Bacterial Dehalogenases: Biochemistry, Genetics

MICROBIOLOGIcAL REVIEWS, Dec. 1994, p. 641-685 Vol. 58, No. 4 0146-0749/94/$04.00+0 Copyright X 1994, American Society for Microbiology Bacterial Dehalogenases: Biochemistry, Genetics, and Biotechnological Applications SUSANNE FETZNER* AND FRANZ LINGENS Institut fiir Mikrobiologie der Universitat Hohenheim, D-70593 Stuttgart, Germany INTRODUCTION ...... 641 DEHALOGENATION MECHANISMS ...... 642 "SPONTANEOUS" OR "FORTUITOUS" DEHALOGENATION REACTIONS...... 643 Dehalogenation after Cleavage of an Aromatic Ring...... 643 Oxygenases...... 644 Laccases, Tyrosinases, and Peroxidases...... 646 Laccases ...... 646 Tyrosinases ...... 646 Peroxidases ...... 646 Hydratases ...... 646 REDUCTIVE DEHALOGENATION...... 647 Haloaromatic Compounds and Some Pesticides...... 647 Haloaliphatic Compounds...... 649 Catalysis by Transition Metal Cofactors (Porphyrins and Corrins) ...... 650 Energy Conservation Coupled to Reductive Dechlorination: Desulfomonile tiedjei DCB-1...... 651 HALOAROMATIC DEHALOGENASES...... 652 Oxygenases...... DEHALOGENATION 652 MonooxygenasesREDUCTIVE ...... ~~~~~~...... 652 Dioxygenases...... 653 Halidohydrolases...... 654 HALOALKANE DEHALOGENASES ...... 656 Oxygenases...... 656 Glutathione S-Transferases...... 656 Haloalkane Halidohydrolases...... 657 Dehydrohalogenases ...... 659 HALOHYDRIN DEHALOGENASES...... 660 HALOACID: _%VsXT A T*'JIVk 1 .60-A

ialoacetate alidonyh rrolases ...... 6...... 6...... 0...... 660 2-Haloalkanoic Acid Halidohydrolases...... 662 Group 1...... 662

Group 2 ...... 662 Group 3.662 Group 4 ...... 663 Group 5.663 3-Haloacyl-CoA Halidohydrolases.664

BIOTECHNOLOGICAL APPLICATIONS OF BACTERIAL DEHALOGENASES...... ooo...... o ...... 664 Microbial Dehalogenases as Industrial Biocatalysts...... 665 Application of Microbial Systems in Environmental Protection Technology and Waste Management...... 665 Bioaugmentation...... 666 (i) Addition of nutrients and/or substrate analogs...... 666...... 666 (ii) Use of microbial inocula...... 66'6 1 ACAC~~~~~t (iii) Bioaugmentation with genetically engineered microorganisms ...... D.. Bioreactors......

ACKNOWLEDGMENTS ...... o... RPF.FliRF.1RNr..

INTRODUCTION hydraulic and heat transfer fluids, plasticizers, and intermedi- Halogenated organic compounds constitute one of the larg- ates for chemical syntheses. Because of their toxicity, biocon- est groups of environmental pollutants as a result of their centration, and persistence, the ubiquitous distribution of widespread use as herbicides, insecticides, fungicides, solvents, halogenated compounds in the biosphere has caused public concern over the possible effects on the quality of life. Concerning toxicity, an important problem is the diversity of * Corresponding author. Mailing address: Institut fur Mikrobiologie toxic compounds and by-products in many formulations of der Universitat Hohenheim (250), D-70593 Stuttgart, Germany. industrial products. This problem applies to mixtures of poly- Phone: 49 711 459 2223. Fax: 49 711 459 2238. chlorinated biphenyls (PCBs) or chlorinated terpenes, poly- 641 642 FETZNER AND LINGENS MICROBIOL. REV.

chloroterphenyls, and polychloroquaterphenyls, formulations negativity of the substituent, and halogenated substances with that may contain up to 105 different isomers, and it is also one or few substituents are thought to be more readily related to many commercially available pesticides. The insec- degradable than the corresponding polyhalogenated com- ticide toxaphene, for instance, is a very complex mixture of pounds. The study of the biochemistry and genetics of micro- more than 177 polychlorinated derivatives. Most of its compo- bial dehalogenases may help us to understand and evaluate the nents are probably isomeric hepta-, octa-, and nonachlorobor- potential for degradation ofxenobiotics in microorganisms and nanes (252). in natural microcosms. Such insight might provide biotechno- However, our contribution to the pool of halogenated logical solutions to deal with environmental problems: in order substances, although having a major impact on the environ- to design effective bioremediation systems, all known param- ment, should be seen against the background of natural abiotic eters influencing biodegradation processes must be taken into (485) and biotic (83, 84, 113, 135, 163, 255, 303, 385,455, 560) consideration. In some cases, it might prove useful to construct production. Volatile halogenated organic compounds are re- bacterial strains with more efficient or even novel catabolic leased into seawater by temperate marine macroalgae at rates activities for the mineralization of environmental pollutants of nanograms to micrograms of each compound per gram of (64, 498). Such genetically engineered microorganisms, de- dry algae per day. Di- and tribromomethane, dibromochlo- signed specifically for controlled, regulated expression of romethane, and several iodo- and bromo-substituted alkyl- whole catabolic pathways, might be used for the treatment of monohalides and alkyldihalides are the predominant volatile specified (industrial) effluents or exhaust gases or might be halogenated organic products of the marine environment. It is suitable for the decontamination of polluted soil or groundwa- very possible that the macroalgae are a major source of volatile ter. However, a case-by-case evaluation of a constructed strain organobromines released into the atmosphere (163). However, in the specific system to which it will be applied in the field is methyl halides, which are the most abundant halohydrocarbon indispensable, since environmental factors might modulate the species in the upper atmosphere, are synthesized not only by biodegradation potential of the strain (453). In addition to marine macroalgae and phytoplankton but also by various these degradative aspects, dehalogenases can be used as wood rot fungi and by the "ice plant," a terrestrial succulent industrial biocatalysts to produce valuable intermediates for growing in saline environments. It has been suggested that the chemical syntheses. Biotransformations of organic compounds methyltransferase catalyzing the synthesis of methyl halides with microbial or enzymic biocatalysts offer new routes for the through an S-adenosylmethionine transfer mechanism is a synthesis of intermediates and novel products, since these constitutive activity in a broad variety of marine algae and biocatalysts possess regiospecificities and chiral specificities white rot fungi (560). Apart from the abundant alkylhalides, that are difficult and expensive to achieve by conventional haloaromatics such as 2,4-dibromophenol were reported to chemistry. occur naturally in some marine sediments. They are produced There are a number of recent review articles dealing with from some invertebrates and are believed to possess antimi- various aspects of microbial dehalogenation, such as degrada- crobial function (255). tion of haloaromatic or haloaliphatic compounds, pesticide Many different genera of wood-rotting fungi produce large degradation, anaerobic and reductive processes, dehalogena- amounts (about 75 mg/kg of wood) of chlorinated anisyl tion mechanisms, and dehalogenation reactions mediated by metabolites in their natural environments. These chloroanisyl distinct classes of organisms (e.g., actinomycetes) (1, 65, 70, derivative-producing fungi are widespread in nature, and a 116, 167, 181, 187, 227, 230, 287, 335, 362, 401, 522, 538, 542, ubiquitous production of chloroanisyl metabolites under nat- 554). This review is a survey of bacterial dehalogenases that ural conditions was proposed by de Jong et al. (84). Chloro- catalyze the cleavage of halogen substituents from haloaromat- anisyl alcohols were shown to be excellent substrates for the ics, haloalkanes, haloalcohols, and haloacids. Biotechnological extracellular aryl alcoholoxidase, generating H202 for the applications of halohydrocarbon-degrading microorganisms, lignin-degrading peroxidases. Thus, chlorinated anisyl alco- especially with respect to bioremediation processes, will also hols, aside from a possible antimicrobial activity, seen to fulfil be discussed. However, because of the abundance of papers on an important physiological function in the fungal ligninolytic dehalogenation and bioremediation, this survey can by no system (83). means be complete. More than 130 chlorine-containing compounds have been isolated from higher plants and ferns. About half of these DEHALOGENATION substances are halogenated polyacetylenes, thiophenes, and MECHANISMS sesquiterpene lactones from members of the family Aster- The carbon-halogen bond is cleaved either by enzymatic aceae. Many compounds are chlorohydrins, which are isolated dehalogenation, where the carbon-halogen bond cleavage is along with the related epoxides (113). In 1973, Siuda and catalyzed by specific enzymes (the dehalogenases), or by DeBernardis (455) reported the structures of more than 200 spontaneous chemical dehalogenation of unstable intermedi- naturally occurring halogenated compounds. By 1986, more ates. In addition, there are enzymes which, because of their than 700 halogenated natural products were identified (361), relaxed substrate specificity, catalyze the conversion of halo- and the list probably is far from complete. Thus, halogenated genated analogs of the corresponding unsubstituted substrate compounds are not uncommon in nature and might well be or of related compounds, which might lead to "fortuitous" important in the adaptation of microorganisms to utilize dehalogenation of the substrate analog. halogenated xenobiotics. Reports on the biodegradation of Concerning the enzymatic cleavage of the carbon-halogen compounds like pentachlorophenol or 2,4,5-trichlorophenoxy- bond, seven mechanisms of dehalogenation are known so far: acetate, formerly classified as recalcitrant, are leading to a (i) Reductive dehalogenation (Fig. la). In the course of a reevaluation of the potential for adaptation of microorganisms reductive dehalogenation, the halogen substituent is replaced to transform or even mineralize such substances. by hydrogen. (ii) Oxygenolytic dehalogenation (Fig. lb). Oxy- The biological recalcitrance of halogenated compounds is genolytic dehalogenation reactions are catalyzed by monooxy- related to the number, type, and position of the halogen genases (or dioxygenases), which incorporate one (or two) substituents. As a general rule, the carbon-halogen bond often atoms of molecular oxygen into the substrate. (iii) Hydrolytic is regarded as increasingly recalcitrant with increased electro- dehalogenation (Fig. lc). In the course of hydrolytic dehalo- VOL. 58, 1994 BACTERIAL DEHALOGENASES 643

t enzyme is a dehalogenase may be a matter of discussion in CcC-'C ' XH- H CC,C Cl-eX CK ' l cC' "la some cases (see the sections on hydratases, and oxygenases, below).

b COOH Dehalogenation after Cleavage of an Aromatic Ring I H _ N+OH + elimination of is an 02 +H + NAD+ +HCL Spontaneous halogen substituents im- ,NADH K+ +C02 portant step in the degradation of various haloaromatic com- c pounds. Many pathways, like the well-studied degradation R-CH2Cl + HW - R-CH20H H%! pathways of 3-chlorobenzoate and 2,4-dichlorophenoxyacetate (2,4-D) converge at a point with chlorocatechols as central d intermediates (for a review, see reference 401). Further exam- CN2Cl * GSH - IGS-CH2II + HCI are the pathways of mono-, di-, tri-, and * ples degradation (GS-CICL I H20 - GS-CH20H * HCI tetrachlorobenzenes 461, GS-CHKH. (C2 * SH (81, 175, 400, 414, 441, 523-525); dichloroaniline (576); some chlorophenols (259); and various mono-, di-, and trihalogenated benzoic acids (110, 186, 202, e 204, 321, 429), which may also proceed via chlorocatechol CH2OH-CHOH-CH2CI - " A * HCI CH"OH- intermediates. Figure 2 illustrates the degradation pathway of 3-chlorobenzoate by Pseudomonas sp. strain B13 (428), often referred to as the modified ortho pathway. Ring cleavage of chlorocatechols is catechol 1,2-dioxygenase II. a> catalyzed by * Ha Compared with catechol 1,2-dioxygenaseI, the type II enzyme Cl Cl Cl a a exhibits a relaxed substrate specificity and high activity with chlorocatechols (99, 100, 322). The products from ortho cleavage of 3- and 4-chlorocatechol are 2- and 3-chloro-cis,cis- muconate, respectively. Muconate cycloisomerase II is proposed to generate 4-carboxychloromethylbut-2-en-4-olide HOOC-CH-CHCt + IOOC-CHfCHOHLCI I from 2-chloro-cis,cis-muconate and 4-chloro-4-carboxymethyl- but-2-en-4-olide from 3-chloro-cis,cis-muconate. These intermediates spontaneously eliminate HCl, yielding the trans and HOOC-CHt-CHO * Ha cis isomers of 4-carboxymethylenebut-2-en-4-olide, respec- FIG. 1. Dehalogenation mechanisms. (a) Reductive dehalogena- tively. Both diene-lactones are substrates for dienelactone tion; (b) oxygenolytic dehalogenation; (c) hydrolytic dehalogenation; hydrolase II, which catalyzes their conversion to maleylacetate (d) "thiolytic" dehalogenation; (e) intramolecular substitution; (f) (278, 401, 426-428, 430). dehydrohalogenation; (g) hydration. The release of one chlorine atom from tri-, di-, or mono- chlorocatechol generally takes place concomitantly with lactonization of the respective chlorinated cis,cis-muconic acid, genation reactions, catalyzed by halidohydrolases, the halogen catalyzed by cycloisomerase II, yielding di- or monochlorinated substituent is replaced in a nucleophilic substitution reaction or unsubstituted maleylacetic acid, respectively (81, 175, 400, by a hydroxy group which is derived from water. (iv) "Thio- 415, 441, 461, 497, 523-525). In contrast to 3-chloro-cis,cis- lytic" dehalogenation (Fig. ld). In dichloromethane-utilizing muconate, which is dehalogenated in the course of cyclo- bacteria, a dehalogenating glutathione S-transferase catalyzes isomerization, 3-fluoro-cis,cis-muconate was not dehaloge- the formation of a S-chloromethyl glutathione conjugate, with nated during or after the cycloisomerase-catalyzed a concomitant dechlorination taking place. (v) Intramolecular lactonization. Instead, 4-fluoromuconolactone was formed by substitution (Fig. le). Intramolecular nucleophilic displace- the muconate cycloisomerases from Alcaligenes eutrophus 335, ment yielding epoxides is a mechanism involved in the deha- A. eutrophus JMP134, and Pseudomonas cepacia. However, logenation of vicinal haloalcohols. (vi) Dehydrohalogenation both dienelactone hydrolase and 3-oxoadipate-enollactone hy- (Fig. lf). In dehydrohalogenation, HCI is eliminated from the drolase were capable to catalyze the conversion of 4-fluoro- molecule, leading to the formation of a double bond. (vii) muconolactone to maleylacetate (426). Hydration (Fig. lg). A hydratase-catalyzed addition of a water In the case of ortho cleavage of di- or trihalogenated molecule to an unsaturated bond can yield dehalogenation of catechols, chloromaleylacetic acids are formed by dienelactone vinylic compounds, such as 3-chloroacrylic acid, by chemical hydrolase II. For instance, 2-chloromaleylacetate is formed decomposition of an unstable intermediate. from 2,4-D, 1,4-dichlorobenzene, 3,5-dichlorobenzoate, or 3,5- dichlorosalicylate. Duxbury et al. (101) detected the enzymatic conversion of chloromaleylacetate to succinate by enzymes of "SPONTANEOUS" OR "FORTUITOUS" an Arthrobacter strain grown on 2,4-D. They proposed a path- DEHALOGENATION REACTIONS way for the conversion of chloromaleylacetate via 2-chloro-4- Spontaneous dehalogenation reactions may occur as a result ketoadipate and chlorosuccinate (101). The same pathway was of chemical decomposition of unstable primary products of an proposed for the 2,4-D-degrading strain Pseudomonas sp. unassociated enzyme reaction. In addition, fortuitous dehalo- strain NCIB 9340 (117), and degradation of 2-chloromaleyl- genation reactions can result from the action of broad-speci- acetate via 2-chlorosuccinate was also proposed for the catab- ficity enzymes, which convert halogenated analogs of their olism of 3,5-dichlorosalicylate by Pseudomonas sp. strain JWS natural substrates. However, there are probably various tran- (425). Whereas Duxbury et al. (101) and Evans et al. (117) sitions, ranging from fortuitous dehalogenation by an enzyme proposed degradation of 2-chloromaleylacetate via 2-chloro-4- with relaxed substrate specificity to a specific dehalogenase- oxoadipate to 2-chlorosuccinate, Chapman (63) suggested catalyzed reaction, and the definition of whether such an chloride elimination from 2-chloro-4-oxoadipate to give ma- 644 FETZNER AND LINGENS MICROBIOL. REV.

COOHCONCIHC21H1 HCLfCl jOOH 2H OOH 00sOH , /, OH F§HOO l O;O Oo 1 2 3 HOHO OH HOOH H§ FIG. 3. Reactions catalyzed by maleylacetate reductase. 1, 2-chlo- C romaleylacetate; 2, maleylacetate; 3, 3-oxoadipate (445).

echols would be unproductive because of suicide inactivation of the meta-cleavage enzyme catechol-2,3-dioxygenase by 3-halocatechols (29, 263). However, elimination of chloride I during meta ring cleavage has been reported by Kersten et al. c (250): 5-chlorovanillate was oxidized by Pseudomonas testosteroni to 5-chloroprotocatechuate, which was subjected to meta cleavage by a 4,5-dioxygenase. The meta cleavage product spontaneously lactonized with concomitant elimination of the chloride ion, yielding 2-pyrone-4,6-dicarboxylate (Fig. 4). The k,Pa chloride elimination spontaneously followed ring opening and did not require an additional cyclizing enzyme, since the I formation of 2-pyrone-4,6-dicarboxylate from 5-chloroprotocatechuate was also catalyzed by homogeneous protocat- [iCY ] [ COON echuate 4,5-dioxygenase alone. Thus, a meta-cleaving dioxygenase fortuitously mediated a dechlorination reaction (see also the following section). a es C-o C-COOH~0 Oxygenases I '-COOH Oxygenolytic dehalogenation of haloaromatic compounds either is catalyzed by specific oxygenases (see the section on haloaromatic dehalogenases, subsection on oxygenases, below) COOH or occurs during a conversion catalyzed by the enzyme for the COOH corresponding unsubstituted substrate. In the degradation of 1,2,4,5-tetrachlorobenzene by Pseudomonas sp. strain PS14, an initial 5,6-dioxygenating attack is followed by the probably spontaneous elimination of HCl during rearomatization of the COOH dihydrodiol (1,3,4,5-tetrachloro-1,2-dihydroxycyclohexane-3,5- C,OOH diene), yielding 3,4,6-trichlorocatechol (414). (For dioxygeno- ol lytic chloride elimination from haloaromatic compounds, see I Succinyt-CaA also the section on haloaromatic dehalogenases, subsection on oxygenases, below.) Succinate Adventitious defluorination of 3-fluoro-substituted ben- £-SCoA zenes involving toluene 2,3-dioxygenase from Pseudomonas sp. OOH strain T-12 is shown in Fig. 5a (402). A similar mechanism was proposed for the benzoate 1,2-dioxygenase-catalyzed defluorination of 2-fluorobenzoate (111, 327). The 1,2-dihydrodiol of 0 - COOH 2-fluorobenzoate is unstable and eliminates fluoride. The H3C-C'SCoA CH resulting 1-ketoacid decarboxylates to catechol (Fig. Sb). T'he tH2 initial oxygenase attack on chlorobiphenyls by biphenyl 2,3- COOH dioxygenase from P. testosteroni B-356 can involve the dehalo- FIG. 2. Modified ortho pathway for the catabolism of 3-chloroben- genation of the molecules. Pseudomonasputida clones carrying zoate by Pseudomonas sp. strain B13 (401, 428). the bphA gene of P. testosteroni B-356 were used to study the metabolite production from 4,4'-, 2,2'-, and 2,4'-dichlorobiphenyl (8). With 2,4'- and 4,4'-dichlorobiphenyl, the oxygen- leylacetate. Pseudomonas sp. strain B13 and A. eutrophus ase-generated metabolites were hydroxylated predominantly at JMP134 form maleylacetate from 2-chloromaleylacetate. Both the 2- and 3-position of the para-substituted ring, with a maleylacetate reductases from strain B13 (241) and from strain concomitant loss of the para chlorine atom. The authors JMP134 (445, 537) reductively dechlorinated 2-chloromaleylacetate to maleylacetate, which subsequently was reduced to 3-oxoadipate. In the conversion of 2-chloromaleylacetate to 3-oxoadipate, 2 mol of NADH were consumed (Fig. 3). Maleylacetate reductase from strain JMP134 converted 2-chlo- protocatechuate NOOt~'~N%~COON j'atOOHHAOOC romaleylacetate and maleylacetate with similar efficiencies HOO 4,5-dioxygenase (445). The enzyme might be referred to as some sort of 2 3 reductive dehalogenase. FIG. 4. Fortuitous dehalogenation subsequent to meta cleavage of The pathways discussed above are "modified ortho path- 5-chloroprotocatechuate. 1, 5-chloroprotocatechuate; 2, putative ring ways" of haloaromatic degradation. meta cleavage of halocat- fission product (enzyme bound); 3, 2-pyrone-4,6-dicarboxylate (250). VOL. 58, 1994 BACTERIAL DEHALOGENASES 645

a R FR R iR H, Ct tO H A spontaneous 0,°C_C .11 H OH Ir~~~OH O~ CL "IH XH X H N0HH1 H NADH.H' NAO* [ K 02+XH2 X +H20 H20 2HQ 6 CO+ HCOOH R = -CH3, -CN, -OCH3, -CF3,-F, -Cl, -Br, -1. WK. C maor a ,- sH sponta-eous HOOC-CHO rea reactions (hydroysis) CHCl2-COOH COOHO [HOOC OH c F O2 43,OH Ct C=C'"H

