review molecular bases of aerobic bacterial degradation of ... · 2002 fig. 2. lateral...

†To whom correspondence should be addressed. Fax: +81-3-5841-8030; E-mail: aseigyo＠mail.ecc.u-tokyo.ac.jpAbbreviations : CAR, carbazole; DD, dibenzo-p-dioxin; DF, dibenzofuran; FN, ‰uorene; IS, insertion sequence; ORF, open reading

frame; PAH, polycyclic aromatic hydrocarbon; PCDD, polychlorinated dibenzo-p-dioxin; PCDF, polychlorinated dibenzofuran; TEQ, tox-icity equivalency quantity; Tn, transposon

Fig. 1. Chemical Structure of Polychlorinated Dibenzo-p-diox-ins (PCDDs) and Polychlorinated Dibenzofurans (PCDFs) andTheir Structural Analogues, and Their Numbering Conventions.

Biosci. Biotechnol. Biochem., 66 (10), 2001–2016, 2002

Review

Molecular Bases of Aerobic Bacterial Degradation of Dioxins:Involvement of Angular Dioxygenation

Hideaki NOJIRI and Toshio OMORI†

Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,Tokyo 113-8657, Japan

In the last decade, extensive investigation has beendone on the bacterial degradation of dioxins and itsrelated compounds, because this class of chemicals ishighly toxic and has been widely distributed in the en-vironment. These studies have revealed the primary im-portance of a novel dioxygenation reaction, called angu-lar dioxygenation, in the aerobic bacterial degradationpathway of dioxin. Accompanied by the electron trans-port proteins, Rieske nonheme iron oxygenase catalyzesthe incorporation of oxygen atoms to the ether bond-carrying carbon (the angular position) and an adjacentcarbon, resulting in the irreversible cleavage of therecalcitrant aryl ether bond. The 2,2?,3-trihydrox-ybiphenyl or 2,2?,3-trihydroxydiphenyl ether derivativesformed are degraded through meta cleavage. In addi-tion to the degradation system of dibenzofuran anddibenzo-p-dioxin (the nonchlorinated model com-pounds of dioxin), those of ‰uorene and carbazole wereshown to function in dioxin degradation. Some dioxindegradation pathways have been studied biochemicallyand genetically. In addition, feasibility studies haveshown that some dioxin-degrading strains can functionin actual dioxin-contaminated soil. These studies pro-vide useful information for the establishment of abioremediation method for dioxin contamination. Thisreview summarizes recent progress on molecular andbiochemical bases of the bacterial aerobic degradationof dioxin and related compounds.

Key words: angular dioxygenation; bacterial degrada-tion; carbazole; dioxin; ‰uorene

Polychlorinated dibenzo-p-dioxins (PCDDs) andpolychlorinated dibenzofurans (PCDFs), called diox-ins, are produced by incineration processes and,unintentionally, as by-products during the synthesisof pesticides and herbicides. Many of their congenersare highly toxic compounds with undesirable eŠectson the environment and living beings, but have beenwidely distributed in the environment. The dioxin-like compounds should not only comprise the so-

called ``dirty dozen'', those PCDDs and PCDFshaving a 2,3,7,8-substitution pattern, but also low-chlorinated and unchlorinated dibenzo-p-dioxins(DD) and dibenzofuran (DF), as well as their bromi-nated analogues. Figure 1 illustrates the chemicalstructure and numbering system of the dioxins and itsrelated compounds cited in this review. Complexmixtures of congeners of PCDDs and PCDFs are fre-quently detected in environmental samples such assediments.1–3) The dioxins are recalcitrant moleculesthat are extremely persistent in soil sediments. Forexample, the half-life of 2,3,7,8-tetrachloro-DD insoils and sediments is 1 to 10 years.4,5)

Recently, bioremediation, the exploitation of thebiodegradative activities of microorganisms to

2002

Fig. 2. Lateral Dioxygenation for Naphthalene (A) and AngularDioxygenation for Dibenzo-p-dioxin (DD) (B).

The product of angular dioxygenation for DD shown inbrackets is unstable, and has not been observed directly.

2002 H. NOJIRI and T. OMORI

remove environmental pollutants and recalcitrantxenobiotics, has been attempted. It has become morepopular as an alternative to physical and chemicalmethods, because of the low cost and small impacton the environment. Among the bioremediation tech-niques, bioaugmentation, the addition of bacteria toa contaminated environment to increase the degrada-tion of pollutants, is attracting attention,6) because insituations where indigenous microorganisms cannotdegrade these xenobiotics, this may be the onlymethod for successful bioremediation. Hence, manyresearchers have focused their studies on the isolationand identiˆcation of the bacteria that can degradexenobiotics, including dioxins.

The initial step in the aerobic microbial degrada-tion of various aromatic compounds is usually the in-troduction of two hydroxyl groups into the benzenering, forming cis-dihydrodiols (Fig. 2(A)). The en-zymes catalyzed the incorporation of both atoms ofdioxygen into aromatic substrates are called diox-ygenases, and this type of dioxygenation is termedthe lateral dioxygenation or cis-dihydroxylation. cis-Dihydrodiols are dehydrogenated by cis-dihydrodioldehydrogenase, resulting in the formation of acatechol moiety. Because the ˆssion of the aromaticring occurs at a catechol moiety in meta or orthofashion, the formation of a catechol moiety is a criti-cal step in the degradation pathways of aromaticcompounds. On the other hand, the elucidation ofthe bacterial degradation pathways of dioxin-relatedcompounds found a new type of oxidative attackwith high regioselectivity and speciˆcity for the angu-lar position (Fig. 2(B)). In this dioxygenation, onecarbon that is bonded to the oxygen atom in DD andDF, and its adjacent carbon in the aromatic ring, areboth oxidized. This reaction, termed angular dioxy-genation, is catalyzed by the Rieske nonheme ironoxygenases, which are termed angular dioxygenases.

