 kb. Restric-
TABLE 2. Complementation of mutants withrecombinant cosmids
0 CCD =,7c.= m- ME a=1 a06a& ficl);bZO IXc XE OX CD U
C
Sp
Ba K
Ba
N
N
E
x
Ba
N
N
FIG. 4. Subcloning of DNT dioxygenase gene(s) (dntA). + +, yellow product (MNC) appeared within minutes in microplate assay andpinkish blue cells on TSA plates; +, yellow product (MNC) appeared within hours in microplate assay and light pinkish cells on TSA plates;-, no activity. The location of the T7 promoter from the vector in each recombinant plasmid is indicated by an arrow.
tion maps of the three subclones are shown in Fig. 4 through6. Further subcloning localized the position of each gene onthe EcoRI fragment. Recombinant clones containing plas-mids with restriction fragments in the opposite orientation(pJS37 and pJS39 for dntA, pJS53 and pJS58 for dntB, andpJS75 and pJS76 for dntD) expressed enzyme activity for all
Plasmid
pJS5O
pJS51
pJS52
pJS53
pJS54
1 kb
0r
Cc< e6 XX< x~)II I I
three genes. This suggests that the DNA inserts all containinternal promoters recognized by E. coli. pJS37 and pJS39,both containing a 6.8-kb NsiI-NsiI DNA insert, have fullyfunctioning dntA (Fig. 4). The recombinant clone containingpJS36 with a 6-kb BamHI-BamHI DNA insert does not haveDNT dioxygenase activity. These results indicate that the
X
It*( I
Xb E
Xbsp
A A
AE
A
pJS55
Activity
+
E
pJS56
pJS57
pJS58
FIG. 5. Subcloning of MNCdetermined by microplate assay.
K A
AL_
A A
monooxygenase gene (dntB). Arrows indicate the location of the T7 promoter. Enzyme activity was
FIG. 6. Subcloning of THT oxygenaseindicate the location of the T7 promoter.determined by microplate assay.
gene (dntD). ArrowsEnzyme activity was
BamHI site in the 6.8-kb NsiI-NsiI DNA fragment is locatedwithin dntA. Furthermore, recombinant clones containingeither pJS34 with a 6.4-kb SphI-XhoI DNA insert or pJS38with a 4.8-kb SphI-NsiI DNA insert have a less active DNTdioxygenase (Fig. 4) than other clones containing dntA.They also produced less indigo when they were grown onTSA plates. Therefore, more than just the 3.5 kb of theSphI-BamHI DNA fragment is required to encode fullyactive DNT dioxygenase.
Preliminary results from analysis of purified MNC mono-oxygenase indicate that the size of the gene is approximately1.6 to 1.8 kb (15). Recombinant clones containing eitherpJS56 with a 1.4-kb KpnI-ApaI DNA insert or pJS57 with a1.5-kb ApaI-XhoI DNA insert have no MNC monooxygen-ase activity. However, pJS53 and pJS58, both containing a2.2-kb ApaI-ApaI DNA fragment, have fully functioningdntB (Fig. 5). This is in good agreement with the predictedsize of dntB. The molecular weight ofTHT oxygenase is stillunknown. Plasmid pJS71 containing a 0.9-kb EcoRI-SmaIfragment does not have THT oxygenase activity (Fig. 6).Plasmid pJS76 has T`HT oxygenase activity and contains a1.2-kb EcoRI-XhoI fragment which is sufficient to encode aprotein with a molecular mass of no more than 44 kDa.