NAOH+H+ NAD+ HF- L i 02 a FIG. 5. Fortuitous defluorination reactions catalyzed by toluene reduction a-c-oI2om 2,3-dioxygenase (402) (a) and benzoate 1,2-dioxygenase (b) (111, 327). ax-c-cs hion

abiohic reactions -c-cOOH {high pH arnd suggested that the peroxide generated by the oxygenase attack temperature) on carbons 2 and 3 creates an environment destabilizing the CHCO3 + HCOOH chlorine atom in the para position, so that some of the destabilized molecules are dehalogenated before hydrogena- FIG. 7. (a) Hypothetical route for the cometabolic degradation of tion of the peroxide occurs. trans-1,2-dichloroethylene by a culture of methanotrophic bacteria. In addition to the dioxygenases mentioned above, monooxy- Concomitant with chloride production from the epoxide intermediate, formation of was genases play a role in fortuitous dehalogenation reactions. glyoxal assumed (redrawn from reference 225). (b) Transformation of TCE and 2,2,2-trichloroacetaldehyde (chloral hy- Salicylate hydroxylase from P. putida catalyzes the hydroxyla- drate) by M. trichosporium OB3b and abiotic reactions of the oxidation tion of 2-iodo-, 2-bromo-, 2-chloro-, 2-nitro-, and 2-aminophe- products. Trichloroethanol and trichloroacetic acid were formed from nol to catechol (480, 488). Halide is released from the ortho- chloral hydrate by M. tnchosponum OB3b expressing soluble methane halophenols. The hydroxylation of ortho-halophenols proceeds monooxygenase. Chloral hydrate was shown to decompose abiotically with the unusual stoichiometry of 2:1:1 for NADH consumed, to chloroform and formic acid at higher pH (pH 9.0) and temperature 02 uptake, and catechol formed; i.e., the dehalogenation (60°C) (redrawn from reference 367). reaction causes a two-fold-higher consumption of NADH than the physiological reaction does. This unusual stoichiometry of NAD(P)H consumption was also found with phenylalanine hydroxylase from rat liver, catalyzing the hydroxylation of ethylene (TCE). Microbial oxidation of TCE has been re- p-fluorophenylalanine to tyrosine and fluoride (469), and with ported to be catalyzed by toluene 2,3-dioxygenase (291, 365, p-hydroxybenzoate 3-hydroxylase from Pseudomonas fluore- 540, 541, 586), toluene 2-monooxygenase (134, 364, 449, 450), scens, catalyzing the oxygenative defluorination of difluoro and toluene 4-monooxygenase (555), phenol hydroxylase and 2,4- tetrafluoro derivatives ofp-hydroxybenzoate (217). To explain dichlorophenol hydroxylase (185), propane monooxygenase the stoichiometry of the reaction, Husain et al. (217) suggested (539), ammonia monooxygenase (18, 531), and methane the formation of a quinonoid species as the primary product monooxygenase (132, 158, 267, 296, 367, 373, 503, 507). The formed upon oxygenative dehalogenation. The additional re- oxygenase with the highest specific activity directed against ducing equivalent is presumably used in a nonenzymatic TCE is the soluble methane monooxygenase from Methylosinus reaction with the quinonoid intermediate, resulting in a dihy- trichosporium OB3b (136). However, TCE oxidation is not a droxy product (Fig. 6). common property of broad-specificity microbial oxygenases Another type of spontaneous dehalogenation reaction is the (539). There probably are different mechanisms of TCE oxi- chemical decomposition of epoxides formed by monooxygen- dation by various oxygenases. Toluene dioxygenase processes ase-catalyzed reactions. The initial step in the aerobic trans- TCE differently from how methane monooxygenase does: formation of halogenated alkenes is generally assumed to be soluble methane monooxygenase from M. trichosporium OB3b an epoxidation of the carbon-carbon double bond (187). The forms epoxides as the major products of the oxygenation, subsequent metabolism of the reactive haloepoxides is not which undergo isomerization or hydrolysis (136). In contrast, known in detail, but extensive dehalogenation is frequently TCE oxidation by toluene dioxygenase of P. putida Fl seems observed. Gratuitous oxidation of organohalides by nonspecific not to involve epoxide intermediates; i.e., TCE monooxygen- oxygenases biosynthesized for other metabolic purposes has ation is not occurring to any significant extent (291). been most extensively studied for the oxidation of trichloro- Soluble methane monooxygenases, e.g., from Methylococcus capsulatus Bath (69, 158, 467), M. trichosporium OB3b (136, 206, 367, 373), Methylobacterium sp. strain CRL-26 (381), or other strains or consortia of methanotrophic bacteria (200, COOH 02 iH, COOH COOH 225), catalyze the oxidation of a variety of hydrocarbons F F including a number of halogenated short-chain alkanes and

F F F -k.('.OH alkenes and even aromatic compounds such as benzene, NADPHOH' NAOP NDP N OH 0 OH toluene, styrene, 3-chlorophenol and 3-chlorotoluene, as well as pyridine and cyclohexane (69, 158, 206). Perchlorinated FIG. 6. Oxygenative defluorination of 2,3,5,6-tetrafluoro-p-hy- such as tetrachloromethane and droxybenzoate by p-hydroxybenzoate 3-hydroxylase from P. fluore- compounds tetrachloroethylene, are not converted methane scens. Evidence for an unstable product with an absorption spectrum however, by monooxygenases. typical of o-quinones suggested that the enzyme catalyzes the forma- Figure 7 shows examples for methane monooxygenase-cata- tion of an o-quinone from the substrate, which is then reduced by an lyzed epoxidations of some chlorinated ethenes and subse- additional molecule of NADPH in a nonenzymatic reaction (217). quent dehalogenation reactions. The purified soluble methane 646 FETZNER AND LINGENS MICROBIOL. REV.

monooxygenase from M. trichosporium OB3b oxidized chlori- rination was accompanied by extensive polymerization reac- nated, fluorinated, and brominated alkenes predominantly by tions. epoxidation, and subsequent hydrolysis yielded stable acidic Tyrosinases. Streptomyces and Actinomyces strains are products (Fig. 7b). Additional aldehydic products resulting known tyrosinase producers. A tyrosinase from Streptomyces from intramolecular halide or hydride migration were ob- euwythernus was shown to oxidize halogen-substituted phenols. served in a yield of 3 to 10% during the oxidation of vinylidene Oxidation was accompanied by partial dehalogenation, and chloride, trifluoroethylene, tribromoethylene, and TCE. Dur- chloride was released from some substrates even in the ab- ing the oxidation of TCE in vitro, a turnover- and time- sence of oxygen consumption (68). dependent inactivation of the protein components of soluble Peroxidases. Arthrobacter and Streptomyces strains produce methane monooxygenase occurred (136). Whereas M. tricho- extracellular peroxidases in soil and when grown on bleach sporium OB3b apparently contains a methane monooxygenase effluents and lignocellulose (see reference 554 and references that specifically oxidizes terminal methyl groups, methane therein). The role of peroxidases in the dehalogenation of monooxygenase from Methylococcus capsulatus Bath catalyzed chlorolignin has still to be investigated. Peroxidases produced both terminal and subterminal hydroxylation reactions. The by soil organisms were shown to catalyze the transformation of enzymes from Methylococcus capsulatus Bath and M tricho- 3,4-dichloroaniline to 3,3', 4,4'-tetrachloroazobenzene. In this sporium OB3b also exhibit different electron donor and inhib- case, dehalogenation did not occur (37). However, a peroxi- itor specificities (69). Methanogens may be suited for the dase(s) secreted by the fungus Phanerochaete chrysosponum controlled biodegradation of TCE in contaminated groundwa- monodechlorinated 2,4-di-, 2,4,5-tri-, 2,4,6-tn-, and pentachlo- ter and soils, since the toxic and carcinogenic compound vinyl rophenol in the para position to yield a para-benzoquinone. chloride, which is a product of anaerobic TCE dehalogenation, The mechanism proposed by Hammel and Tardone (179) is readily oxidized by soluble methane monooxygenase (for a involved the initial formation of a chlorinated phenoxy radical, discussion of TCE bioremediation, see the section on applica- which may be converted to a 4-chlorocyclohexadienone cation tion of microbial systems in environmental protection technol- by a second peroxidase-catalyzed one-electron oxidation. The ogy and waste management, below). However, methanotrophs cation undergoes nucleophilic attack by a water molecule, express soluble methane monooxygenase only under condi- leading to elimination of HCl at the 4-position, which produces tions of copper limitation; this may be a crucial issue for the the p-benzoquinone (179). Phanerochaete chrysospornum was effective use of methanotrophs in bioremediation systems. found to rapidly mineralize 2,4,5-trichlorophenol. Ligninper- However, it was shown that particulate (membrane-associated) oxidase- and manganese peroxidase-catalyzed oxidative de- methane monooxygenases may also oxidize TCE, although the chlorination reactions followed by quinone reduction reactions rates are lower than those observed for the soluble methane yielded the key intermediate 1,2,4,5-tetrahydroxybenzene. monooxygenases (91, 372). Thus, all three chlorine atoms were removed from 2,4,5- Similar to the methane monooxygenases, ammonia mono- trichlorophenol prior to ring cleavage (234). Horseradish oxygenase oxidizes not only TCE but also a variety of mono- peroxidase was also shown to degrade 2- and 4-monochloro- halogenated ethanes and n-chlorinated alkenes. The maximum phenol and to release chloride from higher chlorinated halo- rate of haloethane oxidation increased with decreasing molec- phenols (406). The lignocellulolytic actinomycete Streptomyces ular weight of the halogen from iodoethane to chloroethane. viridosporus produces four extracellular lignin-peroxidase From monohalogenated ethanes, acetaldehyde was the major isoenzymes. Apart from other nonhalogenated substrates, the organic product of oxidation of the halogen-substituted carbon isoform designated P3 also oxidized 2,4-dichlorophenol (392, (C-1), whereas a C-2 attack would produce halogenated alco- 546). It is interesting that lignin-solubilizing peroxidase activ- hols. In contrast to haloethane oxidation, the major products ities in Streptomyces strains are often routinely assayed with of chloropropane or chlorobutane oxidation were the 1- and 2,4-dichlorophenol as substrate (380, 392). However, chloride 2-chlorinated alcohols. Thus, with haloethanes, the haloge- release was not determined in these reports. nated carbon was the preferred position for oxidation, whereas Because of the nonspecificity of the radical-forming fungal with longer-chain haloalkanes, the substituted carbon was and bacterial laccases, tyrosinases, and peroxidases, dehaloge- rarely oxidized (396). nation reactions of halophenols via phenoxyradical formation followed by a nucleophilic substitution releasing halogenide Laccases, Tyrosinases, and Peroxidases may well occur in soil contaminated with haloaromatic compounds. Since laccases are stable and function effectively in an The degradation of halogenated aromatics by actinomycetes immobilized form, they may even be suited for the treatment has been reviewed by Winter and Zimmermann (554). Phe- of toxic chlorophenolic waste. noloxidase (laccase), tyrosinase, and peroxidases are produced not only by white rot fungi but also by Actinomyces and Hydratases Streptomyces strains. These enzymes catalyze one-electron oxidations, often yielding unstable substrate-cation radicals Halogen eliminations have been reported to occur sponta- from a broad range of aromatic compounds. The unstable neously after some hydratase-catalyzed reactions. In the bac- radical intermediates subsequently undergo a variety of non- terial metabolism of 3-chloroacrylic acid by P. cepacia CAA1 enzymatic reactions such as C-C or ether cleavage or oxidative and by the coryneform strain CAA2, hydratases were proposed coupling and polymerization reactions (for reviews on fungal to convert 3-chloroacrylic acid to 3-chloro-3-hydroxypropanoic laccases and peroxidases, see references 55, 257, and 417). acid, which decomposed to malonate semialdehyde and HCl Laccases. Although Actinomyces and Streptomyces strains (189) (Fig. 8a). Although these enzymes from strains CAA1 are known to produce extracellular laccases (phenoloxidases), and CAA2 might be characterized as dehalogenases, Hartmans no Actinomyces laccase is known to be involved in the trans- et al. (189) suggested that the 3-chloroacrylic acid hydratases formation of halogenated compounds. However, direct dechlo- should be classified as hydratases rather than dehalogenases, rination of chlorophenolic compounds by laccases secreted by since the enzymes share more similarities with hydratases like the fungus Trametes (Coriolus) versicolor was demonstrated by fumarase than with dehalogenases like halidohydrolases (see Roy-Arcand and Archibald (406). The laccase-driven dechlo- the section on haloacid dehalogenases, below). However, van VOL. 58, 1994 BACT-ERIAL DEHALOGENASES 647

a Cl H\ ,OOH COOH1 COOH "C CH, CH2 + M(red) / / a M(ox)+CcV 11 HO-C-H H oo'~XH Cl Cl Cl Cl HCL H20 L J QL H Cl ClC + M (red) + H + Cl+M (ox)

F ACOOH COOH COOH IC HO-C-F C=O FIG. 9. Putative two-step mechanism for the reductive dehaloge- c CH2 LM2CH nation of 2,3,4,5,6-pentachlorobiphenyl. This mechanism involves re- / \ Hp0 H+F- HOOC H COOH J COOH duction by an unknown electron donor (M), free radical transition state, and proton abstraction from the solvent. The ultimate source of FIG. 8. Spontaneous halide elimination subsequent to hydratase- electrons is the reduced organic substrate (redrawn form reference catalyzed reactions. (a) 3-Chloroacrylic acid hydratase/dehalogenase 368). (189, 529); (b) fumarate hydratase (315).

171, 216, 477 3-chlorobenzoate, see on Hylckama 424, [for the section Vlieg and Janssen (529) purified two novel inducible energy conservation coupled to reductive dechlorination, be- dehalogenases, one specific for cis- and the other specific for low]), 2,4-dichlorobenzoate (515, 579); a number of chlori- trans-3-chloroacrylic acid, from a coryneform bacterium designated phenols (43, 44, 53, 149-151, 171, 172, 177, 218, 271, 272, nated strain FG41. The enzymes catalyzed the liberation of 283, 306-308, 323, 324, 331, 332, 557, 582); 2,4-dibromo-, chloride from 3-chloroacrylic acid, yielding malonate semial- 2-bromo-, and 4-bromophenol (255); tri- and tetrachlorocat- dehyde. No other substrates were found, and hydration of the echols (363, 557); di-, tri-, and tetrachloroanilines (279, 280, unsubstituted acrylic acid did not occur. This indicates that the 472); 2,4-D (26, 117, 151, 323); 2,4,5-trichlorophenoxyacetic enzymes are not hydratases which, as a result of a broad acid (2,4,5-T) (152, 156, 323, 404, 478); and polychlorinated substrate specificity, cause enzymes must dehalogenation. Both biphenyls (1, 368, 390). Most studies on reductive dehalogena- be classified as a type novel of dehalogenases. The cis-3- tion of haloaromatic compounds were performed with differ- chloroacrylic acid dehalogenase a is 38-kDa homodimer, ent types of microbial consortia. There are only few examples whereas the trans-3-chloroacrylic acid dehalogenase was iso- of pure cultures which accomplish reductive dehalogenation lated as a 50-kDa enzyme composed of 7,400- and 8,400-Da reactions: (i) Desulfomonile tiedjei DCB-1 dechlorinates subunits. These dehalogenases were completely isomer selec- 3-chlorobenzoate, meta-substituted dichlorobenzoates, chloro- tive. Direct proof that the dehalogenation is mechanistically a phenols, and tetrachloroethylene (see the section on energy hydration reaction is still lacking, but a hydration with subse- conservation coupled to reductive dechlorination, below); (ii) quent HCl from elimination the unstable geminal haloalcohol strain DCB-2, which is related to the genus Clostridium, seems plausible (529). preferentially dechlorinates substituents ortho to the phenolic Degradation of 2-chloroallylalcohol by Pseudomonas sp. hydroxyl group of tri- and dichlorophenols (308); (iii) Clostrid- strain JD2 was found to proceed via 2-chloroacrylic acid. The ium rectum S-17, Clostridium sphenoides, several Bacillus dechlorination reaction is not yet fully understood; it is not strains, and members of the family Enterobacteriaceae are clear whether 2-chloroacrylic acid is transformed via hydration involved in lindane dechlorination (173, 201, 223, 305, 369- of the double bond and dechlorination of the intermediate, as 371); (iv) Azotobacter chroococcum MSB-1 reductively dechlo- with 3-chloroacrylic acid, or whether a coenzyme A (CoA) rinates 2,4-D to 4-chlorophenoxyacetic acid (26); (v) Aer- intermediate is formed, as with the p-substituted haloalkanoic obacter aerogenes metabolizes the pesticide DDT (548); and acids (see the section on 3-haloacyl-coenzyme A halidohydro- (vi) the coryneform strain NTB-1 reductively dechlorinates lases, below) (528). A hydratase-type dehalogenation reaction 2,4-dichlorobenzoate at the ortho position (515). Strain NTB-1 was also reported for the conversion of monofluorofumarate (formerlyAlcaligenes denitrificans NTB-1) grows on 2,4-dichlo- by fumarase, yielding ot-fluoromalate, which subsequently de- robenzoate under aerobic conditions. Whereas the first step of composed to oxaloacetate (315) (Fig. 8b). 2,4-dichlorobenzoate degradation involves reductive dechlorination, the second dechlorination step is catalyzed by a hydro- REDUCTIVE DEHALOGENATION lytic dehalogenase system, yielding 4-hydroxybenzoate from 4-chlorobenzoate (see the section on halidohydrolases, below). Haloaromatic Compounds and Some Pesticides Concerning the mechanism of reductive dehalogenation, a Reductive dehalogenation is a two-electron transfer reaction reduced organic substrate or H2 might be the source of both which involves the release of the halogen as a halogenide ion the reducing power and the protons (one-step transfer of two and its replacement by hydrogen. Aerobic microorganisms electrons and one proton). On the other hand, dehalogenation often fail to metabolize the more heavily halogenated com- might occur in a two-step reduction by an electron donor pounds. For highly chlorinated biphenyls, hexachlorobenzene, (reduced organic substrate) and with proton abstraction from and tetrachloroethylene, anaerobic reductive dehalogenation the solvent. Nies and Vogel (368) showed that the source of is the only known biodegradation mechanism (for reviews on the hydrogen added to the aromatic ring in reductive dechlo- reductive dehalogenation, see references 304, 335, 496, and rination of 2,3,4,5,6-pentachlorobiphenyl is a proton from 542). water. They suggested a two-step mechanism, as illustrated in Reductive dehalogenation of haloaromatic compounds was Fig. 9. However, the enzyme catalyzing the reductive dechlo- found in both aerobic and anaerobic microorganisms. There is rination of 2,3,4,5,6-pentachlorobiphenyl has not been de- evidence for the reductive dehalogenation, under methano- scribed yet. genic, sulfidogenic, and even denitrifying (171) conditions, of a Apart from 2,4-D and 2,4,5-T, biotransformation of many number of haloaromatics (75), such as halobenzenes (38, 122, other halogenated pesticides (Fig. 10) was described to involve 211, 393, 505); halotoluenes (393); halobenzoates (149-151, reductive dehalogenation steps. Lists of pesticides and other 648 FETZNER AND LINGENS MICROBIOL. REV.

Cl N02 COOH CH2 0 H-C-CC13 OH COOH r^Cl El cn ClKOCH3 C l Cl cl ct a

3 4 5 Cl

H CH3 H3C\ 5H3 H3CCit YH2IF N, N

CO NH CN CN LO

NH B Br ClN Cl I OH Cl Cl

6 7 9 10

H H Cl3C NyCl Cl NCOOH H3C O0

CLrHj2 CL ° --CH-C2HS H H NH2 0 CH 14 11 12 13

CcllH

15 16 17

R1 R2 L Cl Jc Cltlt-C

18 19 FIG. 10. Some halogenated pesticides. 1, 2,4-D (X = H) or 2,4,5-T (X = Cl); 2, DDT; 3, chloronitrofen (4-nitrophenyl-2,4,6-trichlorophenyl ether); 4, PCP; 5, dicamba (3,6-dichloro-2-methoxybenzoate); 6, bromoxynil (3,5-dibromo-4-hydroxybenzonitrile); 7, TPN (2,4,5,6-tetrachloroisophthalonitrile); 8, diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea]; 9, benthiocarb (thiobencarb; S-4-chlorobenzyl-N,N-diethyl thiocar- bamate); 10, techlofthalam [N-(2,3-dichlorophenyl)-3,4,5,6-tetrachlorophthalamic acid]; 11, nitrapyrin (2-chloro-6-trichloromethylpyridine); 12, picloram (4-amino-2-carboxy-3,5,6-trichloropyridine); 13, bromacil (5-bromo-3-sec-butyl-6-methyluracil); 14, lindane (-y-HCH; y-1,2,3,4,5,6- hexachlorocyclohexane); 15, aldrin (1,2,3,4,10,10-hexachloro-1,4,4a,5,6,7,8,8a-octahydro-1,4-endo,exo-5,8-dimethanonaphthalene); 16, dieldrin (1,2,3,4,10,10-hexachloro-6,6-epoxy-1,4,4a,5,6,7,8,8a-octahydro-1,4-endo,exo-5,8-dimethanonaphthalene); 17, heptachlor (1,4,5,6,7,8,8-heptachlo- ro-3a,5,7,7a-tetrahydro-4,7-methanoindene); 18, mirex (dodecachlorooctahydro-1,3,4-metheno-2H-cyclobuta[cd]pentalene); 19, toxaphene (mixture of isomeric hepta-/octa-/nonachlorobornanes) (toxicant A-1 when R, = CH2Cl and R2 = CHCl2; toxicant A-2 when R1 = CHCl2 and R2 = CH2C1; toxicant B when R1 = CH2Cl and R2 = CH2Cl). anthropogenic compounds which undergo reductive dehaloge- was investigated by Wedemeyer (548). Complete reductive nation were reported by Kobayashi and Rittmann (266) and by dechlorination of the broad-spectrum fungicide 2,4,5,6-tetra- Mohn and Tiedje (335). A metabolic pathway of DDT dechlo- chloroisophthalonitrile (TPN) to isophthalonitrile was ob- rination by Aerobacter aerogenes involving reductive and dehy- served in soil (418). Dicamba (3,6-dichloro-2-methoxybenzo- drochlorination steps, yielding 4,4'-dichlorobenzophenone, ate), after demethylation, was reductively dechlorinated to VOL. 58, 1994 BACTERIAL DEHALOGENASES 649

OH 02 H1.HQt Cl GSH HQt GSH GSSG ct ct \- HCl ct O cl Gt / ac \'S-1 a H 1 2 NADP`H 2 NP OH OH OH 1 +WpB 2 jPC 3

QH 9H GSH FIC OH GSH 6S-SG Cl H H H HH HCH H OH OH

FIG. 11. Hypothetical pathway for the degradation of PCP by Flavobactenum sp. strain ATCC 39723. 1, PCP; 2, tetrachloro-p-hydroquinone; 3, 2,3,6-trichloro-p-hydroquinone; 4, 2,6-dichloro-p-hydroquinone; 5, 2-chloro-p-hydroquinone. PcpB, PCP monooxygenase (565, 567, 568); PcpC, glutathione-dependent reductive dehalogenase (566, 569). Redrawn from reference 569.