The angular dioxygenation has been found to be

involved in the degradation pathways for dioxins (DFand DD), and its structural analogues, ‰uorene (FN),carbazole (CAR), diphenyl ether and its derivatives,and dibenzothiophene. Other biodegradation path-ways for these compounds via lateral dioxygenationor monooxygenation has been well investigated andreviewed.7–10) Diphenyl ether and dibenzothiophenedegradation pathways containing angular dioxygena-tion have not been thoroughly described, but theFN- and CAR-degradation systems function in thedegradation of dioxins. As summarized below, thedegradation pathways of FN and CAR are similar tothose of DF and DD, and the FN- and CAR-degrad-ing bacteria can decompose dioxins through an iden-tical pathway by DF (DD)-mineralizing bacteria.Among the degradation steps of dioxins, angular di-oxygenation is the most important, because this sin-gle-step oxygenation destroys the planar structure ofdioxin from which the dioxin toxicity derives.

In this paper, to outline the progress in thebiochemical and molecular studies on the bacterialdegradation of dioxins, we describe the degradationpathway of dioxin and related compounds via angu-lar dioxygenation, and the features of their degrada-tive genes and enzymes.

Degradation of the Dioxins, FN and CARvia Angular Dioxygenation

(1) Microbial degradation of DF and DDOver the last decade, as listed in Table 1, a number

of bacteria capable of mineralizing DF, DD, or bothhave been isolated by enrichment culture methodsusing a carbon-free mineral medium with DF or DDadded as the sole source of carbon and energy. For-tnagel et al.16) and Engesser et al.11) established thatinitial angular attack could occur on DF at the 4 and4a carbon atoms (Fig. 3). This angular dioxygenationfor DF produces highly unstable hemiacetalproducts, which have not been observed directly. Acompound analogous to DF, 9-‰uorenone, was incu-bated with DF-grown bacteria, and the chemicalstructure of a dead-end product accumulated in thereaction mixture was identiˆed. Based on the massspectrum and nuclear magnetic resonance analyticaldata, the resultant product was identiˆed as 1-hydro-1,1a-dihydroxy‰uoren-9-one11,16,22) (the chemicalstructure is shown in Fig. 3). Thus, formation ofhemiacetals from DFWDD was inferred.

Although, in early works using various DF-degrad-ing bacteria, the formation of a trihydroxyl com-pound was not observed in the culture supernatant,Fortnagel et al.17) reported that a UV-derived mutantstrain (designated HH69-II), which could no longergrow with DF as the carbon source, accumulates2,2?,3-trihydroxybiphenyl as a metabolic intermedi-ate of DF (Fig. 3). From the culture supernatant of

2003

Table 1. Aerobic Bacteria Able to Degrade or Co-Oxidize Dioxins, 9-Fluorenone, or CAR via Angular Dioxygenation

Bacteria Initial dioxygenase Substrate a Reference

Brevibacterium sp. strain DPO220 DF 4,4a-dioxygenase DF, FN Engesser et al.,11) Strubel et al.12)

Terrabacter sp. strain DPO1361 DF 4,4a-dioxygenase DF, FN Engesser et al.,11) Strubel et al.,12,13) Trenzet al.,14) Schmid et al.15)

Terrabacter sp. strain DPO360 DF 4,4a-dioxygenase DF Schmid et al.15)

Sphingomonas sp. strain HH69 DF 4,4a-dioxygenase DF, DD Fortnagel et al.,16,17) Harms et al.,18–20)

Schreiner et al.21)

S. wittichii strain RW1 DF 4,4a-dioxygenase(Dioxin dioxygenase)

DF, DD,9-Fluorenone

Wittich et al.,22) B äunz et al.,23) B äunz andCook,24) Happe et al.,25) Bertini et al.,26)

Wilkes et al.,27) Megharaj et al.,28) Arfmannet al.,29) Aermengaud and Timmis,30,31)

Armengaud et al.,32,33,36) Halden et al.,34)

Keim et al.,35) Yabuuchi et al.37)

Terrabacter sp. strain DBF63 DF 4,4a-dioxygenase DF, FN Monna et al.,38) Kasuga et al.,39,40) Habeet al.,41,42) Nojiri et al.43)

Sphingomonas sp. strain HH19k DF 4,4a-dioxygenase DF Harms and Zehnder44,45)

Sphingomonas sp. strain RW16 DF 4,4a-dioxygenase DF Wittich et al.46)

Pseudomonas sp. strain F274 9-Fluorenone 1,1a-dioxygenase FN, DF Selifonov et al.,47) Grifoll et al.48,49)

P. mendocina strain MC2 9-Fluorenone 1,1a-dioxygenase 9-Fluorenone Casellas et al.50)

Ralstonia sp. strain RJGII.123 CAR 1,9a-dioxygenase CAR Grosser et al.,51) Schneider et al.52)

Pseudomonas sp. strain CA06 CAR 1,9a-dioxygenase CAR Ouchiyama et al.53)

P. resinovorans strain CA10 CAR 1,9a-dioxygenase CAR, DF, DD Ouchiyama et al.,53) Kimura et al.,54) Satoet al.,55,56) Nojiri et al.,57,58,62) Nam et al.,59)

Habe et al.,41,60) Widada et al.61)

P. stutzeri strain ATCC 31258 CAR 1,9a-dioxygenase CAR Hisatsuka and Sato63)

Pseudomonas sp. strain LD2 CAR 1,9a-dioxygenase CAR Gieg et al.64)

P. stutzeri strain OM1 CAR 1,9a-dioxygenase CAR Ouchiyama et al.65)

Sphingomonas sp. strain CDH-7 CAR 1,9a-dioxygenase CAR Kirimura et al.66)

Burkholderia sp. strain JB1 Not identiêd 2-chlorinated DF,2-chlorinated DDb

Parsons et al.67)

Burkholderia sp. strain LB400 Biphenyl 2,3-dioxygenase DF, DD Seeger et al.68)

P. pseudoalcaligenes strain KF707 Biphenyl 2,3-dioxygenasec DF, DD Suenaga et al.69)

a Only the parental compounds which were found to be metabolized via angular dioxygenation are shown, if not otherwise noted.b Angular attack was proposed by the transformation activities of 2-chlorinated DD and DF to 4-chlorocatechol and 5-chlorosalicylic acid, respectively.c The large (a) subunit of the terminal oxygenase component was mutated by random priming recombination.