DISCUSSION
The evidence presented here indicates that the genesencoding enzymes for DNT degradation by Pseudomonassp. strain DNT are encoded on an -180-kb plasmid, pJS1.Recombinant cosmids containing either dntA only or dntBwith or without dntD were isolated, but no recombinantcosmids containing other combinations were isolated. Be-cause partial digestion of EcoRI fragments of total plasmidDNA was used for cosmid cloning, it is likely that dntB anddntD are clustered in pJS1. However, subcloning analysisshowed that dntA and dntB are located in the middle of twodifferent EcoRI fragments (Fig. 4 and 5). These resultsindicate that dntA, dntB, and dntD are not closely linked inpJS1. Furthermore, recombinant clones containing plasmidswith DNA inserts in opposite orientations have enzymeactivity (Fig. 4 through 6). These results suggest that these
three genes for DNT degradation in strain DNT are orga-nized in three different operons. It is unusual but not uniquefor the genes encoding enzymes for a single pathway to belocated in three separate transcription units. The genes for2,4-dichlorophenoxyacetate degradation from Alcaligeneseutrophus are encoded by an 80-kb plasmid, pJP4. Transpo-son mutagenesis and cloning analysis suggested that thesegenes are organized in three operons (8, 9, 38). The genes forbenzoate degradation from Acinetobacter calcoaceticus arealso organized in at least three transcriptional units. How-ever, they are chromosomally encoded and are clusteredwithin a 16-kb DNA fragment (29). Two different operonsoccur in plasmid pWWO (13, 20), NAH7 (46), and pP51 (41)for degradation of toluene, naphthalene, and 1,2,4-trichlo-robenzene, respectively.Although strain DNT can utilize DNT as a sole carbon and
energy source, it grows slowly on the substrate. Transientaccumulation ofMNC and/or HMQ is always detected whencells are grown on DNT (37), and growth is often precededby extended and unpredictable acclimation periods. Tran-sient accumulation of intermediates suggests that the en-zymes for the DNT degradation pathways are sequentiallyinduced and supports the evidence presented here that theyare encoded by three operons.We tentatively designated the gene encoding HMQ reduc-
tase dntC on the basis of the biochemical evidence thatHMQ reductase is inducible (16). E. coli strains have non-specific reductases that convert HMQ to THT (this study).Further studies on the biochemistry and genetics of thissystem are required to demonstrate clearly whether theHMQ reductase in strain DNT is specific for DNT degrada-tion.Comparison of the EcoRI restriction profile of total plas-
mid DNA from strain DNT and the mutant strain S5 showedthat two EcoRI fragments of 14 to 15 kb are lost in S5 (datanot shown). This is consistent with the subcloning analysisfinding that dntA is located in a 14.7-kb EcoRI fragment.Plasmid pJS7 but not pJS6 complemented S5 (Table 2) eventhough both plasmids have the 14.7-kb EcoRI fragment andDNT dioxygenase activity. S5 contains a plasmid with anapproximately 50-kb deletion from pJS1 (Fig. 2). It is likelythat S5 has lost not only the DNA fragment encoding dntAbut also some additional DNA fragments which may encodea regulatory gene for dntA expression in strain DNT. Fur-thermore, S1, a spontaneous mutant of strain DNT, isrevertible and its restriction plasmid profiles are indistin-guishable from those of DNT (data not shown). S1 is alsocomplemented only by pJS7 and not by pJS6. Therefore, S1may be a regulatory mutant and pJS7 may encode dntA anda regulatory gene which is necessary for expression of dntAin strain DNT. Further study is required to test this hypoth-esis.Recombinant E. coli clones containing dnt4 produced
indigo when they were grown on rich medium. This phenom-enon has been reported for a variety of aromatic dioxygen-ase (12, 41, 49) and monooxygenase (27, 47) systems. Allthese aromatic oxygenase systems consist of two or threeprotein components (10, 39, 43, 45, 47). Two to five genesare required to encode these proteins. The size of the DNAfragment encoding the genes is between 2 and 5 kb (34, 39,41, 48). In the naphthalene dioxygenase system, whichconsists of three components, less indigo formation wasdetected in the recombinant clone containing only the oxy-genase component when it was grown on rich medium (11).In the 1,2,4-trichlorobenzene dioxygenase system, recombi-nant clones producing either strong or, weak indigo colora-
tion in indole-agar plates were isolated and at least 5 kb ofDNA is required for full enzyme activity (41). JM109(pJS33)and JM109(pJS35) have less DNT dioxygenase activity andless indigo formation than other recombinant clones contain-ing fully functioning dntA when they are grown on TSA.Subcloning analysis (Fig. 4) indicates that more than 3.5 kbofDNA is necessary to encode completely functioning dntA.By analogy to the systems mentioned above, DNT dioxyge-nase appears to be a multicomponent enzyme system,
In summary, we have described here the cloning andcharacterization of three different genes for DNT degrada-tion from Pseudomonas sp. strain DNT. The DNA frag-ments containing DNT genes from this study will be used asprobes for homologous genes of similar pathways or similarfunctions in aromatic nitro group removal. In addition, wedeveloped a novel screening method for detection of cellsexpressing enzymes of the DNT pathway. It allows rapidand simple screening of a large number of mutants orrecombinant clones. Similar methods can be developed inthe future for other biodegradation pathways that havedistinguishable color differences in substrates and metabolicintermediates. We are currently sequencing dntA, dntB, anddntD in order to study their regulation and function. Over-expression clones for these genes will be used to facilitatestudies on enzyme properties and mechanisms.
ACKNOWLEDGMENTS
We thank Ronald J. Spanggord for providing MNC and THT andBilly E. Haigler, Rakesh Jain, Shirley Nishino, William H. Wallace,and Gerben J. Zylstra for reviewing the manuscript.
This research was supported in part by an appointment to theResearch Participation Program at the Air Force Engineering andService Laboratory administered by Oak Ridge Associated Univer-sities through an interagency agreement between the U.S. Depart-ment of Energy and Tyndall Air Force Base.
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