6-chlorosalicylate by an anaerobic consortium (489). The N- glutathione S-transferases from Proteus mirabilis are also func- heterocyclic herbicide picloram was dechlorinated at the meta tional as dimers (89, 162). In contrast to PcpA (a PCP-induced position under methanogenic conditions, yielding 3,6-dichloro- periplasmic protein [564]) and PcpB (PCP monooxygenase), 4-aminopyridine-2-carboxylic acid (394). Debromination of the PcpC was produced constitutively. The glutathione S-trans- N-heterocyclic bromacil was observed in anoxic aquifer slurries ferase gene pcpC from Flavobacterium sp. strain ATCC 39723 (7). Reductive dechlorination reactions also occurred with had more similarity with two plant glutathione S-transferases diuron (20), techlofthalam (258), benthiocarb (thiobencarb) than with the prokaryotic dichloromethane dehalogenase (337-339), and chloronitrofen (570) in paddy field soils, in soil genes from Methylobacterium strain DM4 and Hyphomicro- suspensions, or in anaerobic enrichment cultures. bium strain DM2. This is not surprising, since the dechlorina- Anaerobic dechlorination and degradation of lindane (-y- tion reaction catalyzed by dichloromethane dehalogenases is hexachlorocyclohexane [-y-HCH]) and the ot and , isomers of mechanistically different from the reductive dechlorination HCH was found with Clostridium spp. and representatives of catalyzed by the Flavobacterium glutathione S-transferase the families Bacillaceae and Enterobacteriaceae (173, 223). (377). Dechlorination of lindane by cell-free preparations of Clostrid- ium sphenoides appeared to be associated with the membrane Haloaliphatic Compounds fraction and required reduced glutathione (201, 305). In this context it is perhaps interesting that reductive dechlorination The mechanisms of reductive dehalogenation of haloali- of tetrachlorohydroquinone by a Flavobactenum sp. is cata- phatic compounds are not fully understood, although there are lyzed by a glutathione S-transferase (see below). Cell extracts a number of reports on the metabolism of halogenated ali- of C. rectum S-17 degraded lindane in the presence of dithio- phatic hydrocarbons under methanogenic, sulfate-reducing, threitol to y-tetrachlorocyclohexene and monochlorobenzene and denitrifying conditions (28, 34, 39-41, 42, 47, 77, 92, (370, 371). Concomitant with the conversion of lindane (or 104-107, 119-121, 138, 145, 210, 212, 274, 289, 326, 384, 491, related compounds), ATP was synthesized. Ohisa et al. (369) 535, 536). The redox potentials of chlorinated hydrocarbon suggested that y-HCH serves as artificial electron acceptor of couples like CCl4/CHCl3, CHCl3/CH2C12, tetrachloroethylene/ the Stickland reaction, an oxidation-reduction reaction of pairs trichloroethylene, or 1,1,1-trichloroethane/1,1-dichloroethane of amino acids. are in the range of Eo' = 0.3 to 0.52 V. This implies that their Reductive dechlorination of the trichloromethyl group of strength as oxidizing agents is between that of oxygen and that nitrapyrin was assigned to ammonia monooxygenase in Nitro- of nitrate, molecules that are readily reduced by microorgan- somonas europaea. It was suggested that the trichloromethyl isms that oxidize organic substrates (230). Redox couples group of nitrapyrin binds at the oxygen-activating iron center Ar-Hal/Ar-H for halogenated benzenes, benzoates, and phe- of the enzyme and so is reduced in place of dioxygen (530). nols are comparable to the redox couple N03-/NO2 (95). Reductive dechlorination of pesticides by porphyrins and For the strictly anaerobic methanogens, Fathepure and corrins under reducing conditions was achieved with hepta- Boyd (120) presented a scheme linking reductive dechlorina- chlor, aldrin, dieldrin (32), DDT (32, 328, 585), lindane (32, tion to methanogenesis; in this scheme, chlorinated ethylenes 310), and the insecticides mirex (32, 214), and toxaphene (252) serve as electron acceptors (Fig. 12). Cell suspensions of four (see also the section on catalysis by transition metal cofactors, different strains of methanogenic bacteria dechlorinated 1,2- below). In the first step of pentachlorophenol (PCP) degradation by the strictly aerobic Flavobacterium sp. strain ATCC 39723, a PCP monooxygenase designated PcpB catalyzes the acetate formation of tetrachlorohydroquinone (565, 567, 568) (see the methanot Methanognss H4 a methylamine acCH, section on haloaromatic dehalogenases, subsection on oxygenases, below). In the next steps, a glutathione-dependent reductive dehalogenase converts tetrachlorohydroquinone to -as ,aL and SXH-/< = 2,3,6-trichlorohydroquinone, 2,6-dichlorohydroquinone, Q.. 2-monochlorohydroquinone (Fig. 11). This dehalogenase PcpC was purified and characterised as a probably homodimeric FIG. 12. Hypothetical scheme for the transfer of electrons to glutathione S-transferase (subunit molecular mass, 30 kDa) tetrachloroethylene during methanogenesis. X is the electron carrier (566, 569). Eukaryotic glutathione S-transferases and the involved in methane production (120). 650 FETZNER AND LINGENS MICROBIOL. REV. dichloroethane via two mechanisms: one pathway involved assmiaon products a dihalo elimination, yielding ethylene, whereas the other pathway proceeded via two hydrogenolytic reactions, yielding CO(- acetyl-CoA - acetate chloroethane and ethane consecutively (210). Subsequently, ca,~ purified methyl-CoM reductase from Methanobacterium thermoautotrophicum AH, together with FAD and a crude "com- CHG3-0CH22- CH3I ponent A" fraction which reduced the nickel of factor F430 in FIG. 13. Reduction of chlorinated methanes by organisms possess- methyl-CoM reductase, was found to convert 1,2-dichloroeth- ing the acetyl-CoA pathway. Upper branch and lower branch, Aceto- ane to ethylene and chloroethane with H2 as the electron bacterium woodii; lower branch, Desulfobactenum autotrophicum. The reductive branch is putatively catalyzed by corrinoid enzymes. CC14 donor (209) (for a discussion of factor F430, see also the and other chlorinated methanes serve as electron acceptors (redrawn following section). Clostridium sp. strain TCAIIB, isolated from reference 107). from a methanogenic mixed culture, transformed 1,1,1-trichloroethane to 1,1-dichloroethane, acetic acid, and other uniden- tified compounds. Reductive dechlorination of tetrachloromethane led to tri- and dichloromethane (145). presence of the CO dehydrogenase/acetyl-CoA pathway for Holliger et al. (212) isolated a gram-negative anaerobic either the utilization or the synthesis of acetate (107) (Fig. 13). bacterium, designated strain PER-K23, which reductively Acetogenic organisms might also be involved in the dehaloge- transformed tetrachloroethylene to ethane via TCE, cis-1,2- nation of tetrachloroethylene (92). Microorganisms possessing dichloroethylene, chloroethylene, and ethylene; in this scheme the acetyl-CoA pathway, like the sulfate reducer Desulfobac- reductive dechlorination was coupled to growth. H2 and for- terium autotrophicum (104, 105, 107) or the acetogenic bacte- mate were the only electron donors that supported growth, and rium Acetobacterium woodii (105, 107), contain high levels of tetrachloroethylene and TCE were the only electron acceptors corrinoid enzymes. Interestingly, heat-killed' cells also showed used by strain PER-K23 (212). Tetrachloroethylene also reductive dechlorination of tetrachloromethane (105, 107, 211, served as an electron acceptor in enrichments on methanol and 273). Evidence that corrinoids or corrinoid enzymes are in- tetrachloroethylene (92) or on benzoate and tetrachloroethyl- volved in reductive dechlorination includes the following. (i) ene (440). Apart from D. tiedjei DCB-1, which was the first The ability of bacteria to dehalogenate, for instance, tetrachlo- organism isolated that couples growth on fumarate, hydrogen, romethane or trichloromonofluoromethane correlates with the or acetate to a reductive dechlorination reaction (see the presence of high concentrations of corrinoids in these micro- section on energy conservation coupled to reductive dechlori- organisms (274, 275). (ii) Corrinoids such as aquocobalamin or nation, below), strain PER-K23 (212) is the only pure culture methylcobalamin catalyze the reduction of tetrachloromethane available for further investigations of such an energy-produc- or trichloromonofluoromethane with titanium(III) citrate or ing mechanism of reductive dehalogenation. with dithiothreitol as electron donors (273, 275, 276). How- Not only obligate anaerobes but also facultative anaerobic ever, a number of metalloporphyrin-containing systems have bacteria were reported to perform reductive dechlorination of been demonstrated to dehalogenate tetrachloromethane, for haloaliphatic compounds. For instance, Escherichia coli K-12 instance, reduced iron(II) porphyrins (10, 60, 61, 264, 543, reduced tetrachloromethane to trichloromethane under fuma- 544), vitamin B12 (208, 275), and the nickel-containing porphi- rate-respiring conditions (78). Reductive transformation of noid factor F430 (273). While factor F430 is unique to meth- tetrachloroethylene to cis-dichloroethylene was achieved in anogens, the cobalt-containing cobalamins and the iron coen- mixed cultures of different aerobic isolates in coculture with a zyme hematin are present in many anaerobic bacteria. Studies Bacillus sp. and a Desulfotomaculum sp. under conditions of on abiotic reductive dehalogenation of halogenated aliphatic limited oxygen supply. This transformation may be catalyzed organics by reduced metals (59) or organometallic complexes by aerobic or facultatively anaerobic bacteria if the redox (25, 178; for a review, see reference 535) support the hypoth- potential drops below physiological values. Dechlorination esis that relatively highly oxidized halogenated compounds occurred when the redox potential decreased to values be- may be fortuitously reduced by the low-potential electron tween -50 and -150 mV and when electron donors were carriers found in anaerobic bacteria. present in excess (235). Respiratory c-type cytochromes were Polyhalomethanes underwent reductive hydrogenolysis shown to be responsible for the dehalogenation of tetrachlo- when incubated with iron(II) deuteroporphyrin IX, rat liver romethane to trichloromethane by the obligate respiratory P-450PB and bacterial P-450CAm. The reductase capacity with bacterium Shewanella putrefaciens 200 (388). A denitrifying these widely divergent P-450 systems was consistent with the Pseudomonas sp. designated strain KC completely degraded chemistry and mechanism established for hemes in homoge- tetrachloromethane to carbon dioxide. Trichloromethane was neous solution (61). Respiratory c-type cytochromes were not produced. Since iron, cobalt, and vanadium inhibited the shown to be responsible for the reductive dehalogenation of transformation of tetrachloromethane, it was hypothesized tetrachloromethane to trichloromethane in Shewanella putre- that the conversion may be linked to (a participant of) a faciens 200, an obligate respiratory bacterium that can utilize a metal-scavenging system, such as an iron(III)-reductase. How- variety of terminal electron acceptors. This dehalogenation did ever, the agent of tetrachloromethane transformation has not not proceed further. Tetrachloromethane transformation rates been identified yet (77, 289, 491). increased with increases in the specific heme c content, and dehalogenation was mediated only by periplasmic and mem- Catalysis by Transition Metal Cofactors brane fractions, not by cytoplasmic fractions of the cells (388). (Porphyrins and Corrins) Apart from polyhalomethanes, other alkyl halides are also converted by heme proteins (57, 544) or transition metal The mechanisms of reductive dehalogenation by transition cofactors. Reductive dechlorination of 1,1,2-trichloroethane metal cofactors have been reviewed recently by Gantzer and yielding vinyl chloride was catalyzed by cytochrome P-450CAM Wackett (147), Wackett and Schanke (542), and Mohn and in P. putida PpG-786 (58). Polychlorinated ethanes with chlo- Tiedje (335). The ability to transform tetrachloromethane or rine substituents on only one carbon underwent consecu- trichloromethane anaerobically has been correlated with the tive reductive dechlorination (without elimination), yielding VOL. 58, 1994 BACTERLAL DEHALOGENASES 651 ethane, when incubated with hematin or vitamin B12 under outside reducing conditions. In contrast, chlorinated ethanes with chlorine substituents at both carbon atoms were converted by reductive elimination by the same catalysts, yielding ethylene (derivatives) (421). Both cobalamin and factor F430 caused the reductive dechlorination of 1,2-dichloroethane by Methanosarcina barken. Co- balamin catalyzed mainly a dihalo elimination, yielding ethylene, whereas factor F430 formed almost equal amounts of COOH ethylene and chloroethane (210). Holliger et al. (209, 213) demonstrated that protein-bound factor F430 was the catalyst, K.a+ H since purified methyl-CoM reductase of Methanobacterium thermoautotrophicum, together with FAD and an additional protein component A [necessary to reduce the nickel of factor COOH F430 to Ni(I)], converted 1,2-dichloroethane to ethylene and + ct- chloroethane in the presence of H2. Free factor F430 was excluded as an in vivo catalyst (209, 213). Another reductive dechlorination by a corrinoid enzyme was shown for homogeneous N5-methyl tetrahydrofolate-homocysteine transmethy- lase of E. coli B. This enzyme catalyzes the cobamide-depen- FIG. 14. Hypothetical scheme for the chemiosmotic coupling be- dent methyl transfer reaction, leading to the synthesis of tween reductive dehalogenation and energy generation in D. tiedjei methionine from homocysteine. By using the purified enzyme, DCB-1 according to Dolfing (redrawn from reference 94). liberation of HCl was observed from tetrachloromethane and trichloromethane (556). Transition metal coenzymes dechlorinate not only aliphatic compounds such as polychlorinated nated tetrachloroethylene to TCE (121). The meta-chlorine of methanes, ethanes, and ethylenes but also halobenzenes (19, the dichlorobenzoate isomers 3,4-/3,5- and 2,5-dichlorobenzo- 147, 211) or, for instance, 2,3,4,5,6-pentachlorobiphenyl (19). ate was also removed by cells of strain DCB-1 (87, 88). In the These data suggest that nonspecific reactions with transition presence of 3-chlorobenzoate as a required inducer, PCP and metal cofactors may also be important in the degradation of other chlorophenols substituted at C-3 (except 3-monochloro- xenobiotic compounds in anaerobic environments. Since cyto- phenol) were dechlorinated. PCP was converted to 2,4,6-tri- chrome P-450CAM may also mediate reductive dechlorination chlorophenol. Bromine and iodine substituents were removed reactions (58), microbial reductive dehalogenation might occur from all positions of halobenzoates and halobenzamides, in the environment even under aerobic conditions. whereas chlorine was only removed from the meta position Wackett and Schanke (542) presented a model for the (331). The tendency for more highly chlorinated phenols to be biologically mediated reductive dehalogenation occurring in more readily dechlorinated by cells of D. tiedjei is consistent the environment. Hydrophobic halogenated organic com- with considerations of thermodynamics and solubility. More pounds may accumulate in biomembranes, becoming available highly chlorinated phenols are in a more highly oxidized state. for reaction with cellular membrane-associated transition Thus, reductive dehalogenation of those congeners has a more metal coenzymes. These coenzymes, which could exist in free negative free energy change. More highly chlorinated phenols or protein-bound form, would be rendered reactive via reduc- are also more lipophilic and are generally more strongly sorbed tion by cellular low-potential reductants such as ferredoxins. by cells (331). Mohn and Tiedje (334) presented evidence for Alternatively, or additionally, microorganisms might produce a chemiosmotic coupling of reductive dechlorination and ATP wide range of specific enzymes catalyzing reductive dehaloge- synthesis in D. tiedjei DCB-1. In this scheme, 3-chlorobenzoate nation reactions (542). serves as a final electron acceptor and energy is generated via the formation of a proton gradient over the membrane coupled Energy Conservation Coupled to Reductive Dechlorination: to a proton-driven ATPase (Fig. 14). The reaction mechanism Desulfomonile tiedjei DCB-1 of the reductive dechlorination of 3-chlorobenzoate has not been elucidated (98). However, Griffith et al. (159) investi- The sulfidogenic bacterium D. tiedjei gen. nov. sp. nov. (86) gated the reductive dechlorination of 2,5-dichlorobenzoate by (formerly strain DCB-1) conserves energy by coupling pyru- cell suspensions of D. tiedjei DCB-1 in D20. The meta- vate, H2, or formate oxidation to reductive dechlorination of dechlorination product of 2,5-dichlorobenzoate was specifi- 3-chlorobenzoate (85, 93, 94, 96-98, 293, 333, 334, 447, 465) cally deuterated at the position of dehalogenation; i.e., only the (Fig. 14). Dehalogenation activity was found to be inducible by proton at the site of dechlorination was derived from the 3-chlorobenzoate. Whereas dehalogenation was stimulated by solvent. These data weigh against a model for dechlorination formate, CO, or H2, it was inhibited by 02. The activity was predicting proton exchange but are consistent with a model membrane bound and was inhibited by sulfite and thiosulfate such as electron addition or nucleophilic attack by the equiv- but not by sulfate (87, 94). Sulfite reduction and dehalogena- alent of a hydride ion on the chlorine position. The authors tion were inhibited by the same respiratory inhibitors (85). suggested that electron addition appears to be the most DeWeerd et al. (85) suggested that reduction of sulfite and plausible mechanism for the dechlorination of meta-chloro- dehalogenation have some common electron carriers. Thus, benzoates by D. tiedjei DCB-1 (159). sulfate respiration and 3-chlorobenzoate respiration might use In analogy to the situation with strain DCB-1, which uses parts of the same electron transport chain. Similarly, a com- 3-chlorobenzoate as an electron acceptor, Holliger et al. (211) petition for H2 between sulfate reduction and dechlorination suggested that in an anaerobic mixed culture which was was suggested for a PCP-degrading, sulfate-reducing metha- reductively dechlorinating 1,2,3-trichlorobenzene to 1,3-di- nogenic enrichment culture (306). chlorobenzene, bacteria were present which used 1,2,3-trichlo- In addition to 3-chlorobenzoate, strain DCB-1 dehaloge- robenzene as the terminal electron acceptor and H2 as the 652 FETZNER AND LINGENS MICROBIOL. REV. electron donor. Strain PER-K23 (212), which couples reduc- monochlorophenol and phenol), removing halogen, nitro, tive dechlorination of tetrachloroethylene to growth, is dis- amino, and cyano groups to produce halide, nitrite, hydroxy- cussed in the section on haloaliphatic compounds (above). lamine, and cyanide, respectively. Elimination of 1 mol of halogen, nitro, or cyano group required 2 mol of NADPH, HALOAROMATIC DEHALOGENASES while only 1 mol of NADPH was required to remove 1 mol of hydrogen (in case of the unsubstitutedpara position) or 1 mol of an amino group. Thus, when the group leaving the benzene Oxygenases ring was an electron-donating group such as an amino group or Monooxygenases. The wood preservative PCP can be me- hydrogen, a hydroxyl group was attached to it, so that hydrox- tabolized both anaerobically (see the section on reductive ylamine or H20 is produced, requiring 1 mol of NADPH per dehalogenation, above) and aerobically. Investigating the fate mol of substrate. In contrast, when the leaving group was an of ['4C]PCP in flooded soil, Weiss et al. (551) found different electron-withdrawing group, it was reduced to the correspond- mechanisms of PCP transformation: a major part of the ing anion. In this case, 2 mol of NADPH was consumed per radioactivity was incorporated into insoluble macromolecules, mol of substrate (567). Such a variation in NAD(P)H stoichi- i.e., bound to humin and humic acid. However, conjugated ometry, depending on the substrate, was also found with other PCPs and methylated derivatives (anisols) were also found, flavoprotein monooxygenases, which, in contrast to PCP and reductive dechlorination to free tetra- and trichlorophe- 4-monooxygenase, mediate fortuitous dehalogenation reac- nols was shown. PCP obviously was also mineralized to carbon tions via oxidized quinones as direct enzyme products, which dioxide in soil. A number of aerobic microbial strains that are reduced by a second NAD(P)H (see the section on metabolized PCP predominantly by acetylation and methyl- oxygenases in spontaneous or fortuitous dehalogenation reac- ation were described by Rott et al. (405). Acetylation and/or tions, above). Owing to its broad substrate range, PCP 4-mono- methylation of the hydroxyl groups, reductive dechlorination oxygenase catalyzed the conversion of the herbicide bromoxy- to tetrachlorophenols, dechlorination and methylation to tet- nil (3,5-dibromo-4-hydroxybenzonitrile) (Fig. 10) to 3,5- rachloroanisols, and hydroxylation to tetrachlorodihydroxy- dibromohydroquinone and cyanide. The rate of bromoxynil benzenes followed by acetylation were the main metabolic turnover was six times that of PCP (501). Since PCP 4-mono- steps of PCP transformation (405). oxygenase from Flavobacterium sp. strain ATCC 39723 utilizes Aerobic degradation of PCP was reported for Pseudomonas such a wide range of substrates, the genepcpB, which encodes strains (391, 481), for various Rhodococcus strains (15, 170), the monooxygenase, might well be a useful candidate for for a number ofArthrobacter strains (66, 67, 422, 423, 463), for constructing novel biodegradative strains by recombinant Flavobacterium strains (409, 464, 500, 565-569), for a Myco- DNA techniques (see the section on application of microbial bacterium sp. (170), and for the 2,4,5-T utilizer P. cepacia systems in environmental protection technology and waste AC1100 (237, 254). A Mycobactenium sp. methylated PCP to management, below). The 'Flavobacterium sp. strain ATCC pentachloroanisole, but this strain was also capable to hydroxy- 39723 pcpB gene was cloned and expressed in E. coli (378). late PCP at the ortho and para positions; this was followed by Sequence analysis and protein database searches revealed successive methylation via tetrachloro-2-methoxyphenol or tet- homologies with other microbial monooxygenases such as rachloro-4-methoxyphenol to tetrachloro-2,4-dimethoxyben- phenol 2-monooxygenase from the white rot fungus Tichos- zene (482). In the first step of PCP degradation by Rhodococ- poron cutaneum and tryptophan 2-monooxygenase from cus, Arthrobacter, and Flavobacterium strains, PCP is converted Pseudomonas syringae. Comparison of nucleotide sequences to tetrachlorohydroquinone (16, 170, 422, 423, 565, 567, 568). showed about 56% similarity to tfdB (localized on plasmid Further degradation of tetrachlorohydroquinone by Rhodococ- pJP4), encoding 2,4-dichlorophenol hydroxylase fromA. eutro- cus and Flavobacterium strains has been suggested to involve phus JMP134, and vanAB, which encodes a Pseudomonas both hydrolytic and reductive dechlorination steps (Fig. 11) vanillate demethylase. ThepcpB gene was not found to be part (17, 169, 566, 569). of an operon or to be present on the 100-kb endogenous The PCP 4-hydroxylase from Arthrobacter sp. strain ATCC plasmid found in Flavobacterium species. A pcpB gene probe 33790 catalyzes a NADPH- and oxygen-dependent dehaloge- hybridized with genomic DNA from Arthrobacter sp. strain nation, suggesting a monooxygenase-type of reaction (422, 423). ATCC 33790 (422, 423) and with DNA from Pseudomonas sp. The PCP 4-hydroxylase from Flavobacterium sp. strain ATCC strain SR3, but there was no hybridization with genomic DNA 39723 was purified. The purified enzyme catalyzed the incor- from Rhodococcus chlorophenolicus PCP-I (378). This is not poration of 180 from 1"02 but not from H2180 into the surprising, since para-hydroxylation of PCP by R chlorophe- reaction product tetrachlorohydroquinone, which unequivo- nolicus PCP-I, as well as by Mycobacterium fortuitum CG-2, is cally proved the monooxygenase mechanism of tetrachloro- catalyzed by a monooxygenase of another type. In both strains, hydroquinone formation (568). PCP 4-monooxygenase is a the dehalogenase activities were membrane associated and monomeric (63-kDa) NADPH-dependent flavoprotein mono- required FAD and NADPH as well as molecular oxygen. oxygenase containing FAD, which is closely related to the Uotila et al. (508, 509) suggested that cytochrome P-450-type external flavoprotein monooxygenases (565). The enzymes of monooxygenases may be involved in PCP para-hydroxylation this family contain a flavin and use aromatic compounds, and by R chlorophenolicus PCP-I and Mycobacterium fortuitum NADPH or NADH are cosubstrates. PCP 4-hydroxylase from CG-2. The product of the para-hydroxylation of PCP, tetra- Flavobacterium sp. strain ATCC 39723 is the first purified chlorohydroquinone, is further ortho-hydroxylated by a soluble flavoprotein monooxygenase which uses halogenated aromatic halohydroquinone dehalogenase in both R chlorophenolicus compounds as natural substrates. It is important to note that PCP-I and Mycobacteriumfortuitum CG-2. This ortho-hydroxy- for every 1 mol of PCP hydroxylated, 2 mol of NADPH is lating dehalogenase was not inhibited by cytochrome P-450 consumed (Fig. 11), whereas the hydroxylation of 2,3,5,6- inhibitors and did not require molecular oxygen. The further tetrachlorophenol requires only 1 mol of NADPH (568). Xun pathway of PCP mineralization proceeds via complete reduc- et al. (567) reported that PCP 4-monooxygenase from Fla- tive dehalogenation (see the section on haloaromatic com- vobacterium sp. strain ATCC 39723 catalyzes thepara-hydroxy- pounds and various pesticides, above) to 1,2,4-trihydroxyben- lation of a very broad range of substituted phenols (except zene, which is mineralized completely (508). VOL. 58, 1994 BACTERIAL DEHALOGENASES 653