2003Involvement of Angular Dioxygenation in Dioxin Degradation

DF-using bacteria, salicylic acid was identiêd.12,17,38)

This indicates that 2,2?,3-trihydroxybiphenyl is metacleaved, and the resultant meta cleavage compound ishydrolyzed to salicylic acid and 2-hydroxypenta-2,4-dienoic acid.13,23) In the DF-degradation pathwayreported in Pseudomonas sp. strain HH69,17) whichwas reassigned to the genus Sphingomonas,20) S.wittichii strain RW1,22) and Staphylococcus auricu-lans strain DBF63,38) which was reassigned to thegenus Terrabacter,39) the metabolic intermediate,salicylic acid, was branched into catechol and gentisicacid degradation pathways.

Initial angular dioxygenation for DD followed byspontaneous ring cleavage resulted in the formationof 2,2?,3-trihydroxydiphenyl ether (Fig. 3).22)

Catechol has also been identiêd by GC-MS analysisas a metabolite of DD. Because the meta cleavageactivity for 2,3-dihydroxydiphenyl ether could be de-tected, Wittich and coworkers proposed that catecholwas formed from 2,2?,3-trihydroxydiphenyl ethervia meta cleavage and subsequent hydrolysis.22)

However, the possible product formed by the hydrol-ysis, 2-hydroxymuconic acid, has not been identiêdas a metabolite of DD. Pfeifer et al.70) reported that

the meta cleavage of 2,3-dihydroxydiphenyl ether by2,3-dihydroxybiphenyl 1,2-dioxygenase leads to theformation of phenol and 2-pyrone-6-carboxylate asproducts of ring ˆssion and ether cleavage withoutparticipation of free intermediates. Thus, it is likelythat only a meta cleavage enzyme is necessary for theproduction of catechol from 2,2?,3-trihydroxy-diphenyl ether as shown in Fig. 3. Similar to thecatechol metabolism in the DF degradation pathway,catechol formed from DD was mineralized throughmeta, ortho, or both cleavage pathways.22)

(2) Microbial degradation of FNFN is an abundant constituent of coal tar and creo-

sote, and one of the simplest polycyclic aromatichydrocarbons (PAHs). FN is one of the 16 PAHs inthe list of priority pollutants compiled by the U.S.Environmental Protection Agency.71) FN-usingbacteria, Pseudomonas sp. strain F274,48) and 9-‰uorenone-using bacteria, P. mendocina strainMC2,50) were isolated from soil severely contaminat-ed with creosote and PAHs, respectively. Several DF-using strains can also grow on FN.14,38)

Based on the identiˆcation of 9-‰uorenol and

2004

Fig. 3. Overview of Bacterial Degradative Pathways for Dibenzofuran (DF), Dibenzo-p-dioxin (DD), Fluorene (FN), and Carbazole(CAR) via Angular Dioxygenation.

The structures shown in brackets have not been characterized. The red, blue, and green arrows show angular dioxygenation, metacleavage, and hydrolysis, respectively. The production of catechol and 2-pyron-6-carboxylic acid from the meta cleavage compound of2,2?,3-trihydroxydiphenyl ether in the DD degradation pathway was proposed by the results reported by Pfeifer et al.70) 2-Pyrone-6-car-boxylic acid is a by-product. The arrows with solid and broken lines indicate the enzymatic and spontaneous conversions, respectively.


9-‰uorenone as metabolic intermediates of FN,monooxygenation at the C9 position to give 9-‰uorenol followed by the dehydrogenation to 9-‰uorenone was shown to be the initial conversionbefore angular dioxygenation of the aromatic ring(Fig. 3).14,38,48) Because 1-hydro-1,1a-dihydroxy-9-‰uorenone was identiˆed as the degradation productof FN or 9-‰uorenone, angular dioxygenation hasbeen established as the crucial step in FN degradation(Fig. 3).11,47,50) Unlike the hemiacetal intermediatesproduced by angular dioxygenation of DF and DD,the angular dioxygenation product of 9-‰uorenone ischemically stable. Dehydrogenation of the 1-sec-hydroxyl group of 1-hydro-1,1a-dihydroxy-9-‰uore-none would yield a b-diketone-type compound,hydrolysis of which at the C9–C1a bond would give2?-carboxy-2,3-dihydroxybiphenyl. 8-Hydroxy-3,4-benzocoumarin was identiˆed as a metabolite ofFN,48,50) but this compound is in fact the d-lactone of

2?-carboxy-2,3-dihydroxybiphenyl and could beformed as a result of a reversible dehydration reac-tion. 2?-Carboxy-2,3-dihydroxybiphenyl was metacleaved at the C1–C2 bond, and subsequent hydroly-sis resulted in the formation of phthalic acid and 2-hydroxypenta-2,4-dienoic acid, as shown in Fig. 3.48)

Phthalic acid is further degraded by Pseudomonassp. strain F274 via protocatechuic acid.48)

(3) Microbial degradation of CARCAR is a nitrogen heterocycle released into the en-

vironment with fossil fuels or their products such ascreosote.72) CAR is used as a chemical feedstock forthe production of dyes, medicines, and plastics, andis known to possess mutagenic and toxic activities73)

and also to be a recalcitrant molecule. As shown inTable 1, several CAR-using bacteria were isolated byenrichment culture, with CAR as a sole source of car-bon and energy, or of carbon, nitrogen, and energy.


Ouchiyama et al.53) isolated Pseudomonas sp.strain CA06 and P. resinovorans strain CA10 fromsoil and activated sludge samples, respectively, whichuse CAR as the sole source of carbon, nitrogen, andenergy. Anthranilic acid and catechol were identiêdas the main metabolites of CAR by HPLC and GC-MS. In addition, meta cleavage activity for 2,3-di-hydroxybiphenyl was strongly induced in strainCA10 cell grown with CAR. These results suggestedthat strain CA10 degrades CAR to anthranilic acidthrough angular dioxygenation, meta cleavage, andhydrolysis. This degradation pathway of CAR bystrain CA10 is homologous to that of DF degrada-tion as shown in Fig. 3. From the extract of culturemedium of various CAR-using bacteria, anthranilicacid was identiêd as a main metabolite,52,63–66) sug-gesting that these CAR-using bacteria degrade CARthrough a pathway similar to that of strain CA10.