fOOH COOH CH2 reduactase QOJ 2 L oxygenase -S HOHHC1t- OHOH [eO2J - 4ADH+H+ 2x 2x FMN le- 2fe-2SI 2xle* 2Fe-2S] COOHNAOH "Rieske Fe~

FIG. 15. Hypothetical scheme for the dehalogenation reaction catalyzed by the two-component 4-chlorophenylacetate 3,4-dioxygenase system from Pseudomonas sp. strain CBS3.

R chlorophenolicus PCP-I not only para-hydroxylated poly- benzoates (Fig. 16) (125, 126). Similar to 4-chlorophenylac- chlorinated phenols but also chlorinated guaiacols and syrin- etate 3,4-dioxygenase, this two-component enzyme system gols (168). Of all strains described so far, Streptomyces rochei constitutes a short electron transfer chain (Fig. 15). 2-Halo- 303 utilized the widest spectrum of chlorinated phenols, benzoate 1,2-dioxygenase consists of a monomeric flavin ade- ranging from 2- and 3-monochlorophenol, various di- and nine dinucleotide- and [2Fe-2S]-containing reductase compo- trichlorophenols, and 2,3,5,6-tetrachlorophenol to PCP. Chlo- nent and a [2Fe-2S]-containing heteromultimeric oxygenase rohydroquinones were formed as intermediates. 2,4,6-Trichlo- component. The enzyme system is structurally related to the rophenol, which was utilized preferentially, and 2,4-dichloro- (chromosomally encoded) benzoate 1,2-dioxygenase (360, phenol were metabolized via initial para-hydroxylation to 571-573) and to the isofunctional TOL plasmid pWWO-en- 2,6-dichloro-p-hydroquinone (157). Analogous results were coded toluate 1,2-dioxygenase (180, 559). Benzoate dioxygen- obtained by Kiyohara et al. (259) in a study on degradation of ase catalyzes the formation of cyclohexadiene-1,2-diol-1-car- 2,4,6-trichlorophenol by three strains ofPseudomonas pickettii. boxylic acid from benzoate, and toluate dioxygenase catalyzes Cells pregrown on 2,4,6-trichlorophenol converted various the formation of the corresponding dihydrodiols from benzo- polychlorinated phenols to the corresponding p-hydroqui- ate and meta- (orpara-) substituted benzoate analogs. Neither nones. Dechlorination by a cell extract of P. pickettii DTP0602 enzyme, however, converts ortho-substituted benzoates. Al- required FAD, NADH, and molecular oxygen, suggesting a though 2-halobenzoate 1,2-dioxygenase from P. cepacia 2CBS monooxygenase-catalyzed formation of the p-hydroquinone exhibited an extremely broad substrate specificity, it preferen- (259). Phenol and 2,4,6-trichlorophenol were the only phenolic tially turned over benzoate analogs with electron-withdrawing compounds which supported growth of Azotobacter sp. strain substituents at the ortho position, especially when the substitu- GP1. This strain possesses two independent pathways for ent was not too bulky (126). Since the enzyme preferred phenol and 2,4,6-trichlorophenol dissimilation. The latter also 2-halobenzoates as substrates, catalyzing a dehalogenation, it was metabolized via 2,6-dichloro-p-hydroquinone (290). 2,4,5- seems justified to call it a dehalogenase, although benzoate is Trichlorophenol, formed from the herbicide 2,4,5-T, is also also converted (yielding the dihydrodiol derivative) but with a degraded further via p-hydroxylation to 2,5-dichlorohydro- significantly lower reaction rate. The halobenzoate 1,2-dioxy- quinone by P. cepacia AC1100 (195, 416). Thus, the hydroqui- genase genes cbdA, cbdB, and cbdC, which were localized to a none pathway of halophenol degradation seems to occur plasmid designated pBaHl, were cloned and sequenced (166). predominantly in microorganisms that utilize more highly The deduced amino acid sequences of CbdABC showed chlorinated phenols like trichlorophenols and PCP, whereas significant homologies to XylXYZ (toluate 1,2-dioxygenase) less highly substituted halophenols usually are degraded via and to BenABC (benzoate 1,2-dioxygenase): amino acid iden- halocatechols (see the section on dehalogenation after cleav- tity between BenA, XylX, and CbdA was 45%; BenB, XylY, age of the aromatic ring, above). and CbdB shared 44% identical amino acids; and amino acid Dioxygenases. 4-Chlorophenylacetate is converted to 3,4- identity between BenC, XylZ, and CbdC was 38% (166). dihydroxyphenylacetate by a two-component dehalogenating Recently, an ortho-halobenzoate 1,2-dioxygenase from P. dioxygenase system from Pseudomonas sp. strain CBS3 (313). aeruginosa 142, which is isofunctional to the two-component The purified enzyme system consists of a monomeric flavin enzyme from P. cepacia 2CBS, was reported to comprise three mononucleotide- and [2Fe-2S]-containing reductase compo- components (403a). A dioxygenase attack on 2-halobenzoates, nent (35 kDa), which transfers electrons from the cosubstrate resulting in the release of the halogen during the decarboxyl- NADH to the terminal oxygenase component. The latter enzyme component is a homotrimeric iron-sulfur protein (144 kDa) containing three Rieske-type iron-sulfur clusters and probably mononuclear iron centers. This mononuclear iron is thought to mediate the electron transfer from the Rieske-type 0 ,1121 iron-sulfur cluster to molecular oxygen, thus activating the X0,2, dioxygen as an iron-peroxo complex. As the active oxygenating ~ ~ ~ Ispontneousj11jO species, [FeO2j+ is presumed to attack the substrate, yielding NADH JW C02+X the dihydroxylated product (Fig. 15) (314, 443). FIG. 16. Formation of catechol from 2-halobenzoates, catalyzed by Another dehalogenating two-component dioxygenase sys- the two-component 2-halobenzoate 1,2-dioxygenase from P. cepacia tem purified is 2-halobenzoate 1,2-dioxygenase from P. cepacia 2CBS (126) and by the three-component ortho-halobenzoate 1,2- 2CBS, which catalyzes the formation of catechol from 2-halo- dioxygenase from P. aeruginosa 142 (403a). 654 FETZNER AND LINGENS MICROBIOL. REV.

ation and rearomatization to catechol, was also assumed to COOH HCt OO occur in Streptomyces violaceoruber (486). x P. putida CLB250 was proposed to possess two benzoate XH2 AOH OH 1,2-dioxygenases with different substrate specificities. The en- COOH zyme present in 2-chlorobenzoate-grown cells preferentially attacked 2-substituted benzoates (112). P. aeruginosa JB2 metabolized to COOH 2-chlorobenzoate 3-chlorocatechol via a 1,6- H '2 X XH2 COOH dioxygenolytic attack, but 2,3- and 2,5-dichlorobenzoate were transformed to 4-chlorocatechol, indicating an initial dehalo- x H H HO l genation. On the basis of detection of spontaneous mutants HO with diminished ability to oxidize 2-chloro-, 2,3-dichloro-, and FIG. 17. Reactions catalyzed by 3-chlorobenzoate 3,4-/4,5-dioxyge- 2,5-dichlorobenzoate, it was suggested that strain JB2 pos- nase (359). sesses, in addition to benzoate dioxygenase, a halobenzoate dioxygenase necessary for the degradation of ortho-substituted benzoates (204). P. putida P1ll is the most versatile strain Halidohydrolases reported to date in the ability to utilize chlorinated benzoates as growth substrates. In strain Plll, dihydrodiol dehydroge- Since delocalization of the w-electrons considerably stabi- nase was induced only by 3- and 4-chlorobenzoate, not by lizes the aromatic ring system, it previously was thought ortho-substituted benzoates (202). The chromosomally en- "unlikely that bacteria have evolved enzymes for the direct coded benzoate dioxygenase from strain Plll catalyzes the hydrolysis of the aromatic carbon-halogen bond" (265). De- formation of the corresponding (chlorinated) dihydrodiols ethylsimazine, a nonhydroxylated s-triazine derivative, has from 3-chlorobenzoate, 4-chlorobenzoate, and benzoate. A considerable aromatic character, but, in contrast to the benze- separate chlorobenzoate 1,2-dioxygenase that converts ortho- noid ring, delocalization of the w-electrons is not complete. chlorobenzoates to the corresponding catechols without the Hydrolytic removal of substituents has been described for need for a functional dihydrodiol dehydrogenase was shown to various s-triazines (70-73, 161, 242). Cook and Hutter (73) be encoded on the 75-kb plasmid pPB111 (48). High frequen- have shown that two isofunctional but different enzyme frac- cies of plasmid loss in the absence of ortho-chlorobenzoates in tions from Rhodococcus corallinus NRRL B-15444R hydrolyti- the culture media were observed with both P. putida P11 (48) cally dechlorinated deethylsimazine to N-ethylammeline (Fig. and P. cepacia 2CBS (165). Cured strains of both Plll and 18). No cofactors were required for dechlorination. This 2CBS were unable to utilize ortho-chlorobenzoates for growth. hydrolytic substitution at the aromatic ring is chemically fea- In conclusion, strains CLB250, JB2, and Plll presumably sible because of the low electron density at the ring carbon harbor two separate enzymes, a 2-halobenzoate dioxygenase atoms. and a benzoate dioxygenase, whereas strain 2CBS possesses A number of reports indicate that hydrolytic dehalogenation the 2-halobenzoate of haloaromatic compounds may occur in the first step of only plasmid-encoded 1,2-dioxygenase. 3-chlorobenzoate degradation (231) and 4-chlorobenzoate The genes encoding the 2-halobenzoate 1,2-dioxygenase degradation (6, 261, 262, 312, 347, 348, 408, 451, 516, 517, 578, components ofP. cepacia 2CBS have been shown to be located 579), and possibly also in the formation of salicylate from on a 60-kb degradative plasmid designated pBaHl (166). In 2-bromobenzoate (207). contrast, the genes specifying the conversion of 2-chloroben- Hydrolytic dehalogenation of 4-chlorobenzoate by Pseudo- zoate and its further degradation by P. cepacia KZ2 were monas sp. strain CBS3 was found to require three proteins, proposed to be chromosomal genes (580). Utilization of ATP, Mg2+, and CoA (108, 109, 300). Elsner et al. (109) 2-chlorobenzoate was found to be plasmid encoded in P. separately cloned the genes encoding two proteins of the cepacia HCV 2,6-DCT (514) and in P. aeruginosa B16 (454), 4-chlorobenzoate dehalogenase system in E. coli. Savard et al. but there are no data on the metabolic route of 2-chloroben- (420), who cloned the genes specifying 4-chlorobenzoate de- zoate catabolism or the enzymes involved. halogenation on a large DNA fragment, suggested that these Alcaligenes sp. strain BR60 (see also the section on applica- genes are located on the chromosome of Pseudomonas sp. tion of microbial systems in environmental protection technol- strain CBS3. In the conversion of 4-chlorobenzoate to 4-hy- ogy and waste management, below) carries an unstable 85-kb droxybenzoate by Pseudomonas sp. strain CBS3, Loffler and plasmid designated pBRC60 (formerly pBR60), which speci- Muller (299) identified 4-chlorobenzoyl-CoA as an intermedi- fies 3- and 4-chlorobenzoate catabolism (562, 563). The chlo- ate in the dehalogenation reaction and proposed the reaction robenzoate-catabolic genes were localized to the 17-kb trans- mechanism shown in Fig. 19. In the first step, a 4-chloroben- poson TnS271, which resides in the degradative plasmid zoate-CoA ligase catalyzes the adenylation of the carboxyl pBRC60 or in the chromosome ofAlcaligenes sp. strain BR60 group of 4-chlorobenzoate, followed by displacement of the (358, 359, 562). Nakatsu and Wyndham (359) proposed a novel AMP with a thiol group from CoA, leading to the formation of mode of dioxygenolytic dehalogenation of 3-chlorobenzoate the thioester 4-chlorobenzoyl-CoA. The formation of the CoA via 3,4-dioxygenolytic attack, yielding protocatechuate, which ester activates the substituent in the para position for a subsequently underwent meta cleavage. The novel dioxygenase attack and enables the was encoded on TnS271. The authors suggested that the en- nucleophilic substitution of the chlorine zyme is a two-component enzyme system similar to other dioxygenases attacking (halo)aromatic compounds (see above). The 3-chlorobenzoate dioxygenase from strain BR60 was not strictly regioselective: it catalyzed both a 3,4-attack, yielding protocatechuate, and a 4,5-attack, yielding 5-chloroprotocat- HA N -CH2-CH3 H20 HQ N-cH2--c3 echuate via an additional dihydrodiol dehydrogenase reaction H (Fig. 17). Thus, the enzyme was designated 3-chlorobenzoate FIG. 18. Dechlorination of deethylsimazine by halidohydrolases 3,4-(4,5-)dioxygenase (359). from R corallinus NRRL B-15444R (73). VOL. 58, 1994 BACTERIAL DEHALOGENASES 655

COOH COSCoA II COSCoA m OOH cated on the 120-kb plasmid pASUl. Similarly, the genes CoASH I specifying the 4-chlorobenzoate dehalogenase of Arthrobacter /-Mg--"\ globiformis KZT-1 were proposed to be located on a 110-kb ATP Yt AM-PPE, OH HIO CoASH plasmid designated pBS1501 (504, 580). There were extensive yIIll' sequence homologies of the putative 4-chlorobenzoate-CoA FIG. 19. Dechlorination of 4-chlorobenzoate to 4-hydroxybenzoate ligase gene (fcbA) and the 4-chlorobenzoyl-CoA dehalogenase by Pseudomonas sp. strain CBS3 (298-301, 435).I, 4-Chlorobenzoate- gene (fcbB) from Arthrobacter sp. strain SU with the corre- CoA ligase; II, 4-chlorobenzoyl-CoA dehalogenase; III, 4-hydroxyben- sponding genes from Pseudomonas sp. strain CBS3. However, zoyl-CoA thioesterase. Redrawn from reference 301. gene fcbC from Arthrobacter sp. strain SU, possibly specifying the thioesterase, showed no homology with the corresponding gene of strain CBS3. The order or the dehalogenation genes by a hydroxy group from water, catalyzed by a dehalogenase. also differed between Arthrobacter sp. strain SU (CoA ligase- No cofactors were required for the dehalogenation reaction. dehalogenase-thioesterase) and Pseudomonas sp. strain From genetic data, it was deduced that 4-hydroxybenzoyl-CoA CBS3 (dehalogenase-CoA ligase-thioesterase) (431). is formed as an intermediate of the reaction sequence, and a The biodegradation of 4-chlorobiphenyl by Alcaligenes sp. third enzyme, 4-hydroxybenzoyl-CoA thioesterase, was sug- strain ALP83 proceeds via 4-chlorobenzoate and 4-hydroxy- gested to be involved in the reaction (435) (Fig. 19). Loffler et benzoate. The dehalogenase activity was localized to a 10-kb al. (298,301) purified the 4-chlorobenzoate-CoA ligase and the fragment carried on plasmid pSS70 (284). pSS70 was similar to 4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp. pSS50 from the 4-chlorobiphenyl-mineralizing strain Alcali- strain CBS3. The ligase was a 115-kDa homodimer which genes sp. strain A5 (448), with pSS70 possessing an additional catalyzed the formation of CoA thioesters with 4-iodo-, 4-bro- 13-kb fragment, which was correlated to the dechlorination mo-, 4-chloro-, and 4-fluorobenzoate. Whereas 4-iodo-, 4-bro- activity. mo-, and 4-chlorobenzoyl-CoA were subsequently dehaloge- Analogous to strains CBS3 and SU discussed above, Acin- nated by 4-halobenzoyl-CoA dehalogenase, 4-fluorobenzoyl- etobacter sp. strain 4-CB1 also degrades 4-chlorobenzoate via CoA was not defluorinated. The dehalogenase, with a 4-chlorobenzoyl-CoA and 4-hydroxybenzoyl-CoA to 4-hy- molecular mass of 120 kDa, was composed of four identical droxybenzoate (74). Dehalogenation of 4-bromobenzoyl-CoA subunits (298, 301). was twice as fast as dehalogenation of 4-chlorobenzoyl-CoA, A fragment of chromosomal DNA of strain CBS3 was while defluorination of 4-fluorobenzoyl-CoA was over 400-fold cloned in E. coli and in P. putida KT2440. Three translation slower than 4-chlorobenzoyl-CoA dehalogenation (79). This products, 57-, 30-, and 16-kDa polypeptides, were identified substrate specificity seems similar to that of the dehalogenase (419). Homodimeric 4-chlorobenzoate-CoA ligase (114 kDa, from Pseudomonas sp. strain CBS3, which does not convert with subunits of 57 kDa), homotetrameric 4-chlorobenzoyl- 4-fluorobenzoyl-CoA (298). Cells of Acinetobacter sp. strain CoA dehalogenase (122 kDa, with subunits of 30 kDa), and 4-CB1 grown on 4-chlorobenzoate cometabolized 3,4-dichlo- homotetrameric 4-hydroxybenzoyl-CoA thioesterase (66 kDa, robenzoate to 3-chloro-4-hydroxybenzoate, which could be with subunits of 16 kDa), were isolated from E. coli clones used as a growth substrate. In both aerobic and anaerobic which expressed the respective genes from Pseudomonas sp. resting-cell incubations, 4-carboxy-1,2-benzoquinone was a fur- strain CBS3 (62). Investigating the ancestry of the 4-chloro- ther intermediate of 3,4-dichlorobenzoate and 3-chloro-4- benzoate dehalogenase system, Babbitt et al. (22) found that hydroxybenzoate catabolism. A hydration mechanism ap- 4-chlorobenzoyl-CoA dehalogenase shows significant sequence peared to be responsible for the conversion of 3-chloro-4- similarities with 2-enoyl-CoA hydratases from rat liver mito- hydroxybenzoate to 4-carboxy-1,2-benzoquinone via an chondria and with the 2-enoyl-CoA hydratase domains of both unstable chlorohydrodiol intermediate. Thus, in contrast to the the multifunctional enzymes from rat liver peroxisomes and E. degradation of 4-chlorobenzoate, 3-chloro-4-hydroxybenzoate coli. These data suggested that the dehalogenase may have a was not metabolized via the protocatechuate pathway (5). common ancestry with the enoyl-CoA hydratases of the fatty Resting cells of the coryneform strain NTB-1, which trans- acid P-oxidation pathway of mitochondrial, eukaryotic, perox- forms 2,4-dichlorobenzoate via initial reductive dechlorination isomal, and bacterial origins (22). Both the 2-enoyl-CoA to 4-chlorobenzoate (see the section on haloaromatic com- hydratase and the dehalogenase activate water for nucleophilic pounds and various pesticides, above), converted 4-chloroben- addition across a carbon-carbon bond that is conjugated with a zoate to 4-hydroxybenzoate under strictly anaerobic conditions CoA thioester group. However, the substrates of the 2-enoyl- in the presence of ferricyanide or nitrate as the electron CoA hydratases were not accepted by 4-chlorobenzoyl-CoA acceptor. 4-Chlorobenzoyl-CoA was suggested as an interme- dehalogenase (62). 4-Chlorobenzoate-CoA ligase showed sig- diate in this anaerobic dehalogenation reaction (160). nificant sequence similarity with other enzymes belonging to It is interesting that in anaerobic systems, the degradation of the large family of acyl-adenyl ligases. Thus, it was proposed aromatic acids via the formation of CoA esters is a common that 4-chlorobenzoate-CoA ligase (as well as 4-chlorobenzoyl- process (13, 35, 116, 148, 193, 318, 330, 424, 434, 584). CoA dehalogenase) may have evolved from the P-oxidation However, the aerobic metabolism of phenylacetate in a variety pathway (22). However, 4-chlorobenzoate-CoA ligase was of microbial strains also proceeds via a CoA thioester (124, inactive with aliphatic carboxylic acids, thus being catalytically 316, 317, 534). The aerobic oxidation of benzoate by the distinct from the enzymes involved in fatty acid P-oxidation denitrifying Pseudomonas strain KB740 was shown to proceed (62). There were no proteins homologous in sequence to the via benzoyl-CoA and 3-hydroxybenzoyl-CoA. Strain KB740 4-hydroxybenzoate-CoA thioesterase, suggesting that the thio- possesses (i) an aerobically induced benzoate-CoA ligase, (ii) esterase may have been recruited from a different pathway an aerobically induced 3-hydroxybenzoate-CoA ligase, (iii) an (22). aerobically induced 2-aminobenzoate-CoA ligase, (iv) an The genes encoding the 4-chlorobenzoate dehalogenase anaerobically induced 2-aminobenzoate-CoA ligase, (v) an system ofArthrobacter sp. strain SU (347) also were cloned and anaerobically induced phenylacetate-CoA ligase, and (vi) an expressed in E. coli (431). In strain SU, the three genes anaerobically induced 4-hydroxyphenylacetate-CoA ligase encoding CoA ligase, dehalogenase, and thioesterase are lo- (12-14, 329, 330). Thus, degradation of aromatic acids via CoA 656 FETZNER AND LINGENS MICROBIOL. REV.

reactions, and identity of the 15 N-terminal amino acids (268). CH202 (SCH2C /_ - 16S-CH2CI0i2H GSH HCI H20 HCt t These observations suggested that either the dehalogenase was horizontally distributed among the methyl- glutathione CH20 + GSH structural gene S -transferase otrophic bacteria studied or the dehalogenases evolved very FIG. 20. Hypothetical pathway for the formation of formaldehyde recently from a common ancestral gene. Concerning horizon- and inorganic chloride from dichloromethane by Hyphomicrobium sp. tal gene transfer, however, the dichloromethane utilization strain DM2 (146, 270). genes of Methylobacterium sp. strain DM4 were shown to be located either on the chromosome or on a still undetected megaplasmid (144). Three cryptic plasmids carried by strain esters appears not to be restricted to anaerobic systems but is DM4 are unrelated to dichloromethane metabolism. LaRoche also involved in aerobic degradation processes. and Leisinger (281, 282) determined the nucleotide sequence of the dichloromethane dehalogenase structural gene (dcmA) HALOALKANE DEHALOGENASES and of the regulatory gene (dcmR) governing the expression of dichloromethane dehalogenase from Methylobacterium sp. strain DM4. dcmR encodes a trans-active protein (repressor) Oxygenases controlling dcmA expression as well as its own synthesis (282). As a general rule, haloalkanes with long carbon chains, such Alignment of the deduced amino acid sequences of function- as 1,9-dichlorononane, are dehalogenated oxidatively (374, ally related eukaryotic glutathione S-transferases with dcmA 375, 574), whereas haloalkanes with short carbon chains are revealed three regions containing highly conserved amino acid dehalogenated by a glutathione-dependent reaction or hydro- residues, which indicated that dcmA is a member of the lytically (see below). Oxygenase-catalyzed dehalogenation re- glutathione S-transferase supergene family. Dichloromethane actions of halogenated methanes, ethanes, and ethylenes are dehalogenase from Methylobacterium sp. strain DM4 showed due to multifunctional enzymes with broad substrate specificity some sequence similarities to mammalian glutathione S-trans- like methane monooxygenase or involve enzymes from aro- ferases belonging to the theta class (319). Mammalian gluta- matic degradative pathways. Oxidation can lead to dehaloge- thione S-transferases formerly were grouped into three distinct nation as a result of the formation of labile products that classes, designated alpha, mu, and pi (309). Theta was de- undergo chemical decomposition (see the section on oxygen- scribed as a novel class of glutathione S-transferases by Meyer ases in spontaneous or fortuitous dehalogenation reactions, et al. (319). The mammalian enzymes belonging to the theta above). There are no reports on oxygenolytic dehalogenases class show little relatedness to the enzymes of the alpha, mu, or specific for halogenated alkanes. pi classes. Further studies are necessary to investigate the relationship between bacterial glutathione S-transferases (dichloromethane dehalogenases) and the mammalian glutathi- Glutathione S-Transferases one S-transferases of this novel class, theta. Dichloromethane is converted to formaldehyde and inor- In 1988, Scholtz et al. (439) isolated Methylophilus strain ganic chloride by various Pseudomonas strains (51, 143), DM11, which, because of the presence of a highly active Hyphomicrobium strains (268, 270, 474), and Methylobacterium dichloromethane dehalogenase, grows faster than the original sp. strain DM4 (143, 268). An inducible dichloromethane DM isolates Hyphomicrobium strain DM2 and Methylobacte- dehalogenase was purified from Hyphomicrobium sp. strain num strain DM4. Characterization of the structural and kinetic DM2. It was characterized as a 195-kDa homohexameric properties of the DM11 enzyme suggested that there are two protein with a subunit molecular mass of 33 kDa, which groups of dichloromethane-dehalogenating glutathione catalyzes the formation of formaldehyde from dichlorometh- S-transferases, which are immunologically distinct, show dif- ane in the presence of glutathione (270, 288). A glutathione ferent reaction rates, and have different N-terminal amino acid S-transferase from rat liver cytosol also was reported to sequences. Group A enzymes are represented by the dehalo- catalyze the conversion of dihalomethanes to formaldehyde. genases from Hyphomicrobium strain DM2 and Methylobacte- The reaction proceeded via an S-halomethylglutathione inter- rium strain DM4 (268). They have a subunit molecular mass of mediate (9). To differentiate between nucleophilic mechanisms 33 kDa and an a6 structure, whereas the group B enzyme from and a potential deprotonation/halide elimination sequence to a Methylophilus strain DM11 has a subunit mass of 34 kDa and carbene intermediate, dideuterodichloromethane was incu- an a2 structure. There is no evidence for the presence of metals bated in the presence of glutathione with cell extracts of strain or other prosthetic groups in dichloromethane dehalogenases. DM2 (146) and with dichloromethane dehalogenase from All dichloromethane dehalogenases exhibit stringent substrate strain DM11 (538). In both cases, dideuteroformaldehyde was specificity, since only dihalomethanes serve as substrates. found as a product. The deuterium retention in the product However, the enzymes from both strains DM4 and DM11 were argues for a direct halide displacement and excludes elimina- inhibited by low concentrations of haloacetonitriles like chlo- tion-addition or oxidation-reduction reactions in the formation roacetonitrile, which appear to compete with the substrate for of formaldehyde from dichloromethane. These results support binding to the same site (302). Despite structural and kinetic a mechanism involving a nucleophilic substitution by glutathi- differences between the group A and B enzymes, there is one, yielding an S-chloromethyl glutathione conjugate. This potentially some evolutionary relatedness between group A chloromethyl thioether is likely to undergo rapid hydrolysis in and group B dichloromethane dehalogenases, since a gene an aqueous environment. The hemimercaptal thus formed is in probe prepared from cloned DNA of strain DM4 hybridized equilibrium with glutathione and formaldehyde (Fig. 20) (146, with DNA from strain DM11 (439). 538). A presumably hydrolytic mechanism of dichloromethane Comparison of dichloromethane dehalogenases from two dechlorination by a strictly anaerobic, fermentative bacterium Hyphomicrobium strains, one Pseudomonas sp., and Methyl- (strain DMA) was proposed by Braus-Stromeyer et al. (47). obacterium sp. strain DM4 revealed similar subunit molecular However, this hydrolytic dehalogenation reaction appeared to masses, similar substrate ranges, strong immunological cross- be different from the glutathione-dependent dehalogenation VOL. 58, 1994 BACTERLAL DEHALOGENASES 657

CH2Ct-CH2CI H A) 100 R hatc alkane halidohydrolase enz- RI RI H + ee400 -0 21 R2 CH2a-CH2OH R2 X-, H+

atc ohol dehydrogenase B) POQH2 __H R, RO,. CH2CI-CHO enz-BH: H R2 \ enz- + H H 4.HOH IH20+NAD R2 x- R2 aldehyde dehydrogenase INADH- H' FIG. 22. Possible mechanisms for the reaction catalyzed by haloal- CH2C(-COOH kane dehalogenase from strain GJ70. (A) Nucleophilic catalysis involv- H 20 ing a carboxylic residue; (B) general base catalysis with activated water hatoalkanoic acid halidohydrolase HCt as the nucleophile (redrawn from reference 224). CH20H-COOH (137) (Fig. 22A). Incubation of DhlA with 1,2-dichloroethane central metabolic pathwas in the presence of H218O, which resulted in the incorporation FIG. 21. Proposed route for the degradation of 1,2-dichloroethane of 180 into 2-chloroethanol and into the carboxylate group of byX autotrophicus GJ10 according to Janssen et al. (229). Apart from Asp-124, supported the proposed mechanism via a covalent the haloalkane and haloalkanoic acid dehalogenase activities, the ester intermediate. The role of Asp-124 also was investigated presence of 2-chloroethanol dehydrogenase suggested the catabolism by site-directed mutagenesis. Mutant enzymes with Ala, Gly, or of 1,2-dichloroethane to carboxylic acids (redrawn from reference Glu instead of Asp at position 124 showed no activity toward 229). 1,2-dichloroethane and 1,2-dibromoethane (389a). Final proof for the proposed reaction mechanism was obtained by crystallographic analysis of different reaction intermediates in crystals mechanism of the aerobic dichloromethane-utilizing bacteria of haloalkane dehalogenase DhIA (532, 533). Crystal struc- discussed above. tures of DhIA were determined in the presence of the substrate 1,2-dichloroethane. The substrate was bound to the active site, Haloalkane Halidohydrolases which is located between the two domains of the protein in an internal, predominantly hydrophobic cavity. Refinement of the Direct hydrolytic dehalogenation of haloalkanes was first crystal structure at 1.9-A (0.19-nm) resolution showed that the found with the haloalkane dehalogenase from the nitrogen- nucleophilic residue Asp-124 and the two other active-site fixing hydrogen bacterium Xanthobacter autotrophicus GJ10 residues, His-289 and Asp-260, are situated in this internal (229). Because of the presence of two halidohydrolases, strain cavity. Asp-124 performs a nucleophilic attack on the sub- GJ10 is capable of rapid utilization of 1,2-dichloroethane (Fig. strate, resulting in an ester intermediate. The crystal structure 21). Both dehalogenases in X autotrophicus GJ10 are synthe- of the enzyme alkylated at Asp-124 by a substrate molecule was sized constitutively. The haloalkane dehalogenase gene dhlA also obtained. In the second step of the catalytic mechanism, was cloned and sequenced (228). It appeared to encode a the alkyl-enzyme intermediate was hydrolyzed by a water 310-amino-acid polypeptide (molecular mass, 35,143 Da). In molecule, which is activated by the His-289-Asp-260 pair in the 1991, Tardif et al. (490) isolated a 200-kb plasmid designated active site. Verschueren et al. (533) investigated the three- pXAU1 from X autotrophicus GJ10. This plasmid was shown dimensional structure of the dehalogenase with chloride bound to contain the dhA gene coding for haloalkane halidohydro- in the active site after the alcohol product was released. Thus, lase, whereas the gene for 2-haloalkanoic acid halidohydrolase, Asp-124, His-289, and Asp-260 apparently form a catalytic the second dehalogenase in strain GJ10, seems to be located triad, with the triad residues occurring in the same topological on the chromosome (see the section on 2-haloalkanoic acid positions as in other members of the ao/1-fold hydrolase family. halidohydrolases, below). Plasmid pXAU1 is the first plasmid Corresponding catalytic triads are described for a number of reported to be involved in haloalkane degradation (490). such hydrolases, for instance serine proteases (36, 292, 397, Haloalkane dehalogenase from strain GJ10 dehalogenated 398), lipases (46, 442, 553), phospholipase A2 (90), acetylcho- C-1 to C-4 a-halogenated n-alkanes and a,w-dihalogenated line esterase (479), and dienelactone hydrolase from Pseudo- n-alkanes, but it was inactive toward halogenated carboxylic monas sp. strain B13 (382, 383). Considering the two domains acids. Since no oxygen and no cofactors were required for of haloalkane dehalogenase, the main domain shows structural activity, nucleophilic displacement with water was suggested as similarities with other hydrolytic proteins of the ao/,-fold type. the mechanism of halide release. Thiol reagents strongly The second domain, designated the cap domain, forms a inhibited the enzyme. Therefore, it was first assumed that a separate part of the enzyme that is absent or different in other cysteine is located in the active center, and a reaction mecha- hydrolytic proteins of the a/a-fold type; this suggests that the nism involving a thioether intermediate, as proposed by Gold- cap domain functions as an activity-modifying domain. Van der man (153), was suggested (251). To obtain more insight into Ploeg et al. (526) showed that the substrate specificity of the the mechanism of the catalytic reaction, haloalkane halidohy- haloalkane dehalogenase DhlA from strain GJ10 was modified drolase DhlA from strain GJ10 was purified, characterized, by mutations in the cap domain. Such a strategy might also be and crystallized, and its three-dimensional structure was deter- useful for the construction of strains for the bioremediation of mined (137, 251, 407). The structural information on haloal- recalcitrant compounds. Strain construction may involve two kane halidohydrolase, however, seemed to rule out the possi- strategies, namely, (i) the combination of existing catabolic bility that a cysteine is involved in the reaction mechanism. routes in a single organism and (ii) the development of From the structural analysis, it was concluded that Asp-124 is catabolic enzymes with activities that are new or much higher the nucleophilic residue attacking the substrate. The assumed than the activities found in natural isolates (526) (see also the covalent intermediate would be an ester, which must subse- section on application of microbial systems in environmental quently be cleaved by a water molecule, releasing the alcohol protection technology and waste management, below). 658 FETZNER AND LINGENS MICROBIOL. REV.

TABLE 1. Haloalkane halidohydrolases

Source Name Mol(Da)mass Substrates Reference(s) X autotrophicus GJ10 DhlA 35,143 C-1 to C-4 1-chloro-n-alkanes, C-1 to 227, 251 C-12 1-bromo-n-alkanes, C-2 to C-3 a,o-dihalo-n-alkanes, 1-iodopropane Strain GJ70 28,000 Halomethanes, C-2 to C-5 1-halo-n- 224,226 alkanes, C-3 to C-5 2-bromo-n- alkanes, C-2 to C-9 a,w-dichloro-n- alkanes, 1,2-dibromopropane, C-2 to C-6 halogenated alcohols, bis(2-chloroethyl)ether Corynebacterium sp. strain m15-3 36,000 Bromoethane, 1,2-dibromoethane, C-2 575 to C-9 1-chloro-n-alkanes, C-2 to C-9 a,c-dichloro-n-alkanes, C-3 to C-4 .a-chloro-w-alkanols R erythropolis Y2 34,000 C-3 to C-16 1-chloro-n-alkanes, C-2 to 413 C-10 a,w-dichloro-n-alkanes, C-2 to C-4 a-chloro-w-alkanols, bis(2-chloroethyl)ether Rhodococcus sp. strain HAl 1-Chlorohexane 36,000 C-1 to C-7 1-iodo-n-alkanes, C-1 to C-9 436, 438 (formerly Arthrobacter sp. halidohydrolase 1-bromo-n-alkanes, C-2 to C-10 strain HA1)a 1-chloro-n-alkanes, C-2 to C-10 a,w-dibromo-n-alkanes, C-3 to C-6 aL,w-dichloro-n-alkanes, C-4 to C-6 aL-chloro-w-alkanols P. paucimobilis UT26 LinB 33,100 1-chlorobutane, 1-chlorononane, 352 1-chlorodecane, 2-chlorobutane, 2-chlorooctane, 3-chlorohexane

Analogous to X. autotrophicus GJ10, Ancylobacter aquaticus suggested a periplasmic hydrolytic dehalogenase (452), but the AD20, AD25, and AD27 and other facultative methanotrophs enzyme has not been characterized yet. also degrade 1,2-dichloroethane via initial hydrolytic dechlori- A number of haloalkane halidohydrolases have been found nation to 2-chloroethanol. The haloalkane dehalogenase from in gram-positive bacteria. The 1-chlorobutane-utilizing Coryne- strains AD25 and AD27 also catalyzed the dehalogenation of bacterium sp. strain m15-3 contains an inducible haloalkane different ,B-chloroethers such as 2-chloroethylvinylether (518). dehalogenase, which hydrolyzes 1-halo-n-alkanes and a,w- The nucleotide sequences of the haloalkane dehalogenase dihalo-n-alkanes (chain length, C-2 to C-9) and some chloroal- genes of strains AD20 and AD25 were the same as the cohols (574, 575). The haloalkane halidohydrolase from the sequence of dhlA from X autotrophicus GJ10 and GJ11, and 1-chlorohexane-utilizing organism Arthrobacter sp. strain HAI the genes were located on identical restriction fragments. The is a 36- to 37-kDa monomer which, in contrast to the dehalo- organisms may have obtained the dhL4 gene by horizontal gene genases from strain GJ10 or GJ70 (described above), showed transmission (520). low sensitivity toward sulfhydryl reagents (436). However, as Janssen et al. (224) investigated the haloalkane dehaloge- yet there are no data on the mechanism of the catalytic nase from strain GJ70 (formerlyAcinetobacter sp. strain GJ70 reaction. The biochemical characteristics of the haloalkane [226]). This dehalogenase was found to be an inducible mono- halidohydrolase from Rhodococcus erythropolis Y2 closely re- meric enzyme (28 kDa) showing a broad substrate range (see sembled those of the enzymes from Corynebacterium sp. strain Table 1). Optically active 2-bromobutane was converted with m15-3 and Arthrobacter sp. strain HAI. Moreover, the 17 first inversion of configuration at the chiral carbon atom, suggesting amino acid residues of the N-termini of the Rhodococcus and that the dehalogenase reaction proceeds by a nucleophilic Arthrobacter enzymes were identical (413). This degree of substitution reaction involving a carboxyl group or a general homology raises interesting questions about the evolution of base catalysis (Fig. 22B). these enzymes, since these actinomycetes were isolated from A wide range of 1-bromoalkanes were biodegraded by two geographically separate industrial sites. Pseudomonas isolates, designated ES-1 and ES-2 (452). Strain Haloalkane-degrading organisms usually possess a single ES-2 grew on 1-bromoalkanes containing 6-to 18 carbon atoms enzyme active toward a broad range of haloalkanes. However, and on 1-chloroalkanes with 10 to 18 carbon atoms. Utilization Scholtz et al. (437) presented evidence for the presence of of water-insoluble haloalkanes, such as 1-bromooctane, ap- three inducible haloalkane dehalogenases in Arthrobacter sp. peared to proceed via extracellular emulsification by a consti- strain HAl, indicating that haloalkane utilizers may also tutively excreted broad-spectrum surface-active reagent. The possess multiple enzyme forms, as demonstrated for the chemical nature of this excreted surface-active compound 2-haloacid-utilizing bacteria (see the section on 2-haloalkanoic remained undetermined. Dehalogenation was probably associ- acid halidohydrolases, below). All haloalkane dehalogenases ated with the outer surface of the cell membrane. The authors known so far are composed of a single polypeptide chain of VOL. 58, 1994 BACTERIAL DEHALOGENASES 659

product 1,2,4-trichlorobenzol (Fig. 23) (219, 220, 352). This Cl dehydrochlorination will be discussed in the following section. a I 1,3,4,6-Tetrachloro-1,4-cyclohexadiene is metabolized to 2,5- dichloro-2,5-cyclohexadiene-1,4-diol by two steps of hydrolytic LinA VHa dehalogenation via the chemically unstable intermediate 2,4,5- 2 trichloro-2,5-cyclohexadiene-1-ol (Fig. 23). The hydrolytic dehalogenation is catalyzed by a halidohydrolase designated LinB. ThelinB gene, which is located on the chromosome of LinA [-HCl strain UT26, encodes a 33.1-kDa protein. The deduced amino