When anthranilic acid was used for a growth sub-strate by strain CA10, catechol and a small amountof cis,cis-muconic acid were detected.53) These resultsindicate the possibility that anthranilic acid is con-verted to catechol by anthranilate 1,2-dioxygenase,and that the catechol formed is mineralized via orthocleavage by strain CA10. On the other hand, P. stut-zeri strain OM1 was found to metabolize catechol viameta cleavage.65)

Biochemical and Genetic Characterizationof the Dioxin Degradation Pathway

The genes for the meta cleavage enzyme involvedin DF and DD degradations were isolated by shotguncloning with the enzyme activity used to screen forthe positive clone.15,25,39) This method has been usedto clone the aromatic compound degradation genes,because a colony of Escherichia coli cells having themeta cleavage enzyme gene has a bright yellow colorfrom the meta cleavage compound when an ap-propriate substrate having the catechol moiety issprayed on. In addition, angular dioxygenase, metacleavage enzyme, and hydrolase were puriêd fromthe DF-degrading bacteria.15,23,24) The degradativegenes involved in the degradation pathways of easilydegradable aromatic compounds, such as naphtha-lene, biphenyl, toluene, and phenol, often have awell-clustered gene organization. Thus, based on thegenetic information on the meta cleavage enzyme andpuriêd enzyme, attempts to clone the other degrada-tive genes from DF- and DD-degrading bacteria bythe gene-walking method have been done. However,these attempts almost completely failed, because theDF- and DD-degradative genes reported so far aredispersed in the genome and do not constitute a sim-ple gene cluster.32,40,43) This is why the dioxin degrada-tion pathway has not been well characterized. In con-trast, CAR and dioxin degradation pathway of P.

resinovorans strain CA10 has been characterizedreasonably well at the genetic level.55,56,58,62)

(1) Dioxin degradation pathway of S. wittichiistrain RW1

S. wittichii strain RW1 is the most well-character-ized DFWDD-degrading bacterium. Since Wittichet al.22) reported the isolation of the DF and DD-degrader, strain RW1, there have been intensive at-tempts to isolate the enzymes and genes involved inthe DF and DD degradation. Two isofunctional metacleavage compound hydrolases (hydrolase H1 andH2), and respective components of the initial angulardioxygenase system, DF 4,4a-dioxygenase (dioxindioxygenase) were puriêd from strain RW1 cells byCook and coworkers.23,24) The hydrolases H1 and H2were presumed to be monomeric. There was a 50zidentity between the N-terminal amino acid se-quences of the two hydrolases, but no similar proteinsequences were observed by data base search analy-sis.23) The DF 4,4a-dioxygenase system was found toconsist of a terminal oxygenase that has a heter-otetrameric structure (a2 b2) and an electron-transfersystem.24) An atypical electron transfer chain inter-acting with the terminal oxygenase component of thedioxin dioxygenase system, consisting of a 12-kDaputidaredoxin-type [2Fe–2S] ferredoxin and twoisofunctional monomeric ‰avoproteins (ferredoxinreductase A1 and A2), has been puriêd, and thesequences of the N-termini of the polypeptides havebeen analyzed.24) The characteristics of the electrontransport chain indicate that the dioxin dioxygenasesystem of strain RW1 is a class IIA dioxygenase sys-tem according to the classiˆcation scheme of Batieet al.74)

As described above, the dbfB gene encoding2,2?,3-trihydroxybiphenyl 1,2-dioxygenase, the metacleavage enzyme involved in DF degradation, wascloned from the genomic cosmid library of strainRW1.25) Comparison of the amino acid sequence ofDbfB showed signiˆcant similarities to the metacleavage enzyme involved in the biphenylWpolychlori-nated biphenyl degradation pathway. Unlike mostmeta cleavage enzymes, which have an oligomericquaternary structure, the DbfB of strain RW1 is amonomeric protein. 1H NMR analysis of puriêdDbfB showed that the high spin Fe(II) iron present inthe active form of the enzyme is coordinated by atleast two His residues.26)

Using the N-terminal sequence of puriêdputidaredoxin-type [2Fe–2S] ferredoxin and the con-served stretch of only four amino acids at the C-ter-minal of the ferredoxin, Armengaud and Timmis30)

designed a nested-PCR strategy to obtain a speciˆcprobe for the ferredoxin gene. With this probe, theauthors identiêd the ferredoxin gene, fdx1. Fer-redoxin Fdx1 of strain RW1 shares about 40z identi-ty with several [2Fe–2S] ferredoxins involved in elec-

2006

Fig. 4. Genetic Organization of Loci Encompassing the Genes Involved in DF and DD Degradation by S. wittichii Strain RW1.This ˆgure is adapted from Armengaud et al.32,33) The pentagons and triangles in the physical map indicate the size, location, and the

direction of transcription of the ORFs derived from the DNA sequence analysis.


tron transfer to bacterial monooxygenase systems.UV and visible spectrophotometry and electronparamagnetic resonance spectroscopy using thehyperexpressed Fdx1, it was found that Fdx1 con-tains a putidaredoxin-type [2Fe–2S] cluster. Fdx1 isthus related to the group of ferredoxins that typicallydonate electrons to monooxygenases, namedcytochrome P450, and that are an atypical electrontransfer system for the Rieske nonheme ironoxygenase system. The redA2 gene encoding the fer-redoxin reductase A224) was also cloned by PCR.31)

From the nucleotide and deduced amino acid se-quences, as well as from the biochemical data ob-tained from a recombinant form of this reductase, itis evident that this ‰avoprotein is similar to class-Icytochrome P450-type reductase.75) Indeed, the twoproteins of the electron transport system, RedA2 andFdx1, are similar to their counterparts supplying elec-trons to the well-characterized cytochrome P450cam,76)

a monooxygenase from P. putida, which is able tooxidize camphor. The genes encoding the terminaloxygenase component of dioxin dioxygenase werealso isolated by a PCR-based technique.32) ThedxnA1 and dxnA2 cistrons, encoding the large (a)and small (b) subunits of the terminal oxygenasecomponent, were just upstream of the hydrolase genedxnB encoding hydrolase H123) (Fig. 4). In the 4.5 kbupstream region of the dxnA1 gene, the dbfB genewas located in the opposite direction (Fig. 4). Recent-ly, Armengaud et al.33) reported that there are nineadditional genes, dxnC to dxnI, in the downstreamregion of dxnB, constituting the same operon withdxnA1A2B (Fig. 4). DxnDFEGHI were found toform a complete 4-hydroxysalicylic acidWhydroxy-quinol-degradative pathway. A homology searchanalysis found an additional putidaredoxin-type

ferredoxin gene ( fdx3) in this operon (Fig. 4). Fdx3functions as an electron transport protein fromRedA2 to DxnA1A2 in the dioxin dioxygenase sys-tem.36) Thus, S. wittichii degrades DF and DD by theenzymes as shown in the Fig. 5, and the respectiveenzymes was encoded in the physically distinct loci,dxnA1A2Bfdx3, fdx1, redA2, and dbfB in the RW1genome. Aermengaud et al.32) reported that, while theexpression of the dbfB gene was constitutive, that ofdbfA1A2 is modulated according to the availablecarbon source. However, the detailed transcriptionalcontrol of dioxin degradation genes of strain RW1has not been reported.

(2) Dioxin degradation pathway by Terrabactersp. strain DBF63

Two genes encoding meta cleavage enzymes [dbfBand open reading frame K1 (ORFK1)] were clonedfrom Terrabacter sp. strain DBF63 by shotgun clon-ing on the basis of the expressed activity of metacleavage enzymes.39) Based on the comparison of thesubstrate speciˆcity, it was suggested that, at leastDbfB is involved in the DF degradation by strainDBF63. Nucleotide sequence analysis showed dbfBand dbfC cistrons encoding a meta cleavage enzymeand a meta cleavage compound hydrolase, respec-tively.

Recently, Kasuga et al.40) succeeded in cloning thedbfA1 and dbfA2 genes, which encode the large (a)and small (b) subunits of the terminal oxygenasecomponent of the DF 4,4a-dioxygenase system,respectively, from strain DBF63 genome by a PCR-based method. DbfA1 and DbfA2 showed moderatehomology to the respective subunits of other ring-hydroxylating dioxygenases (less than 40z), andsome motifs such as the Fe(II) binding site and the

2007

Fig. 5. DF Degradation by the Enzyme Systems Harbored by S. wittichii Strain RW1, Terrabacter sp. Strain DBF63, or P. resinovoransStrain CA10.

The reactions done by the multi-component ring-hydroxylating dioxygenase (iron-sulfur protein; terminal oxygenase component) andtheir electron transfer system, meta cleavage enzyme, and hydrolase are shown. Chemical designations: I, dibenzofuran (DF); II, 2,2?,3-trihydroxybiphenyl; III, 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid; IV, salicylic acid.


Rieske-type [2Fe–2S] cluster ligands77) were con-served in DbfA1. DF 4,4a-dioxygenase activity wasconˆrmed by the resting cell reaction of E. coli cellscontaining the cloned dbfA1 and dbfA2 genes withthe complementation of nonspeciˆc ferredoxin andferredoxin reductase components of E. coli. It wasalso found that DbfA1A2 can catalyze the angulardioxygenation for 9-‰uorenone (Habe et al.,unpublished results) and 9-hydroxylation for FN.40)

Phylogenetic analysis showed that DbfA1 formed abranch with a recently reported large (a) subunit ofPAH dioxygenase from Gram-positive bacteria.40)

The cistron dbfA1A2 is a part of an about 7.3-kb-long operon consisting of 8 ORFs. Although a geneencoding a putative [3Fe–4S] ferredoxin was ob-served in this operon, the ferredoxin reductase genehas not been identiêd (Habe et al., unpublishedresults). The enzymes involved in the dioxin degrada-tion by strain DBF63 are summarized in Fig. 5.

By the pulsed êld gel electrophoresis andSouthern hybridization, two linear plasmids, pDBF1and pDBF2, about 160 kb and 190 kb long, respec-tively, were detected in strain DBF63, and thedbfA1A2 cistron was shown to be located on thesetwo plasmids.43) On the other hand, dbfBC geneswere located on the chromosome.43) Although theregulator protein and eŠector molecule have notbeen identiêd, reverse transcription-PCR analysisshowed that dbfA1A2 and dbfBC genes are stronglyexpressed in the DF-grown strain DBF63 cells.43)

These results suggest that dbfA1A2 and dbfBC genesare involved in the DF degradation, and that thesedegradative genes are dispersed on the strain DBF63genome.

(3) Degradation pathway for CAR and dioxin ofP. resinovorans strain CA10

The transposon mutagenesis of P. resinovoransstrain CA10 indicated a putative gene cluster in-volved in the CAR degradation including the metacleavage enzyme gene.54) On the basis of these results,we cloned the intact CAR-degrading car gene clusterfrom the wild-type strain CA10 by shotgun cloning,using the meta cleavage activity for 2,3-dihydrox-ybiphenyl.55,56) As a result of the nucleotide sequenceanalysis of the cloned DNA fragment, homologysearch analyses, and the measurment of the enzymeactivities, the genes encoding angular dioxygenasefor CAR (CAR 1,9a-dioxygenase; CarAaAcAd),meta cleavage enzyme (CarBaBb), and meta cleavagecompound hydrolase (CarC) were identiêd, asshown in Fig. 6(A). It was found that the 1,263-bpDNA region containing the entire carAa gene wastandemly duplicated except for one base, and thatthere were only three bases (GGC) between these tan-demly-linked DNA regions.56) To our knowledge,such gene structure has never been observed in otherdegradative gene clusters. The signiˆcance of thisduplication of carAa genes in strain CA10 cells isunknown. Recently, we succeeded in isolating severalCAR- and dioxin-degrading bacteria, some of whichhave a homologous car gene cluster containing a sin-gle copy of the carAa gene (Widada et al., unpub-lished results). Comparison of the CAR- and dioxin-degrading capacity between bacteria having diŠerentcopy numbers of the carAa gene might provide us in-sight on the signiˆcance of this gene duplication. Inaddition, the genes encoding the terminal oxygenasecomponent (CarAa) of CAR 1,9a-dioxygenase areseparated from the genes encoding the ferredoxincomponent (CarAc) and ferredoxin reductase com-

2008

Fig. 6. Physical Maps of the 44,266-base Pair EcoRV-EcoRI DNA Region Containing car and ant Genes55,56,58) (A) and 8,918-base PairEcoRI-NotI DNA Region Containing catRBCA Genes62) (B), That are Involved in the Carbazole (CAR) Degradation by P. resinovo-rans Strain CA10.