6 acid sequence showed significant similarity (29.3% overall spontaneous to the [ aCct ] Cl c identity) plasmid-encoded haloalkane halidohydrolase H20 - HQ DhlA from X. autotrophicus GJ10 (see above). Sequence LinB- --HQl similarities were also found to DmpD (2-hydroxymuconic clN LOH spontaneous Cl (OH semialdehyde hydrolase from Pseudomonas strain CF600) and TodF (2-hydroxy-6-oxo-2,4-heptadienoate hydrolase from P. _ Cl Cl HCt putida Fl), suggesting that there may exist a common ancestral LinB J-HCl "hydrolase domain" found in a variety of hydrolases. This includes not only haloalkane halidohydrolases but also some Cl2OH haloacid halidohydrolases (see the section on 2-haloalkanoic acid hydrolases [below] for further discussion). LinB may I catalyze hydrolytic dehalogenation via the same mechanism as DhlA, as follows. Two of the three deduced active-site residues FIG. 23. Proposed pathway for the metabolism of lindane (-y- are conserved between and and hexachlorocyclohexane) in P. paucimobilis UT26 (352). 1, -y-Hexachlo- DhlA LinB, the residues rocyclohexane; 2, y-pentachlorocyclohexene; 3, 1,3,4,6-tetrachloro-1,4- surrounding Asp-124 and His-284 of DhlA and Asp-108 and cyclohexadiene (chemically unstable); 4, 2,4,5-trichloro-2,5-cyclo- His-271 of LinB are strongly conserved. Glu-243 of LinB may hexadiene-1-ol (chemically unstable); 5, 2,5-dichloro-2,5-cyclohexa- play the role corresponding to Asp-260 of DhlA. Thus, amino diene-1,4-diol; 6, 1,2,4-trichlorobenzene; 7, 2,5-dichlorophenol. LinA, acid residues putatively forming a catalytic triad are also -y-hexachlorocyclohexane dehydrochlorinase; LinB, halidohydrolase. present in LinB. LinB catalyzed the dechlorination of a wide Redrawn from reference 352. range of monochloroalkanes including 1-chlorodecane, 1-chlorononane, 1-chlorobutane, 2-chlorobutane, 2-chlorooctane, and 3-chlorohexane. Some of these compounds are poor substrates for other dehalogenases (352). molecular mass between 28 and 36 kDa. Table 1 shows a list of the haloalkane dehalogenases known to date. Haloalkane Dehydrohalogenases halidohydrolases are of similar size to 2-haloacid halidohydrolases, but there are no common substrates and no immunolog- As mentioned above, P. paucimobilis UT26 converts y-HCH ical cross-reactivities between the haloalkane dehalogenases by two steps of dehydrochlorination to the chemically unstable and 2-haloacid dehalogenases tested so far (181, 251). Thus, intermediate 1,3,4,6-tetrachloro-1,4-cyclohexadiene, which is haloalkane halidohydrolases form a distinct class of enzymes. the substrate for the hydrolytic dehalogenase LinB discussed It is interesting that most haloalkane halidohydrolases are also above (Fig. 23). The elimination of HCl from both -y-HCH and active toward haloalcohols. However, this type of dehalogena- ,y-pentachlorocyclohexene is catalyzed by a dehydrochlorinase tion, yielding alcohols, differs mechanistically from the ep- designated LinA. Imai et al. (219) have cloned and sequenced oxide-forming reaction catalyzed by haloalcohol dehaloge- the linA gene from P. paucimobilis UT26. Southern hybridiza- nases, which form a distinct class of dehalogenases (see the tion analyses revealed no linA-homologous sequences in other section on halohydrin dehalogenases, below). Pseudomonas strains. When the nucleotide sequence of the -y-HCH, known as the insecticide lindane, is degraded by linA gene was compared with gene sequences from databases, various aerobic microorganisms. Aerobic degradation of lin- again no homologous sequences were found. A eukaryotic dane via y-pentachlorocyclohexene by various soil organisms dechlorinase known since 1959 (294, 295) is DDT dehydro- was found by Haider et al. (173, 174) and by Tu (506). An chlorinase from Musca domestica (the house fly), which cata- investigation of the biodegradation of a- and p-HCH under lyzes the monodehydrochlorination of 1,1,1-trichloro-2,2-bis different redox conditions revealed that aerobic conditions (4-chlorophenyl)ethane (DDT) and probably also of y-hexa- were best for the conversion of a-HCH in a soil slurry, whereas chlorocyclohexane and -y-pentachlorocyclohexene. This en- under denitrifying and sulfate-reducing conditions, no signifi- zyme belongs to the glutathione S-transferase family (221). In cant bioconversion of a-HCH was observed. The P isomer was a comparison of the amino acid sequence of LinA with the recalcitrant at all of the redox conditions studied (23, 24). sequences of several eukaryotic and bacterial glutathione However, a Pseudomonas sp. has been reported to degrade a-, S-transferases, no homology was found. Moreover, purified P-, and -y-HCH under aerobic conditions. Degradation of all LinA did not show glutathione S-transferase or DDT dechlo- three isomers led to the release of chloride in stoichiometric rinase activity in the presence of glutathione (219). Nagata et amounts. y-Pentachlorocyclohexene was formed as an inter- al. (351) purified LinA from an E. coli clone expressing the linA mediate of -y-HCH degradation (412). Conversion of hexachlo- gene from P. paucimobilis UT26. LinA catalyzed the release of rocyclohexane isomers by anaerobic bacteria has also been three chloride ions per molecule of y-HCH. Its substrate reported (see the section on haloaromatic compounds and specificity was very narrow: ox-HCH, y-HCH, 8-HCH, at-pen- various pesticides, above). In Pseudomonaspaucimobilis UT26, tachlorocyclohexene, and y-pentachlorocyclohexene were the ,y-HCH is converted by dehydrochlorination via y-pentachlo- only substrates converted. It was concluded that LinA catalyzes rocyclohexene to the unstable intermediate 1,3,4,6-tetrachloro- the stereoselective dehydrochlorination of HCH with a trans 1,4-cyclohexadiene, which may decompose to the dead-end and diaxial pair of hydrogen and chloride. LinA was composed 660 FETZNER AND LINGENS MICROBIOL. REV.

gene from Corynebacterium sp. strain N-1074. The enzyme had H2C-CH-CH2CO a molecular mass of 105 kDa. It consisted of four identical H20 ADl: epoxide hydrolase subunits (28 kDa) and contained no metals. No significant A02 chemical conversion inhibition was caused by thiol reagents or carbonyl reagents CH2OH-CHOH-CH2Cl (350). In cell extracts of the wild-type Corynebacterium strain AD1, AD2: haloalcohod dehatogenase N-1074, two 1,3-dichloro-2-propanol dehalogenases (Ia and Ib) and two epichlorohydrin hydrolases (IIa and IIb) were l HCl found. Ia and IIa showed only low enantioselectivity for each CH20H-CH-CH2 reaction, whereas Tb and IIb exhibited considerable enantio- H20 ADl: epoxide hydrolase selectivity, yielding R-3-chloro-1,2-propanediol (see also the 1 AD2: chemicat conversion section on microbial dehalogenases as industrial biocatalysts, CH20H-CHOH-CH20H below). Both dehalogenases were isolated. The 108-kDa homotetrameric enzyme Ia seemed to be identical with the FIG. 24. Proposed routes for the degradation of epichlorohydrin by was already from a E. coli clone sp. strain AD2 (517). enzyme which purified Pseudomonas sp. strain AD1 and Arthrobacter carrying a gene from Corynebacterium strain N-1074. The 115-kDa halohydrin hydrogen halide lyase Ib molecules were tetramers, composed of different combinations (4:0/3:1/2:2/1: of four identical 16.5-kDa subunits, and it did not require any 3/0:4) of a 32-kDa and a 35-kDa polypeptide. Ta and Ib both cofactors. Thus, it appeared to be different from the two other catalyzed the transformation of several halohydrins into the dehydrochlorinases described to date, namely, the glutathione- corresponding epoxides, with liberation of halide and its dependent DDT dehydrochlorinase from the house fly, and the reverse reaction, i.e., transformation of epoxides into the 3-chloro-D-alanine dehydrochlorinase from P. putida, which corresponding halohydrins in the presence of halide. Since Ia requires pyridoxal 5'-phosphate (351). and Ib differed with respect to enantioselectivity for 1,3- Johri et al. (232) reported an HCH-dechlorinase activity in dichloro-2-propanol conversion, substrate specificity, molecu- Pseudomonas ovalis CFT1 and Pseudomonas tralucida CFT4. lar mass, subunit composition, and immunological properties, Strain CFT1 harbours a 50-kb plasmid, which was suggested to the halohydrin hydrogen halide lyases of strain N-1074 appear contain the determinants of HCH degradation, whereas the to be phylogenetically different (355). However, the N-terminal degradation genes in strain CFT4 were proposed to be located amino acid sequence of halohydrin hydrogen halide lyase Ta either on a large plasmid or on a transposable element. from Corynebacterium sp. strain N-1074 was quite similar to However, as yet there are no data concerning the product(s) of that of the halohydrin dehalogenase from Arthrobacter sp. this dechlorination reaction or the mechanism of dehalogena- strain AD2 (350). tion. HALOACID DEHALOGENASES HALOHYDRIN DEHALOGENASES

In 1968, Castro and Bartnicki (56) discovered a dehaloge- Haloacetate Halidohydrolases nase (halohydrin epoxidase) from a 2,3-dibromo-1-propanol- utilizing Flavobacterium sp., which catalyzed the formation of Haloacetate halidohydrolases, which form a subgroup of the epoxides from a number of vicinal halohydrins (31, 56). In 2-haloacid halidohydrolases, are characterized by their relative 1989, van den Wijngaard et al. (517) reported the degradation inactivity toward halopropionates. The haloacetate halidohy- of epichlorohydrin and halohydrins by Pseudomonas sp. strain drolases have been classified into two types. The first type AD1, Arthrobacter sp. strain AD2, and a coryneform strain comprises enzymes which are active in the cleavage of the AD3 (Fig. 24). In strains AD1 and AD2, a halohydrin deha- carbon-fluorine bond of fluoroacetate, yielding fluoride and logenase activity which catalyzed the formation of epoxides glycolate, whereas the enzymes of the second type do not from a number of mono- and dihalogenated alcohols and catalyze the hydrolytic defluorination of fluoroacetate. Table 2 ketones was detected. The enzyme catalyzing the dehalogena- shows a list of the type 1 and type 2 haloacetate halidohydro- tion of vicinal haloalcohols to their corresponding epoxides lases described so far. was purified fromArthrobacter sp. strain AD2 (519). The native The type 1 haloacetate dehalogenases from Pseudomonas sp. protein is a dimer with a subunit molecular mass of 29 kDa. (153) and Pseudomonas sp. strain A (244) and the type 2 Halohydrin dehalogenase from strain AD2 converted C-2- and enzymes from Pseudomonas dehalogenans NCIB 9062 (80) and C-3- bromo- and chloroalcohols and was also active with Moraxella sp. strain B (247) are strongly inhibited by sulfhydryl chloroacetone and 1,3-dichloroacetone, yielding epoxides as reagents, which might suggest the participation of a sulfhydryl products. Neither cofactors nor oxygen was required for the group in catalysis (153). Au and Walsh (21) investigated the dehalogenation. Thus, the reaction mechanism is likely to stereochemical course of the fluoroacetate halidohydrolase proceed via intramolecular substitution, and the halohydrin reaction in Pseudomonas sp. strain A by using both enanti- dehalogenases seem to constitute a unique type of dehaloge- omers of 2-fluoropropionate. The reaction proceeded via nating enzymes. This conception was confirmed by immuno- inversion of configuration (compare with the following sec- logical studies: the enzyme from strain AD2 showed no tion). immunological cross-reactions with 2-haloalkanoic acid deha- Fluoroacetate halidohydrolase from Pseudomonas sp. strain logenases from Pseudomonas sp. strain 113, Pseudomonas sp. A was suggested to be encoded by a multicopy plasmid (21). strain GJ1, or X autotrophicus GJ10, and there was also no Both DehHl and DehH2 from Moraxella sp. strain B were immunological relatedness to haloalkane dehalogenase DhLA shown to be specified by a 65-kb conjugative plasmid desig- from strain GJ10 (519). nated pUO1, which was isolated and characterized (246, 249). An enzyme catalyzing the conversion of 1,3-dichloro-2- A 64.5-kb plasmid (pUO2) was detected (243) in Pseudomonas propanol to epichlorohydrin was purified from a recombinant sp. strain Cl, which was very similar to the pUO1 plasmid E. coli, which carried the halohydrin hydrogen halide lyase harbored by Moraxella sp. strain B. Strain Cl also produced VOL. 58, 1994 BACTERIAL DEHALOGENASES 661

TABLE 2. Haloacetate halidohydrolases

i/ Inhibiton by SH- Native/subunit mol Reference(s) Source Name Type blockng agents mass (kDa) Pseudomonas sp. 1 i + NDb 153 Pseudomonas sp. 1 i ND ND 499 Pseudomonas sp. strain A 1 c + 42/33 244 Moraxella sp. strain B DehHl 1 i ND ND/33.3 246, 248 Pseudomonas sp. strain C HI 1 c NDc ND/33.3C 243 P. dehalogenans NCIB 9062 2 ND + ND 80 Moraxella sp. strain B DehH2 2 c + 43/26 247, 248 Pseudomonas sp. strain C H-2 2 c +d 43/26d 243 a i, inducible; c, constitutive. b ND, not determined. c Enzyme identical to DehHl from Moraxella sp. strain B. d Enzyme identical to DehH2 from Moraxella sp. strain B. both an Hi- and H2-type dehalogenase, and the authors contents. Sequence analysis revealed no homology between suggested that the corresponding enzymes of the two organ- dehHl and dehH2. Considering that the enzymes DehHl and isms were identical. Thus, it might well be that horizontal gene DehH2 belong to different mechanistic groups (Table 2), this transfer is not an uncommon mechanism for spreading such lack of homology is not surprising. The G+C contents of both plasmid-borne dehalogenase genes. Plasmid pUOl was found dehHl and dehH2 were higher than that of the genomic DNA to be structurally unstable, with a 5.8-kb sequence apparently of the genus Moraxella, and the codon usage also differed. capable of spontaneous excision (249). Concomitant with the These data may indicate that the genes originated in different loss of this 5.8-kb sequence was the loss of the DehH2 enzyme. organisms and that their presence in strain B may be the result The dehH2 sequence was flanked by two repeated sequences of acquisitive evolution. Two amino-terminal regions of pro- about 1.8 kb long (248), which might play a part in the frequent tein DehHl showed similarity to the amino-terminal amino spontaneous deletion of dehH2 from the plasmid, suggesting that the segment might be a transposon. However, transposon acid sequenceof haloalkane hiAof atro- activity was not proven definitely (245). These findings raise phicus GJ1O (see the section on haloalkane halidohydrolases, the question of a possible evolutionary relatedness between above) and of three hydrolases of Pseudomonas strains. These such a spontaneously excised sequence harboring a haloacetate similar regionS might be common domains derived from an dehalogenase gene and the transposons harboring 2-haloal- ancestral Pseudomonas hydrolase. DehH2, which belongs to kanoic acid dehalogenase genes from P. putida PP3 (457) (see mechanistic group 2 of the haloacetate halidohydrolases, the following section). In Moraxella sp. strain B, the genes showed homologies to the group 1 haloalkanoic acid halidohy- dehHl and dehH2 are closely linked on plasmid pUOl. How- drolases DehCI, DehCII, HdlIVa, DhlB, and HadL, which are ever, the genes have different codon usages, sizes, and G+C discussed in the following section (248).

TABLE 3. Mechanistic groups of 2-haloalkanoic acid dehalogenasesa Group Characteristics Organism (enzyme) Reference(s) 1 Reaction results in inversion of configuration Pseudomonas sp. (HI and HII) 154 L-2MCPAb is converted; D-2MCPA is not converted P. dehalogenans NCIB 9061 297 Reaction is unaffected by sulfhydryl-blocking agents Rhizobium sp. (HI) 11, 286 Pseudomonas sp. strain CBS3 (DehCI and DehCII) 260, 341, 432, 433 P. cepacia MBA4 (HdlIVa) 349, 502 X autotrophicus GJ10 (DhlB) 527 P. putida AJ1 (HadL) 30, 233 P. putida 109 (DehH109) 247a 2 Reaction results in inversion of configuration P. putida PP3 (DehII) 550 Both L-2MCPA and D-2MCPA are converted Pseudomonas sp. strain 113 342, 343 Reaction is unaffected by sulfhydryl-blocking agents Rhizobium sp. (HII) 11, 286 3 Catalysis with retention of configuration P. putida PP3 (DehI) 493, 494, 550 Both L-2MCPA and D-2MCPA are converted Reaction is inhibited by sulfhydryl-blocking agents 4 Reaction results in inversion of configuration Rhizobium sp. (HIII) 286 D-2MCPA is converted; L-2MCPA is not converted P. putida AJ1 (HadD) 30, 233, 458 Reaction is unaffected by sulfhydryl-blocking agents 5 Enzymes with elevated activity toward 181 trichloroacetate a Groups are those of Hardman (181) and Weightman et al. (550). b MCPA, monochloropropionate. 662 FETZNER AND LINGENS MICROBIOL. REV.

HH.R ~~HR R H nase gene (dhlB) from strain GJ10 (527). However, the H X- __¢x --*-H.X HO- + N-terminal amino acids of DehCI and DehCII showed a partial sequence homology with a stretch of amino acids FIG. 25. 2-Haloalkanoic acid halidohydrolase mechanism sug- around Asp-124 of the GJ10 haloalkane halidohydrolase gested by Little and Williams (297), involving general base catalysis DhIA. Asp-124 has been suggested to be the active-site residue with activated water as the nucleophile (compare Fig. 22B). Redrawn from reference 297. of the GJ10 DhlA enzyme (see the section on haloalkane halidohydrolases, above). Site-directed mutagenesis was used to substitute Asp-10 in DehCI by Ala, resulting in complete loss of dehalogenating activity. It was suggested that Asp-10 2-Haloalkanoic Acid Halidohydrolases might be the nucleophilic residue essential in the active site of From consideration of the biochemical features of the the 2-haloalkanoic acid halidohydrolase I from Pseudomonas 2-haloalkanoic acid halidohydrolases described to date, sp. strain CBS3 (433). Thus, the mechanism of the 2-haloal- Weightman et al. (550) and Hardman (181) suggested that they kanoic acid halidohydrolases from strain CBS3 might corre- be placed into five mechanistic groups (Table 3). spond to the mechanism postulated for haloalkane dehaloge- Group 1. The 2-haloalkanoic acid halidohydrolases belong- nase from strain GJ10. The hdlIVa gene, which encodes the ing to mechanistic group 1 are active toward the L-isomer of group 1 halidohydrolase HdlIVa from P. cepacia MBA4, was 2-monochloropropionic acid, yielding D-lactate as the product sequenced. Comparison of the amino acid sequence of HdlIVa of the reaction. Group 1 enzymes are relatively insensitive to with the enzymes of strain CBS3 revealed a high degree of sulfhydryl-blocking agents. Goldman et al. (154) suggested two homology with DehCI (67% identity, 81% similarity) but also possibilities to explain the observed inversion of configuration significant homology with DehCII (37% identity, 56% similar- during the reaction of L-2-monochloropropionate to D-lactate: ity) (349). P. putida AJ1 possesses two haloalkanoic acid (i) simple displacement of the halide by a hydroxyl ion, and (ii) dehalogenases, the group 4 enzyme HadD (see below), and the action of the carboxyl group of the enzyme as the nucleophile group 1 enzyme HadL. There is no homology between HadL to displace the halide, with subsequent hydrolysis of the ester and HadD, suggesting different ancestral origins. In contrast to (compare Fig. 22A). Little and Williams (297) studied the type the homodimeric enzymes DehCI, DehCII, and HdlIVa, HadL 1 2-haloalkanoic acid halidohydrolase from Pseudomonas de- is a tetramer. HadL is highly homologous to DehCII (51% halogenans NCIB 9061, which was unaffected by sulfhydryl- identity) and DehCI (38% identity) from Pseudomonas sp. blocking agents. They proposed a general base catalysis mech- strain CBS3 and to DhlB from X autotrophicus GJ10 (43% anism possibly involving a His residue as the active nucleophile identity [287]). The G+C content and codon usage of the hadL (Fig. 25). X autotrophicus GJ10 constitutively produces two gene differed from the P. putida genome, which again might halidohydrolases, one specific for 1,2-dichloroethane and re- indicate gene acquisition (compare DehHl and DehH2 [see lated haloalkanes (DhlA [see the section on haloalkane ha- the previous section]) (233). The plasmid-encoded 2-haloal- lidohydrolases, above]) and the other specific for halogenated kanoic acid halidohydrolase DehH109 from P. putida 109 (344) alkanoic acids, belonging to mechanistic group 1. Haloalkanoic recently was shown to possess significant homologies to the acid dehalogenase from strain GJ10 was purified, and its other group 1 enzymes and also to haloacetate dehalogenase structural gene (dhlB) was cloned and sequenced (527). There DehH2 from Moraxella sp. strain B (247a). Originally, mech- was no sequence homology of dhlB with dhL4, the gene anistic group 1 was defined to involve enzymes which are active encoding haloalkane dehalogenase from the same organism. toward the L-isomer only of 2-monochloropropionate, yielding However, dhlB showed considerable homology with the se- D-lactate, and which are not affected by sulfhydryl-blocking quences of dehCI and dehCII, which encode the two group 1 agents. However, the group 1 enzymes DhlB, DehCI, DehCII, haloalkanoic acid dehalogenases from Pseudomonas sp. strain HdlIVa, HadL, and DehH109 apparently are not only mech- CBS3 (432). Thus, haloalkanoic acid and haloalkane halidohy- anistically but also evolutionary related. drolases appear not to be evolutionary closely related, al- Group 2. The second mechanistic group comprises 2-haloal- though they mechanistically catalyze an analogous dehaloge- kanoic acid halidohydrolases which are unaffected by sulfhy- nation reaction. In accordance with Goldman et al. (154), van dryl-blocking agents and which also catalyze halide hydrolysis der Ploeg et al. (527) suggested two alternative mechanisms for with inversion of product configuration. However, these en- hydrolytic dehalogenation accompanied by inversion of the zymes are active toward both L-2-monochloropropionate and configuration, i.e., (i) nucleophilic attack by activated water D-2-monochloropropionate. The dehalogenase from P. puitda (Fig. 25) or (ii) attack by a carboxylate group of the enzyme 113 has been found to be representative of this type of followed by ester hydrolysis (compare Fig. 22A). The three- 2-haloalkanoic acid halidohydrolase (342, 343). P. putida PP3, dimensional structure of the haloalkane halidohydrolase from isolated by Senior et al. (446) from a microbial community strain GJ10 supported the latter mechanism for this enzyme growing on the herbicide dalapon (2,2-dichloropropionic acid), (see the section on haloalkane halidohydrolases, above), and was shown to harbor two halidohydrolases which differ funda- genetic data on the two 2-haloalkanoic acid halidohydrolases mentally in their reaction mechanism (456, 549, 550). The from Pseudomonas sp. strain CBS3 also suggested a mecha- fraction II halidohydrolase (DehII) formed L- and D-lactate nism involving a carboxylate group (432), as follows. Pseudo- from D- and L-2-monochloropropionate, respectively, thus be- monas sp. strain CBS3 possesses two isofunctional group 1 longing to the group 2 halidohydrolases. 2-haloalkanoic acid halidohydrolases (260, 341). DehCI and Group 3. The fraction I enzyme (DehI) from P. putida PP3 DehCII were homodimeric proteins with subunit molecular also used both D- and L-2-monochloropropionate as substrate. masses of 25,401 Da (DehCI) and 25,683 Da (DehCII). However, the product of the reaction retained the same optical Schneider et al. (432) have sequenced the dehCI and dehCII configuration as the substrate provided (550). DehI was con- genes. Comparison of the two sequences revealed 45% homol- siderably more sensitive to sulfhydryl-blocking agents than was ogy at the DNA level. No homology of dehCI and dehCII with DehIl. In 1965, Goldman (153) had suggested a mechanism the haloalkane dehalogenase gene (dhLA) from X autotrophi- involving an S-carboxymethyl intermediate to account for the cus GJ10 could be detected, but there was 61% (dehCI) and inhibition of halidohydrolases by sulfhydryl reagents. Such a 60.5% (dehCII) similarity to the haloalkanoic acid dehaloge- concept had been criticized, since thioethers are inherently VOL. 58, 1994 BACTERLAL DEHALOGENASES 663

H R R H R-..... 6H above), DehCI, DehCII, HadL, HdlIVa, DhlB, and HadD. On .. the basis of the amino acid sequences, the enzymes appear to fall into three classes (classes I, II, and III), which show low (12 to 21%) identity to each other. The haloacetate dehalogenase DehHl from Moraxella sp. strain B (see the section on haloacetate halidohydrolases [above] and Table 2) forms a separate class (class I). The second class of haloacid dehalogenases (class II) comprises six enzymes: the group 2 haloac- FIG. 26. 2-Haloalkanoic acid halidohydrolase mechanism involving etate halidohydrolase DehH2 from Moraxella sp. strain B (see a thioether intermediate and a double inversion (550). Redrawn from the section on haloacetate halidohydrolases [above] and Table reference 550. 2) and the five group 1 haloalkanoic acid dehalogenases, DehCI, DehCII, HdlIVa, DhlB, and HadL (Table 3) that have been sequenced so far. The enzymes in this class share 33 to resistant to hydrolysis (297). However, in 1982, Weightman et 67% amino acid identity. DehH109 from P. putida 109 appar- al. (550) proposed an analogous mechanism involving a double ently also belongs to this class of enzymes (247a). The third inversion and a thioether intermediate, which is hydrolyzed, to class (class III) is represented by the HadD dehalogenase ofP. account for the observed retention of configuration (Fig. 26). putida AJ1, which belongs to mechanistic group 4 (Table 3). Group 4. In a Rhizobium sp., three 2-haloacid halidohydro- None of the haloacid dehalogenases in the classes II and III lases were found (11, 286). The HIlI dehalogenase represents shows homologies to haloalkane dehalogenases or to other a unique type of 2-haloacid halidohydrolase, being active only sequences stored in the databases (287). toward the D-isomer of 2-monochloropropionate. The reaction A number of organisms have been shown to possess more results in inversion of product configuration (286). Another than one 2-haloalkanoic acid halidohydrolase (11, 30, 154, 183, D-2-halopropionate dehalogenase, designated HadD, was 233, 286, 502, 549, 550). These isofunctional enzymes were found in P. putida AJi (30, 458, 459). This enzyme is a distinguished by their different electrophoretic mobilities (for tetrameric protein composed of four identical subunits. Smith reviews, see references 181 and 183). Pseudomonas sp. strain et al. (458) suggested a base catalysis for the mechanism of E4, for instance, may contain three dehalogenases. Dehaloge- dehalogenation by the D-2-haloacid halidohydrolase HadD, a nase III was detected only when strain E4 was grown under His residue possibly acting as the basic group which promotes substrate-limited conditions (184). Similarly, in addition to the nucleophilic attack (Fig. 25). The genes hadD and hadL (see monobromoacetate halidohydrolase HIVa, a second dehalo- the discussion of group 1, above) are closely linked in an genase was found in P. cepacia MBA4 when cells were grown operon of strain AJ1. HadD has no significant homology with in chemostat culture (502). Thus, expression of dehalogenases any of the other haloalkanoic acid halidohydrolases sequenced appeared to be regulated by growth conditions. The physiology to date, and no significant matches were discovered in DNA or of halidohydrolase isoenzyme expression was discussed by protein databases. Thus, HadD seems to be a unique protein Hardman (181). The identification of so many electrophoreti- (30). cally distinct isofunctional proteins raises the question of their Group 5. A possible fifth group of 2-haloacid halidohydro- environmental significance and their evolutionary relatedness. lases may be represented by enzymes with elevated activity However, the role of 2-haloalkanoic acid halidohydrolases in toward trichloroacetate, catalyzing the complete hydrolytic the environment is not known. It is not clear whether these dechlorination of this substrate (181). enzymes are expressed in the environment as dehalogenases or Table 3 provides a survey of the mechanistic groups of whether they function as unspecific hydrolases. Evolution of 2-haloalkanoic acid dehalogenases, and Table 4 shows the halidohydrolase activities might involve (i) the modification of molecular masses and structures of the 2-haloalkanoic acid existing hydrolase activities (gene duplication and/or muta- halidohydrolases characterized to date. In their review on tional changes); (ii) decryptification of silent genes (hydrolase hydrolytic dehalogenases, Leisinger and Bader (287) presented genes?) by transposition events; and (iii) acquisitive evolution a table listing the amino acid identities between the haloac- by horizontal gene transfer. Concerning the three Rhizobium etate and haloalkanoate halidohydrolase sequences DehHd dehalogenases and the two P. putida PP3 dehalogenases, (see the section on haloacetate halidohydrolases, above), fundamental differences in the mechanisms suggest that dupli- DehH2 (see the section on haloacetate halidohydrolases, cation of ancestral genes and subsequent mutational changes

TABLE 4. Some 2-haloalkanoic acid halidohydrolases

Name Native/subunit mol Structure Source Group mass (Da) Reference(s) P. dehalogenans NCIB 9061 1 15,000 297 X autotrophicus GJ10 DhlB 1 36,000/28,000 (27,433)b a 527 P. putida 109 DehH109 1 34,000/25,000 (25,231)b ax 344, 247a P. putida US2 NDa 30,000/28,000 a 471 P. cepacia MBA4 HdlIVa 1 45,000/25,900 aC2 349, 502 P. putida AM HadL 1 79,000/25,700 (25,687)b a4 (or a3) 233 Pseudomonas sp. strain CBS3 DehCI 1 41,000/28,000 (25,401)b a2 260, 341,432, 433 DehCII 1 64,000/29,000 (25,683)b a2 Pseudomonas sp. strain 113 2 68,000/35,000 a2 343 Pseudomonas sp. strain E4 HI ND 46,000/23,000 a2 502 P. putida AJ1 HadD 4 135,000/31,800(33,601)b Oa4 30,458 a ND, not determined. b Deduced from nucleotide sequence analysis. 664 FETZNER AND LINGENS MICROBIOL. REV. did not originally give rise to the various enzyme forms. fH3 Hardman et al. (182) isolated four Pseudomonas spp. and two CG-CHCH-8-H Alcaligenes spp. from soil; they were shown to contain large ATP,CoASH plasmids ranging from 149 to 285 kb in size. These plasmids Mg2* were associated with the ability to utilize 2-monochloropropi- AMP + PP1 onate and monochloroacetate. pUU204 was shown to be CH3 unstable, and the dehalogenase gene associated with it ap- a-cHw -C-SCOA peared to be readily transferred to plasmid R68-45. However, Ecd3 oro-2-etlA- conjugability of these plasmids was not demonstrated. There HCIl pvroprytGCkAdalogenese was no further evidence that the dehalogenase genes on these CH3 plasmids were located on transposons. Brokamp and Schmidt HO-CHr- H-j-SCoA (50) investigated the horizontal transfer of plasmid pFL40 harboring the haloalkanoic acid halidohydrolase gene dhlC fromAlcaligenes xylosoxidans ABIV. This plasmid was horizon- NAOH H 0 tally transferred to P. fluorescens in sterilized soil and also to CH3 different soil bacteria in nonsterilized soil. Thus, dissemination OHC-CH--So of halidohydrolase genes in situ may be important for spreading biodegradative capacities in soil ecosystems. The halidohydrolase genes from strain PP3 were seen to be chromosomally located. Beeching et al. (33) demonstrated the CoASHo transposition of the halidohydrolase I gene from P. putida PP3 CH3 to plasmid R68.44. Analysis of five classes of PP3 mutants led OHC-CH-C-OH Slater et al. (457) to suggest that a separate permease gene was CoASH.ND associated with each of the halidohydrolases and that the genes were carried on three transposons: Tn-dehlA, encoding DehI C02,NADH+ and permease I, Tn-dehB, encoding DehII, and Tn-dehC, H3C-CH2-C-SCoA encoding permease II. The authors speculated that the deha- 0 logenase genes may be cryptic genes which are silenced if their FIG. 27. Degradation of 3-chloro-2-methylpropionic acid by Xan- expression is disadvantageous but which are activated under thobacter sp. strain CIMW99. The outlined pathway was suggested by environmental conditions if it is beneficial. The silenced genes Smith et al. (460) on the basis of the demonstration of the enzymatic would be located in a region of the chromosome that prevents activities involved in the degradation of 3-chloro-2-methylpropionic expression, and a replication-transposition event could put a acid to propionyl-CoA. Redrawn from reference 460. copy of the genes into a position permitting expression. Thus, translocation of a transposon might be responsible for expression of previously cryptic halidohydrolase genes. In 1992, 3-Haloacyl-CoA Halidohydrolases Thomas et al. (493, 494) characterized a transposon-like element from P. putida PP3, designated DEH, which is located In the degradation of 3-chloro-2-methylpropionic acid by on the chromosome. This unusual mobile genetic element Xanthobacter sp. strain CIMW99, hydrolytic release of chloride carries the gene dehI, which encodes the group 3 haloalkanoic was observed only from the CoA ester of 3-chloro-2-methyl- acid halidohydrolase DehI. The DEH element also contains a propionic acid, not from the free acid (460) (Fig. 27). The regulatory gene dehRI adjacent to dehI; this regulatory gene reaction appeared not to be stereospecific. Similarly, Kohler- encodes an activator protein, a regulated promoter for the Staub and Kohler (269) demonstrated that the dechlorination transcription of dehI, and functions associated with recombi- of 3-chlorocrotonic acid and the dechlorination of 3-chlorobu- nation. In addition, DEH may carry genes encoding a per- tyric acid by cell extracts from Alcaligenes sp. strain CC1 both mease for transmembrane transport of haloalkanoates. The required that the substrates be converted to the corresponding DEH element was not classified as a conventional transposon, CoA derivatives. These dehalogenases might constitute a because it did not move as a discrete DNA fragment: dehI- separate class of halidohydrolases catalyzing the dehalogena- containing fragments inserted into different plasmid DNA tion of P-halogenated aliphatic acyl-CoA esters. However, the targets varied in size between 6 and 13 kb. Insertion of DEH enzymes are not isolated yet, and further studies are necessary appeared to be promiscuous. The DEH element was highly to characterize these enzymes with respect to their biochemical mobile in the chromosome of P. putida PP3, and movement of properties and the mechanism of the catalytic reaction. It DEH was associated with genetic switching of dehI. Despite its might be interesting to compare these 3-haloacyl-CoA deha- unusual features, DEH can be classified as a transposable logenases with 4-chlorobenzoyl-CoA dehalogenase, which is element, since it is capable of combining at high frequencies the only other dehalogenase described so far acting on a CoA with plasmid and chromosomal DNA targets (493, 494). An- ester (see the section on halidohydrolases, above). other dehalogenase suggested to be located on a mobile DNA element is the plasmid-encoded haloacetate halidohydrolase BIOTECHNOLOGICAL APPLICATIONS OF BACTERIAL DehH2 from Moraxella sp. strain B. However, direct evidence DEHALOGENASES for transposition has not been provided in this case (see the previous section). The 17-kb transposon Tn5271, which is Technological applications of bacterial transformations of present on plasmid pBRC60, specifies the genes for 3-chloro- halogenated substances can be considered with respect to two benzoate degradation by Alcaligenes sp. strain BR60. The different objectives: (i) synthesis, i.e., microbial strains or dehalogenating 3-chlorobenzoate 3,4-/4,5-dioxygenase is lo- biocatalysts may be used for the biosynthetic generation of cated on this transposon (see the section on haloaromatic (halogenated) synthetic intermediates or novel compounds; dehalogenases, subsection oxygenases, above). and (ii) degradation, i.e., halohydrocarbon-degrading organ- VOL. 58, 1994 BACTERIAL DEHALOGENASES 665 isms or microbial dehalogenases may be used to detoxify and highly pure optically active (R)- as well as (S)-3-chloro-1,2- to mineralize environmental pollutants. propanediol. From (RS)-3-chloro-1,2-propanediol, the Pseudo- monas strain stereospecifically dehalogenated the (S) enanti- Microbial Dehalogenases as Industrial Biocatalysts omer, whereas the (R) enantiomer rested unchanged. By contrast, the Alcaligenes strain dehalogenated and assimilated Currently, halogenated compounds are synthesized predom- the (R) enantiomer, leaving the (S) enantiomer. From the (R)- inantly by chemical techniques. However, biotransformations and (S)- enantiomers of 3-chloro-1,2-propanediol, which were could provide economic and versatile alternatives. Esters of unassimilatable by the respective strains, highly pure (R)- and 2-monochloropropionic acid are used as intermediates in the (S)-glycidol was prepared chemically. Optically active glycidol production of pharmaceutical products, and short-chain is an important C-3 building block for chiral pharmaceuticals 2-halocarboxylic acids are key intermediates in the synthesis of such as,3-adrenergic blockers and cardiovascular drugs (483). phenoxypropionate herbicides. If racemic 2-monochloropropi- Nakamura et al. (356) produced optically active 3-chloro- onate is used for chemical syntheses, racemic products are 1,2-propanediol from prochiral 1,3-dichloro-2-propanol by us- generated. However, in most cases only one of the isomers is ing several microbial isolates from soil. One of these strains, biologically active, and half of the racemic product would be Corynebacterium sp. strain N-1074, which possesses an ep- expensive inactive chiral ballast or would even lead to toxic oxide-forming halohydrin dehalogenase (see the section on side effects. Selective dehalogenation of one of the isomers of halohydrin dehalogenases, above) catalyzing the conversion of 2-monochloropropionate, exploiting the chiral specificity of prochiral 1,3-dichloro-2-propanol to epichlorohydrin, was used 2-haloalkanoic acid dehalogenases, is used to remove the L- or to produce optically active (R)-3-chloro-1,2-propanediol (357). D-isomeric form from a chiral mixture of 2-monochloropropi- Since halohydrin hydrogen halide lyases from Corynebacterium onate (345). ICI Biological Products (U.K.) uses P. putida sp. strain N-1074 also catalyze the reverse reaction, i.e., AJ1/23 to produce L-2-monochloropropionate for use in her- formation of halohydrins from the corresponding epoxides bicide manufacture from racemic 2-monochloropropionate (355), halohydrin hydrogen halide lyaseIa from strain N-1074, (492). The process has now reached commercial scale. This purified from a recombinant E. coli clone, was applied to the chiral specificity of the 2-haloalkanoic acid dehalogenases has transformation of epoxides into useful compounds for further also been used to produce both the enantiomers of lactic acid syntheses: epoxides were transformed into the corresponding from racemic 2-monochloropropionic acid in optically pure 13-hydroxynitriles in the presence of cyanide (354). form and at high yield (346). By using the L-2-haloacid dehalogenase from P. putida (344), a simple method for the Application of Microbial Systems in Environmental total conversion of racemic 2-monochloropropionate into D- Protection Technology and Waste Management lactate was developed: L-2-monochloropropionate was fully converted to D-lactate by L-2-haloacid dehalogenase, and the During industrial processes, end-of-pipe or in-process treat- unreacted D-2-monochloropropionate was dehalogenated ment of toxic substances should become a matter of course to chemically, retaining its configuration, to produce D-lactate avoid the production of hazardous waste. However, for pesti- (194). Similarly, the production of optically active 3-halolactate cides, their direct application can lead to contamination not from 2,3-dihalopropionate was performed with 2-haloalkanoic only of soil but also of groundwater and aquifers as a result of acid halidohydrolase from P. putida (353). leachates. Moreover, there is the legacy of contaminated Takahashi et al. (487) reported a method for the production industrial sites, landfill sites, and landfill leachates as a result of of (R)-3-chloro-1,2-propanediol by enantioselective microbial the improper disposal techniques of the past. Since civilization degradation of (R,S)-3-chloro-1,2-propanediol. Kasai et al. will most probably continue to be accompanied by the produc- (238) have shown that optically active 2,3-dichloro-1-propanol tion of hazardous waste, it is necessary to develop efficient and 3-chloro-1,2-propanediol could be prepared from racemic strategies for waste management. In future technologies, mi- 2,3-dichloro-1-propanol by using Pseudomonas sp. strain OS- crobial systems like the bacterial dehalogenases might be K-29, which is capable of assimilating (R)-2,3-dichloro-1-pro- potent tools to deal with environmental pollutants. panol as the sole source of carbon, leaving the (S) enantiomer Biotechnology for hazardous-waste management involves unchanged. Cells of Pseudomonas sp. strain OS-K-29 immobi- the development of systems that use biological catalysts to lized in calcium alginate were used to obtain optically pure detoxify, degrade, or accumulate environmental pollutants. (S)-2,3-dichloro-1-propanol, which, upon treatment with aque- Biotechnology offers a number of strategies for waste treat- ous NaOH, yielded highly pure (R)-epichlorohydrin (239). In ment: (i) improvement of existing processes by application of contrast to Pseudomonas strain OS-K-29, which removes the adapted or engineered microbial strains (e.g., the municipal (R) isomer from racemic 2,3-dichloro-1-propanol, an Alcali- treatment of wastewaters); (ii) use of adapted or genetically genes strain stereospecifically assimilates (S)-2,3-dichloro-1- engineered microorganisms to treat contaminated soil, ground- propanol. This strain was used to obtain optically pure (R)-2,3- water, or aquifers; (iii) construction of bioreactors containing dichloro-1-propanol, which upon alkaline treatment yielded biofilms of suitable organisms or use of immobilized biocata- highly pure optically active (S)-epichlorohydrin (240). Opti- lysts in the detoxification of environmental chemicals; (iv) cally active epichlorohydrin is a useful intermediate for the development of biosensors to detect trace amounts of toxic synthesis of chiral pharmaceuticals such as ,B-adrenergic block- organics or heavy metals; and (v) recovery of products from ers, platelet-activating factor, L-carnitine, vitamins, and pher- wastes (e.g., recovery of metals by using metal-accumulating omones and also for the synthesis of agrochemicals and bacteria) (54). A number of reviews dealing with various ferro-electric liquid crystals (239, 240). aspects of bioremediation have been published (45, 127, 167, Crude extracts of Alcaligenes sp. strain DS-S-7G stereospe- 266, 285, 340, 399, 495). Biotechnological approaches which cifically degraded (R)-3-chloro-1,2-propanediol, yielding (S)-3- have been applied successfully so far involve bioaugmentation chloro-1,2-propanediol from the racemate in 38% yield. (S)- to cope with contaminated landfill sites, industrial sites, and Glycidol was subsequently synthesized by alkaline treatment groundwater, and the development of bioreactors to deal with (484). Using both a Pseudomonas and an Alcaligenes strain, specific contaminants. Suzuki and Kasai (483) developed a method of producing There are several possible reasons for the persistence of a 666 FETZNER AND LINGENS MICROBIOL. REV. pollutant in the environment; these include (i) the absence of enrichment with phenol was applied successfully in laboratory microorganisms with the capability of degrading the com- microcosms and in an in situ groundwater test site to cometa- pound; (ii) the presence of unfavorable environmental condi- bolically degrade TCE and cis-dichloroethylene. Oxygen was tions for biodegradation (environmental factors which may also injected into the test site. However, phenol addition to influence biodegradation include permeability of the subsur- aquifers may be questioned. Nevertheless, in comparison with face for air and water, temperature, pH, salinity and water other oxygenase systems evaluated to date, phenol has been content [water activity], oxygen tension [redox potential], shown to be relatively effective at removing TCE and cis- availability of nutrients, presence of inhibitory chemicals, and dichloroethylene (215). Alternatively, cometabolism of TCE by presence of competing organisms or of predators [protozoa] methanotrophs (which possess methane monooxygenase [see grazing on the degradative microorganisms); (iii) unfavorable the section on oxygenases in spontaneous or fortuitous deha- substrate concentration (the pollutant may be present in too logenation reactions, above]) may be applicable for in situ high a concentration, leading to toxicity, or in too low a bioremediation of TCE-contaminated groundwater or soil concentration, failing to induce the degradative enzymes or (136, 552). Application possibilities in this case are best if being below the threshold concentration Smin [see below]); and dilute contaminations have to be treated, such as with ground- (iv) lack of bioavailability of the pollutant because of incorpo- water cleanup, since some strains of methanotrophic bacteria ration into humic substances or strong adsorption to soil may be rather sensitive to inactivation by TCE (372). Soluble particles. It is the aim of biological remediation methods to methane monooxygenase, which is induced under conditions of optimize the environmental conditions so that indigenous or copper limitation only, is thought to be responsible for TCE inoculated organisms can degrade the contaminant at the degradation in most cases. It is not known whether these maximum possible rate. conditions of extreme copper limitation required for the Bioaugmentation. Bioaugmentation can be achieved by induction of soluble methane monooxygenase are ever found three methods (181): (i) stimulation of existing organisms by in nature. However, membrane-associated (particulate) meth- optimizing their nutrient supply or by addition of substrate ane monooxygenase was recently shown to also oxidize TCE, analogs (analog enrichment technique); (ii) removal of indig- although at lower rates than those observed for the soluble enous organisms from contaminated sites, enrichment and enzyme (91, 267, 372). selection of adapted organisms, and return of these organisms A major problem of biological remediation is that microbial as inocula to the contaminated site; (iii) addition of genetically agents are not capable of reaching the "nine-nines" engineered organisms. In the first two methods, bioaugmenta- (99.9999999%) efficiency obtained with physicochemical treat- tion may accelerate what would eventually happen as a result ment processes. In biological systems, there is a minimum of the indigenous microbial population. Because of concerns concentration below which compounds are not degraded any about the construction and the environmental release of more. This threshold concentration (Smin) depends mainly on genetically engineered microorganisms, the third option is still the kinetic parameters of growth and metabolism (substrate at the laboratory stage in most countries (54, 256, 547). affinity constant) but also on the thermodynamics of the overall (i) Addition of nutrients and/or substrate analogs. Methods transformation reaction (581). Indeed, the most important used for in situ site cleanup involve the provision of inorganic parameter with respect to the biodegradation of contaminants nutrients, oxygen, or organic growth substrates or the enhance- to very low concentrations is the substrate affinity constant. ment of cometabolism by the supply of the primary growth This problem was evaluated by Hartmans et al. (190) with substrate (analog enrichment). Competition for nutrients (oth- respect to the removal ofvinyl chloride and 1,2-dichloroethane er than carbon) may limit biodegradation: low phosphate to the required low levels of 5 mg m-3 by means of a mixed concentrations in water of a creek in spring and summer were culture ofMycobacterium aurum Ll andX autotrophicus GJ10. thought to limit the growth of the chlorobenzoate-degradative Typical Snin, for aerobic systems are in the range of 0.1 to 1.0 population of Alcaligenes sp. strain BR60 and to prevent the mg liter-', but the desired end concentrations often are 1 ,ug removal of the contaminant (563). The concept of analog liter-' or less. Oligotrophic bacteria have been defined as enrichment was initially designed by Focht and Alexander organisms that can live under conditions of very low carbon (128, 129). Treatment of soil with growth substrates that are flux (<1 mg liter-' day-'); i.e. the Smin needed for growth is structurally analogous to xenobiotic compounds offers an lower than that required for eutrophic (high-nutrient) organ- approach for biodegradative cleanup. Analog enrichment with isms. Since oligotrophic species have a large advantage at low aniline was used successfully as a method for the removal of substrate concentrations because of their high affinity for 3,4-dichloroaniline from contaminated soil. The effect of ani- substrate, they are potentially useful for the removal of trace line was due to the induction of pathways that cometabolize contaminants from water, soil, or waste gases. Unfortunately, 3,4-dichloroaniline (576). PCBs in soil were mineralized by oligotrophs and their biodegradative potential are relatively analog enrichment with biphenyl and inoculation with the unstudied (266). However, to design efficient waste treatment cometabolizing strain Acinetobacter sp. strain P6. Strain P6 is methods, it is always important to have kinetic data on growth not able to grow on any of the PCBs, except 4-chlorobiphenyl, and degradation as well as data on substrate affinity. because the ring fission products are not dehalogenated. (ii) Use of microbial inocula. The addition of competent Mineralization was more rapid in soils amended with both wild-type or mutant microorganisms to contaminated sites has biphenyl and P6 inoculum, but the final levels of PCB miner- now become established as an in situ technology. Stimulation alized were similar to those found in soils enriched with the of indigenous microbial populations and augmentation with analog (biphenyl) alone (52, 130). adapted strains have been used successfully to detoxify com- TCE and tetrachloroethylene are among the most pervasive plex industrial-waste sites (468). Since many wastes or contam- pollutants of groundwater. Tetrachloroethylene was rapidly inated sites contain very complex mixtures of diverse com- converted to TCE under methanogenic conditions, but further pounds, the use of tailor-made cultures in the effort to abate degradation was restricted (476). Since a major problem with pollution often is seen with scepticism. However, microbial anaerobic catabolism of chloroethylenes is the incomplete strains may be applied in the treatment of special industrial degradation leading to toxic products like vinyl chloride, wastewaters or waste gases with a defined composition, con- aerobic treatment processes would be preferable. Thus, analog taining relatively few substrates and possibly offering condi- VOL. 58, 1994 BACTERIAL DEHALOGENASES 667 tions (pH, temperature, mineral content) which are highly pathway, showed that they did not perform equally well in soil selective for a narrow range of specialized microorganisms. (222). Examples are the treatment of 2,4-D- and 2,4-dichlorophenol- To enhance mineralization of polychlorinated biphenyls in containing effluents from the manufacture of the herbicide soil, Hickey et al. (205) used the chlorobenzoate utilizers P. 2,4-D (127) and the elimination of dichloromethane from aeruginosa JB2 and P. putida Plll and the biphenyl utilizer waste gases, which is described in the following section. A Pseudomonas sp. strain PB133 as inocula. The strains were simple method of amendment of a contaminated site is the use applied solely and in combination. Mineralization of Aroclor of sludge as a kind of inoculum. Addition of sewage sludge to 1242 was greatest in soils inoculated with the chlorobenzoate PCB-contaminated soil increased mineralization (118). PCP degraders alone, showing that chlorobenzoate catabolism was degradation rates in soil were greatly enhanced by a combina- the limiting factor in Aroclor 1242 removal. The authors tion of bioaugmentation with anaerobic sewage sludge and speculated that this effect might be due to the role of chloro- induced anaerobiosis, which was achieved by flooding the soil benzoates as inhibitor precursors: chlorobenzoates may be with water. However, this anaerobic treatment resulted in the metabolized to chlorocatechols, which are potent inhibitors of accumulation of less highly chlorinated phenols (325). Syn- the meta-cleaving 2,3-dihydroxybiphenyl dioxygenase (205). To thetic wastewater containing some sugars and PCP was treated biodegrade 3-chlorobenzoate in soil, inoculation with P. alcali- with activated sludge. Additional continuous feeding of the genes C-O was suggested as a viable strategy, since the growth sludge unit with a PCP-metabolizing Arthrobacter sp. from a rate of this strain in soil was comparable to its growth rate in chemostat resulted in immediate acclimation of the system to culture and since relatively small inocula (102 cells g-1) were PCP and improved the stability of the system to shock loads sufficient for 3-chlorobenzoate removal (131). Tetrachlo- (103). Such a use of chemostat cultures as "continuous inocu- romethane was removed by the addition of Pseudomonas sp. la" to industrial treatment processes will probably become strain KC to groundwater and soil slurries. Under denitrifying more widespread, since chemostats generally respond well to conditions, strain KC converted tetrachloromethane to carbon shock loads (127). dioxide. The activity was hampered by ferric iron, cobalt, The applicability of specialized strains as inocula for the vanadium, and oxygen (see the section on haloaliphatic com- bioremediation of contaminated soil and groundwater was pounds, above). However, application of strain KC in field studied extensively with various PCP-degrading strains. PCP environments is favorable if the pH is properly controlled: was removed from a variety of soils by inoculation with a adjustment to pH 8.2 decreased iron solubility and avoided Flavobacterium sp. (76). However, inoculum size was critical, copper toxicity (77, 289, 491). and in some soil samples, several inoculations of the Flavobac- In general, the use of defined strains as inocula is a safe terium sp. over a period of several months were required for strategy, because the intermediates and final degradation substantial removal of PCP. The reason for the rapid decline in products are known, whereas the stimulation of the natural soil the number of Flavobacterium organisms in soil may be their microflora by addition of nutrients might lead to unpredictable inability to compete with the indigenous microflora. Briglia et products, e.g., 0-methylated compounds, or radical interme- al. (49) demonstrated that cells of Rhodococcus chlorophenoli- diates, which might react with unsoluble polymers or condense cus PCP-1 immobilized on polyurethane foam and introduced with humic substances. into soil maintained high PCP-mineralizing activity for 200 A number of factors, in particular migration and survival, days, whereas the population of immobilized Flavobactenium influence the biodegradative effectivity of an inoculum. Con- sp. declined by 7 orders of magnitude within 60 days. The cerning migration, inoculated cells must come into contact sensitivity to and degradation of PCP by Flavobacterium cells with the contaminant; i.e., they have to move through soil to were greatly influenced by a number of factors, including the sites containing the chemical. However, unicellular organ- inoculum density, pH, temperature, other available carbon isms can move only within water films, and migration is sources, and the presence of other pollutants (500). Increased restricted by filtration effects and by sorption to clay minerals. soil water content enhanced biodegradation of PCP by Fla- Concerning survival, the competitiveness of an introduced vobacterium sp. strain ATCC 39723, whereas increased chlo- strain is a major factor in determining its applicability as an ride ion concentrations and anoxic conditions were inhibitory inoculum. Inoculated organisms may be killed by antibiotic- or (444). Compared with the PCP-degrading Flavobactenium sp. toxin-producing or lytic microorganisms or by predating pro- as inoculum, inocula of Arthrobacter sp. strain ATCC 33790 tozoa. They may die if they are unable to sequester nutrients (102) and of R. chlorophenolicus PCP-1 (49,320,512) appeared competitively or if they are unable to metabolize relatively high to achieve a more efficient removal of the pesticide from soil. or very low substrate concentrations. Abiotic stress (pH, The genus Arthrobacter is well known for the ability of its cells temperature, water content, oxygen tension, redox potential) to withstand desiccation and starvation without loss of viability. also affects survival. Whereas some genera are relatively Similarly, cells of R. chlorophenolicus PCP-1 were shown to resistant to starvation and desiccation, others rapidly die (see survive for several months in various soils (320). Polyurethane- the discussion of PCP-utilizing Flavobacterium, Arthrobacter, immobilized cells of strain PCP-1 can also be used for the and Rhodococcus strains, above [49]). Introduced organisms bioremediation of contaminated groundwater, which is low in may fail to degrade or may even lose degradative activities in additional carbon sources compared with waste effluents (513). soils if they preferentially utilize more readily degradable However, amendment of soil contaminated by polychlorinated carbon sources. Several laboratory studies have monitored the phenoxyphenols with R. chlorophenolicus led to biomethyla- survival of microorganisms in the environment. Predation by tion, but there was no further metabolism of the respective protozoa was proved to be a major factor limiting growth and polychlorinated phenoxyanisols (511). Repeated applications causing the decline of various bacterial populations introduced ofP. cepacia AC1100 were needed to maintain biodegradation into soil amended with readily available organic nutrients (2, 3, of 2,4,5-T in soil, and the titer of P. cepacia AC1 00 cells in soil 155). Heitkamp and Cerniglia (196) showed that organic rapidly fell in the absence of 2,4,5-T (253). Mineralization of nutrient enrichment of sediment and water microcosms inoc- 2,4-D in soil was achieved by inoculation with a mixture of ulated with a Mycobacterium sp. hindered the mineralization of three different 2,4-D-degrading organisms. Comparison of the polycyclic aromatic hydrocarbons because of overgrowth of three strains, which harbor the identical 2,4-D degradation indigenous bacteria. Similarly, without the use of an inoculum, 668 FETZNER AND LINGENS MICROBIOL. REV.