The pentagons and triangles in the physical map indicate the size, location, and the direction of transcription of the ORFs derivedfrom the DNA sequence analysis. This ˆgure is adapted from Nojiri et al.58,62) Restriction site designations are: A, ApaI; C, ClaI; EI,EcoRI; EV, EcoRV; H, HindIII; K, KpnI; N, NotI; P, PstI; Sl, Sal I; Xh, XhoI.


ponent (CarAd) by a DNA fragment of about 2-kbencoding carBaBbC (Fig. 6(A)). These ˆrst three en-zymes in CAR degradation have been found to func-tion in dioxin degradation as the counterparts of thecorresponding enzymes in DF (DD)-degrading bac-teria.39,41,56,57) The functions of the Car enzymes ofstrain CA10 in DF degradation may be as shown inFig. 5.

On the basis of the identiˆcation of theoxygenation product after a biotransformation ex-periment, CAR 1,9a-dioxygenase was shown to cata-lyze the angular dioxygenation for CAR, DF, DD,xanthene, and phenoxathiin, but not for 9-‰uore-none or dibenzothiophene,56,57) indicating that the an-gular dioxygenation occurs at the angular positionadjacent to an oxygen or nitrogen atom more prefer-ably than that adjacent to a sulfur or carbon atom.The three components of the CAR 1,9a-dioxygenasesystem have been puriêd (Nam et al., in prepara-tion). Based on gel-ˆltration analyses, CarAa,CarAc, and CarAd were trimer, monomer, andmonomer proteins, respectively. In the puriêd His-tagged CarAd, 0.6 mole FAD per mole of proteinwas detected. UV and visible absorption spec-troscopy indicated that CarAa, CarAc, and CarAdproteins have the Rieske-type,78) Rieske-type,78) andplant-type79) [2Fe–2S] clusters, respectively. Thesefacts are in agreement with the functions suggestedfrom the deduced amino acid sequences.56)

The Rieske nonheme iron oxygenase systems areclassiêd into three classes, classes I, II, and III, interms of the number of constituent components and

the nature of the redox centers.74,80) Further subdivi-sion is based on the type of ‰avin in the reductase (Iaand IB), or the coordination of the [2Fe–2S] clusterin the ferredoxin (IIa and IIb). Based on this classiˆ-cation system, CAR 1,9a-dioxygenase seems to havea class III electron transport chain like a well-knownnaphthalene dioxygenase. On the other hand, Namet al.59) proposed a new classiˆcation scheme for theoxygenase components based on their amino acid se-quence similarity in the a subunits of terminal oxyeg-nase component, because the Batie classiˆcation sys-tem could not cover all of the dioxygenase systems.On the basis of Nam's scheme, terminal oxygenaseswere classiêd into four groups, I–IV. The oxygenasecomponents included by group I in Nam's classiˆca-tion have only the identical subunit, and most of theterminal oxygenase components of class IAoxygenases in the Batie classiˆcation belong to thisgroup I. The terminal oxygenase components of classIB, class II, and class III oxygenase systems in theBatie classiˆcation are mainly classiêd into group II,group IV, and group III, respectively, in Nam's clas-siˆcation system. The phylogenetic study found thatCarAa has a closer relation to the terminal oxygenasecontained by class IA dioxygenase systems, andCarAa was classiêd into group I in Nam's classiˆca-tion.59)

Recently, Schmidt and Shaw81) suggested that theterminal oxygenase and ferredoxin components con-taining a Rieske-motif form a superfamily with oneancestor. To investigate the evolutionary relation-ships of CarAa and CarAc with other Rieske-type

2009

Fig. 7. Phylogenetic Tree of Ferredoxin and Terminal Oxygenase Components of Oxygenase Systems.The scale bar denotes 0.1 substitution per site. The tree was constructed with the Clustal W package. The name of each bacterial strain

and its DDBJWEMBLWGenBank or SWISSPROT accession number are shown after the enzyme name in parentheses. Grouping intoGroups I–IV of oxygenase proteins is based on the scheme of Nam et al.59) This ˆgure is adapted from Nam et al. (in preparation). Theterminal oxygenase (CarAa) and ferredoxin (CarAc) of CAR 1,9a-dioxygenase from P. resinovorans strain CA10 are shaded.


proteins, a phylogenetic tree was established (Fig. 7,Nam et al., in preparation). As shown in Fig. 7, theferredoxins involved in the monooxygenase systemand dioxygenase system constitute the distinctiveclusters, and CarAc was contained in the ferredoxincluster of dioxygenase system. As for the oxygenasecomponents, the distant a‹liations of the oxygenaseswere observed between group I and groups II, III,and IV in Nam's classiˆcation. These phylogenies in-dicate that group I oxygenases are evolutionallydiŠerent from group II, III, and IV oxygenases. Inaddition, among the oxygenases classiˆed into groupI, CarAa of strain CA10 and OxoO of strain 86 werephylogenetically distant from other group Ioxygenases (Fig. 7). On the other hand, the

phylogenetic tree of ferredoxin-NADP+ reductase-type reductases showed that CarAd of strain CA10 ismore closely related to the reductase components ofsome monooxygenase systems, such as toluene 4-monooxygenase, tolueneWo-xylene monooxygenase,and alkene monooxygenase, than those of class IBand III dioxygenase systems (Fig. 8) (Nam et al., inpreparation). The distant phylogenetic a‹liations ofrespective components of CAR 1,9a-dioxygenase in-dicate the possibility that some of these three compo-nents have been recruited from diŠerent oxygenasesystems.

We have analyzed the nucleotide sequence of the27,939-bp-long upstream and 9,448-bp-long down-stream regions of carAaAaBaBbCAc(ORF7)Ad

2010

Fig. 8. Phylogenetic Tree of the Reductase Components of Oxygenase Systems.The scale bar denotes 0.2 substitution per site. The tree was constructed by the neighbor-joining method (Phylip package version

3.573c). The name of each bacterial strain and its DDBJWEMBLWGenBank or SWISSPROT accession number are shown after the en-zyme name in parentheses. Grouping into Classes IB and III is based on the scheme of Batie et al.74) This ˆgure is adapted from Namet al. (in preparation). The ferredoxin reductase (CarAd) of CAR 1,9a-dioxygenase from P. resinovorans strain CA10 is shaded.


genes.58) Thirty-two ORFs were identiˆed, and the cargene cluster was found to consist of ten genes(carAaAaBaBbCAcAdDFE ), as shown in Fig. 6(A),encoding the enzymes for the ˆrst three-step conver-sions of CAR to anthranilic acid and the degradationof 2-hydroxypenta-2,4-dienoic acid. The unusualgene structure and the higher phylogenetic related-ness of CarFE to the enzymes involved in 3-(3-hydroxyphenyl)propionic acid degradation indicatedthat carFE has been recruited from the other locus.The operonic structure and detailed expressionmechanism of car genes have not been reported, butthe strong expression of these genes as several tran-scriptional units was observed in CAR- and an-thranilate-grown strain CA10 cells (Urata et al., un-published results). In the 21-kb upstream region fromcarAa, the genes encoding Rieske nonheme ironoxygenase (ORF26, ORF27, and ORF28) were found(Fig. 6(A)). Inductive expression in CAR- and an-thranilate-grown cells and the results of homologysearching indicate that these genes encode the an-thranilate 1,2-dioxygenase involved in CAR degrada-

tion.58) Therefore, these ORFs were designatedantABC.

Four similar IS5-related insertion sequences (ISs;three copies of ISPre1 and one copy of ISPre2,formerly designated as IS5car1 to IS5car4) wereidentiˆed in neighboring regions of car and ant loci(Fig. 6(A)).58) The nucleotide sequence of ISPre2showed 81.6z identity with that of ISPre1, althoughit was 98z identical with the IS-like sequence con-taining tnpA1 from P. stutzeri strain AN10.82) Com-parison of the nucleotide sequences indicated that theone-ended transposition of ISPre1 together with the5?-portion of antA into the immediate upstream ofcarAa had resulted in the formation of additionalcopies of ISPre1 and ORF9.58) In addition to the IS-dependent recombination, gene duplications (ORFs11 and 12, and two copies of carAa gene) had oc-curred.

In addition, the car gene clusters were found on the199-kb circular plasmid, pCAR1.58) Because catecholformed from CAR is degraded through the orthocleavage pathway encoded by the chromosomal cat


operon (Fig. 6(B)),62) it can be considered that strainCA10 had acquired a metabolic capacity for dioxinand CAR by recruitment of pCAR1. In other bacter-ia having the ability to degrade both dioxin andCAR, we have also detected the pCAR1-like plasmidcontaining the car gene cluster homologue, althoughchromosomally encoded car gene clusters were foundas well (Widada et al., unpublished results). Thisphenomenon suggests that pCAR1 is important inthe distribution of the degradation capacity for diox-in and CAR in the environment. Very recently, theentire nucleotide sequence (199,035 bp) of pCAR1was analyzed (Maeda et al., unpublished results).The presence of the homologous genes to the trh andtra genes involved in the conjugal transfer of theplasmids Rts1 from Proteus vulgaris83) and R27 fromSalmonella strains84) suggested the possibility thatpCAR1 is a self-transmissible plasmid. Interestingly,it was also shown that the entire car and ant loci arecontained in a 73-kb-long transposon, Tn4676. Thetransposase and cointegrate resolution proteins areclosely related to those of the toluene- and xylene-degrading transposon, Tn4651,85) identiˆed from theTOL plasmid pWW0, although the replication andmaintenance systems of pCAR1 has no relationshipto those of pWW0. Because several bacterial strainshave Tn4676 inserted on their chromosome (Shintaniet al., unpublished results), both the plasmid pCAR1and Tn4676 are important in the distribution of thedegrading capacity for dioxin and CAR in the en-vironment.

Microbial Degradation of Chlorinated DFand DD

(1) Aerobic degradation of chlorinated DFs andDDs

Although the nonhalogenated basic structure DFand DD is eŠectively mineralized by appropriate bac-terial strains as mentioned above, their polychlori-nated derivatives are not. The abilities of DF (DD)-or CAR-mineralizing bacteria to oxidize mono- totrichlorinated congeners of DD and DF were ana-lyzed.19,27,35,41) Harms et al.19) reported that a mutantstrain Pseudomonas (Sphingomonas ) sp. strainHH69-II, which is defective in 2,3-dihydrox-ybiphenyl 1,2-dioxygenase, led to the formation ofan equal amount of 4?-chloro-2,2?,3-trihydrox-ybiphenyl and 4-chloro-2,2?,3-trihydroxybiphenylfrom 3-chloro-DF. Wilkes et al.27) demonstrated thatmost mono- and dichlorinated DFs and DDs testedwere degraded to the corresponding mono- anddichlorinated salicylic acids and catechols, respec-tively, together with salicylic acid and catechol.Wittich et al.46) also reported that the conversion of 3-chloro-DF in the presence of an inhibitor of metacleavage by Sphingomonas sp. strain RW16 led to the

accumulation of two metabolites that are likely to be4?-chloro-2,2?,3-trihydroxybiphenyl and 4-chloro-2,2?,3-trihydroxybiphenyl. These results indicatedthat angular dioxygenase from these bacteria attack-ed both the substituted and the nonsubstitutedaromatic nuclei of monochlorinated DFs and DDsnonspeciˆcally. However, in these studies, the con-centrations of the target substrates were from 100to 1,000 ppm. On the other hand, Parsons et al.67)

proposed that the initial dioxygenation took place onthe nonchlorinated ring during the degradation of alow concentration (0.3 ppm) of 2-chloro-DF and 2-chloro-DD by the biphenyl-using Burkholderia sp.strain JB1, since 5-chlorosalicylic acid and 4-chlo-rocatechol as well as only one isomer each of chlori-nated 2,2?,3-trihydroxybiphenyl and 2,2?,3-tri-hydroxydiphenyl ether were detected in respectivebiotransformation experiments. In our experimentson dioxin degradability by the CAR 1,9a-dioxy-genase from strain CA10 and the DF 4,4a-dioxyeg-nase from strain DBF63, we found that these two an-gular dioxygenases act mainly on the nonsubstitutedaromatic nucleus of chlorinated DFs and DDs(10 ppm), although both enzymes were able to act onthe chlorine-substituted aromatic rings.41) Wilkeset al.27) also suggested that at least the initial conver-sion rates for the chlorinated DFs and DDs decreasedwith increasing chlorine substitution. In addition,based on a biotransformation experiment by usingparental bacterial strain, strains CA10 and DBF63,and E. coli transformants expressing the metacleavage enzyme and hydrolase in addition to the ini-tial angular dioxygenase, Habe et al.41) suggested thatthe in‰uence of the chlorine substitution pattern ofmono- to trichlorinated DFs and DDs on the forma-tion of corresponding chlorocatechol and chlorosali-cylic acid may not only depend on the substratespeciˆcity of the initial angular dioxygenases but alsoon those of subsequent acting enzymes, especiallyhydrolase. Two DF (DD)-degrading bacteria, S. wit-tichii strain RW1 and Sphingomonas sp. strainRW16, can transform monochlorinated DF by angu-lar dioxygenation, meta cleavage, and hydrolysis, butthe resultant products, corresponding monochlori-nated salicylic acids, were accumulated in the cul-ture.27,29,46) Wittich and co-workers reported the com-plete mineralization of monochlorinated DF by theconsortiums consisting of a DF-degrader and a chlo-rosalicylic acid-degrading bacteria.29,46)

An extensive study of the in‰uence of the substitut-ed pattern on the depletion of many of the 210 con-geners of polychlorinated DFs and DDs by severalbacterial strains was reported by Schreiner et al.21) Inthe study, the DF-degrader Sphingomonas (formerlyPseudomonas17)) sp. strain HH69 degraded 31zof 2,3,7,8-tetrachloro-DF and 15z of 2,3,7,8-tetrachloro-DD within 84 days. Surprisingly, 1,2,4,5-tetrachlorobenzene-mineralizing Burkholderia


(formerly Pseudomonas86)) sp. strain PS12 degraded64z of 2,3,7,8-tetrachloro-DF in the same time, and100z of 2,3,7,8-tetrachloro-DD within 25 days.Chlorine substitution in the 1-, 4-, 6-, or 9-positiongenerally retarded the biodegradation.21)

(2) Bacterial biodegradation of dioxins in soilsand sediments

Polychlorinated DDsWDFs are often bound to soilorganic matter and surfaces of particles. The reducedbioavailability of halogenated DDWDF, therefore, isof interest and was examined by Harms andZehnder.44,45) Megharaj et al.28) reported that cultur-ing the S. wittichii strain RW1 ˆrst in soil extractmedium prolonged the survival of this strain in soil.Halden et al.34) reported the removal of DF, DD, and2-chloro-DD (10 ppm each) from a soil microcosm bythe addition of strain RW1. The density of strainRW1 cells and the contents of soil organic matter in-‰uenced the rate and extent of removal. Recently, wetried experimentally to bioremediate an actual diox-in-contaminated soil that was contaminated mainlyby tetra- to octachlorinated dioxins, by using the soilslurry system and P. resinovorans strain CA10cells.60) The soil slurry (ratio of soil:water, 1:5,wtWvol) prepared with dioxin-contaminated soil col-lected at an incinerator site was incubated with CAR-grown strain CA10 cells. During the 7-day incuba-tion, the total amount of chlorinated DD and DFcongeners and toxicity equivalency quantity (TEQ)decreased from 725 to 665 ngWg soil and 11 to 9.4 ngTEQWg soil, respectively, by a single inoculation ofthe CAR-grown strain CA10 cells (1011 CFUWg drysoil). Although the degradation rate of total dioxinwas 8.3z, strain CA10 had a potential to transformtetra- to heptachlorinated congeners including themost toxic compound, 2,3,7,8-tetrachlorinated DD.With a similar slurry system, a preliminary bio-remediation experiment with Terrabacter sp. strainDBF63 cells was done.42) From this experiment, wedetected approximately 10z depletion of tetra- tohexachlorinated congeners by 7 days of incubationwith DF-grown strain DBF63 cells (108 CFUWg drysoil).

Conclusions and Prospects

Because angular dioxygenation for dioxin resultsin the spontaneous cleavage of the three-ring struc-ture, the single-step oxidation destroys the planarstructure from which the toxicity of dioxin originat-ed. This fact indicates the primary importance of ini-tial angular dioxygenation in decontamination of di-oxin. Recently, biphenyl 2,3-dioxygenase, the initialdioxygenase in the biphenyl degradation pathway byBurkholderia sp. strain LB400, has been reported tocatalyze the angular dioxygenation for DD and DF.68)

Suenaga et al.69) also reported that the biphenyl 2,3-

dioxygenase with point mutation(s) introduced of P.pseudoalcaligenes strain KF707 catalyzed the angulardioxygenation for DD and DF. These results indicatethat this novel dioxygenation is one of the reactionsoriginating from the ``relaxed'' substrate speciˆcityof the Rieske nonheme iron oxygenase super family.Recently, the three-dimensional structure of the ter-minal oxygenase component of naphthalene 1,2-di-oxygenase has been reported.77,87) The structure of theangular dioxygenase will provide useful informationon the ``relaxed'' substrate recognition and activa-tion of the reaction.

On the basis of biochemical and genetic informa-tion, the extension of the substrate range of dioxin-degrading bacteria, higher expression of dioxindegrading enzyme(s), and the construction of thehybrid metabolic pathway allowing the improvedmineralizing capacity for dioxins will probably beachieved in the near future. Although, to optimizethe biodegradation conditions, a laboratory scale-microcosm study, a feasibility study with a pilotplant, and a ˆeld trial on a long-term basis are stillnecessary, bacteria harboring the dioxin-degradingcapacity are promising for the bioremediation of di-oxin contamination.

The well-clustered genetic organization of entirepathways or independently functioning pathway seg-ments developed through exposure to correspondingcompounds for a long time. On the other hand, thedegradative genes involved in the degradation ofhigher recalcitrant compounds seemed to be disor-dered or separated. In fact, dioxin-degrading geneswere separated from each other on the genomes ofS. wittichii strain RW132) and Terrabacter sp. strainDBF63.43) Dioxin- and CAR-degrading car genes ofP. resinovorans strain CA10 are disordered and havea novel mosaic structure.58) Perhaps the diŠerentlevels of maturation in genetic structures resultedfrom the diŠerences in the length of exposure toxenobiotics or rarity of the gene(s) indispensable fordegradation. That is, the genetic analysis of thedegradative genes for dioxin and CAR will provideuseful information on the maturation (evolution) ofxenobiotic-degrading gene clusters.

Acknowledgments

A part of this work was supported by the Programfor Promotion of Basic Research Activities for In-novative Biosciences (PROBRAIN) in Japan.

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