the addition of an auxiliary carbon source showed repressive the degradation pathway of 2,3,5-T by strain AC1100. Thus, effects of aerobic biomineralization of ot-hexachlorocyclohex- since the combined cultures failed to degrade mixtures of ane in contaminated soil (24). Factors found by Zaidi et al. 2,4-D and 2,4,5-T, a new derivative of strain AC1100 was (577) to limit the success of an inoculum in enhancing biodeg- constructed by the transfer of the 2,4-D-degradative plasmid radation of organic chemicals in lake water included low pJP4 from strain JMP134 into strain AC1100. The new strain, concentration of the organic pollutant (carbon source near or designated RHJ1, which efficiently degraded mixtures of 2,4-D below the threshold for growth, Smin), the presence of other and 2,4,5-T, may be suitable for the remediation of waste readily degradable carbon sources, the concentration of inor- materials contaminated with both 2,4-D and 2,4,5-T (195). ganic nutrients, the effect of season (influence of rainfall on the Since many sites polluted with organic compounds were also concentration of inorganic nutrients in the lake water), and found to contain inorganic pollutants, such as heavy metals, grazing by protozoa. Thus, inoculation of contaminated sites Springael et al. (462) constructed heavy-metal-resistant with (halo)hydrocarbon-degrading bacteria often meets with haloaromatic-degrading A. eutrophus strains which were capa- limited success. However, specific modification and regulation ble of degrading various PCB isomers and 2,4-D in the of the environmental conditions in the contaminated site, the presence of 1 mM nickel or 2 mM zinc: A. eutrophus AS use of highly competitive and resistant wild-type strains, or the harboring pSS50 (4-chlorobiphenyl degradation) andA. eutro- use of specifically engineered strains as inocula may help to phus JMP134 harboring pJP4 (2,4-D degradation) were mated develop effective bioremediation methods. with A. eutrophus strains carrying megaplasmids conferring (iii) Bioaugmentation with genetically engineered microor- multiple resistance toward heavy metals. ganisms. The biodegradation potential of microorganisms may The hybrid Pseudomonas sp. strain B13 FR1(pFRC20P) was be modified by genetic engineering techniques. Directed mu- designed from three different bacterial strains to form a new tations may broaden the substrate or effector specificity of catabolic pathway which enables the recombinant strain to degradative enzymes, or the regulation of a degradative path- simultaneously degrade chloro- and methylaromatics (403). way may be modified (395). To achieve more effective or even This hybrid strain was shown to survive in laboratory micro- novel degradation capacities, hybrid strains may be con- cosms containing different aquatic sediments. The genetically structed by restructuring or combining various catabolic path- engineered microorganism did not measurably influence eco- ways. Such specifically designed strains might be used for the system parameters such as photosynthesis, community struc- treatment of specified industrial effluents or exhaust gases or ture of heterotrophic bacteria, or respiratory activity, and it for the decontamination of sites polluted with defined sub- efficiently enhanced the degradation of chloro- and methylaro- stances. matics in the sediments (389, 545). To cope with PCB contamination of soils and aquifers, Whereas these studies demonstrate the potential of geneti- different approaches were made to construct suitable microbial cally engineered organisms to perform bioremediation of strains. Site-directed mutagenesis of the bphA gene of Pseudo- environmentally hazardous compounds, there is also an exam- monas sp. strain LB400 (encoding the large subunit of the ple of a genetically engineered microorganism adversely affect- oxygenase component of biphenyl dioxygenase) resulted in a ing the indigenous microbial community. P. putida PP301 novel dioxygenase, which combines the broad substrate speci- (pRO103), when inoculated into aridic soil, accumulated 2,4- ficity of the LB400 enzyme with the superior ability of the dichlorophenol from 2,4-D, leading to a marked reduction in homologous enzyme from P. pseudoalcaligenes KF707 to de- soil respiration and a decline in the number of fungal prop- grade some double para-substituted PCB congeners. The agules in the soil. However, in agricultural soil, accumulation mutant strain might be suitable for the development of an of 2,4-dichlorophenol was not observed. This demonstrates the effective bioremediation process for sites contaminated with a need for a case-by-case evaluation of the suitability of geneti- mixture of (highly chlorinated) PCB congeners (115). Other cally engineered microorganisms for the specific system to attempts to achieve efficient degradation of PCBs involved the which it will be applied in the field. In this genetically construction of hybrid strains by mating between (chloro-) engineered strain PP301(pRO103), 2,4-dichlorophenol con- biphenyl- and chlorobenzoate-utilizing microorganisms (4, version was the bottleneck of the degradation pathway, since 203, 336). Not only are strains "constructed" in the laboratory, the tfdB gene encoding 2,4-dichlorophenol hydroxylase was but also horizontal gene transfer occurs in natural ecosystems. regulated by the product of another gene, whereas all other For instance, the catabolic plasmid pBRC60 from Alcaligenes catabolic genes were expressed constitutively (453). This dem- sp. strain BR60, which carries the chlorobenzoate-catabolic onstrates that genetically engineered microorganisms should transposon Tn5271 (see the section on haloaromatic dehalo- be designed specifically for controlled, regulated expression of genases, subsection on oxygenases) was demonstrated to be whole catabolic pathways and that their applicability for biore- mobile in a groundwater bioremediation system and in natural mediation purposes should be evaluated separately for each bacterial communities such as a groundwater community and system, since environmental factors might modulate their sediments and waters of a flowthrough freshwater mesocosm biodegradative behavior. (140, 141, 561). Populations of TnS271-carrying bacteria were Since there often is public concern about the environmental significantly higher in microcosms dosed with 3-chlorobenzo- release of genetically engineered microorganisms, their use as ate, 3-chlorobiphenyl, and 4-chloroaniline, which apparently inocula under field conditions is likely to be restricted by exerted selection pressure to maintain Tn5271 in the micro- political considerations in many countries. Alternatively, con- cosm (141). structed strains may be used as inocula in more defined or, if Mixtures of the herbicides 2,4-D and 2,4,5-T were toxic to possible, closed systems, e.g., in bioreactors designed to handle the 2,4,5-T degrader P. cepacia AC1100 alone and to combined specific wastes. For instance, calcium alginate-entrapped cells cultures of strain AC1100 and the 2,4-D degraderA. eutrophus of the constructed strain Pseudomonas sp. strain USlex (410) JMP134. 2,4,5-T was proposed to competitively inhibit the in a fluidized-bed column reactor were used for the continuous initial step of 2,4-D degradation in strain JMP134. Strain dehalogenation of a mixture of the herbicide 2,4-D and all AC1100 formed 2,4-dichlorophenol and 2-chlorohydroqui- three isomeric monochlorobenzoates (411). none from 2,4-D. The latter was inhibitory to the transforma- Bioreactors. An important strategy for end-of-pipe treat- tion of 2,4,5-trichlorophenol to 2,5-dichlorohydroquinone in ment or waste management is the development of bioreactors VOL. 58, 1994 BACTERIAL DEHALOGENASES 669

to deal with specific environmental pollutants. A bioreactor tri- and dichlorinated products with acetate as the electron may contain inoculations of competent microorganisms, often donor in an anaerobic biofilm column. The effluent of this immobilized as biofilms, or immobilized biocatalysts (e.g., anaerobic column was fed directly into a second, aerobic dehalogenases or corrinoids). There are as yet only few column containing microorganisms from activated sludge, bioreactors in which the second strategy is used: Marks and which metabolized the partially dechlorinated intermediates. Maule (311) used immobilized porphyrins and corrins to Such a two-stage sequential anaerobic-aerobic biofilm reactor dehalogenate organohalides such as lindane from an aqueous system may be suitable for treating groundwaters and indus- solution under reducing conditions. Tetra- and dichlorometh- trial effluents (123). ane also were efficiently degraded when supplied to the Combined systems involving first anaerobic and then aerobic immobilized system in gaseous form. Concerning the pump- treatment may be optimal for the remediation of wastes and-treat detoxification of contaminated groundwater or water containing highly chlorinated aliphatic and aromatic hydrocar- used to wash out pollutants from contaminated soil, the water bons, because reductive dechlorination is relatively rapid for can be passed through a continuous bioreactor for removal of chemicals with a large number of chlorine substituents. The the pollutants. TCE, for instance, is a significant groundwater resulting less highly chlorinated products usually are more pollutant. To avoid the problem of toxic products such as vinyl susceptible to a hydrolytic or oxidative process and less sus- chloride, which is generated by anaerobic TCE degradation, ceptible to further reduction. Consequently, two-stage systems Nelson et al. (366) developed a bench-scale continuous-treat- were successfully developed for the degradation of highly ment system for complete aerobic degradation of TCE by P. chlorinated phenols, especially PCP. Chloroguaiacols, chlo- cepacia G4, which utilizes enzymes of an aromatic degradative roveratroles, chlorocatechols, and chlorophenols were not pathway (presumably toluene 2-monooxygenase) to oxidize completely mineralized but were converted to less highly TCE. Interactions between substrates influence the kinetics of chlorinated compounds in an upflow anaerobic sludge blanket TCE removal: strain G4 requires toluene, o-cresol, m-cresol, reactor (332, 557). In contrast, complete anaerobic mineraliza- or phenol to induce the catabolic enzymes required for both tion of 3,4,5-trichlorophenol and all three isomeric monochlo- phenol and TCE degradation, but a competitive inhibitory rophenols in upflow anaerobic reactors was reported by interaction between the "natural" substrate and inducer phe- Krumme and Boyd (277), and PCP also turned out to be nol and the "fortuitous" substrate TCE affects the kinetics of removable by anaerobic fixed-film and upflow anaerobic sludge TCE removal. This effect must be considered in bioremedia- blanket reactors, as well as in an anaerobic semicontinuous- tion or in bioreactor design. To efficiently remove TCE with flow, stirred tank reactor (164, 197, 199, 558). By using a strain G4, phenol might be replaced by a noninhibitory, fixed-film reactor filled with ceramic Raschig rings and inocu- gratuitous inducer, or the catabolic genes might be placed lated with anaerobic digested sewage sludge, the influence of under alternative genetic regulation by using genetic engineer- an additional easily degradable carbon source on the anaerobic ing techniques. Alternatively, a bioreactor may be designed in dehalogenation of PCP and its metabolites was investigated. a way that maximizes enzyme production but minimizes inhib- The degradation of PCP, tetrachlorophenol, and trichlorophe- itory interactions between the inducer and TCE. Pursuing the nol was significantly enhanced by the presence of glucose latter approach, Folsom and Chapman (133) constructed a (198). Whereas the above-mentioned systems all were bench- bench-scale recirculating bioreactor. P. cepacia G4 grown with scale reactors, both a laboratory-scale and a pilot plant of the phenol in this bioreactor degraded 0.7 g of TCE per g of cell "Enso-Fenox reactor" were successfully used to treat bleach- protein per day. Two different bench-scale reactor types, i.e., a ing effluent from kraft pulping and debarking effluent and continuous-recycle, expanded-bed reactor (387) and a fixed- mixed effluent from thermodynamically pulping. In the first film, packed-bed trickling filter (470) were used to investigate stage, an anaerobic fluidized-bed reactor removed toxic chlo- the degradation of TCE by methanotrophic microbial consor- rophenolic compounds, which, together with other aerobically tia. In the continuous-recycle reactor, the threshold TCE degradable hydrocarbons, were mineralized in the second concentration (Smin) was 0.5 mg liter-', whereas in the trick- stage, an aerobic trickling filter (176). This combination of an ling-filter reactor, when the effluent was recycled, the TCE anaerobic fluidized bed and an aerobic trickling filter demon- threshold concentration was 50 to 100 ,ug liter-'. However, strated the superiority of two-stage anaerobic-aerobic treat- considering the technical feasibility of TCE bioremediation by ment processes, especially for bioremediation of complex methanotrophic microorganisms, colonization by protozoa wastes. 2,4,6-Trichlorophenol also was mineralized completely grazing on methanotrophs might be a problem of bioreactors in a sequential anaerobic-aerobic treatment process. Mineral- run with contaminated waters. ization was achieved by simply shifting an anaerobic popula- Tetrachloroethylene is resistant to biodegradation in aerobic tion, which produced 4-chlorophenol from 2,4,6-trichlorophe- environments but is biotransformed under anoxic conditions. nol, to aerobic conditions without any additional inoculation With benzoate as the electron donor, fermentative bacteria in (236). an anoxic fixed-bed reactor reductively dehalogenated tetra- Several bioreactors containing immobilized specific strains chloroethylene to cis-1,2-dichloroethylene. Tetrachloroethyl- were designed. A treatment system containing an immobilized ene was used as the electron acceptor and was dechlorinated in Flavobacterium strain to clean up PCP-contaminated ground- the mixed anaerobic culture without methanogenesis (440). water was described by Pflug and Burton (386). Polyurethane- Tetrachloroethylene was only partially mineralized in a con- immobilized cells of Flavobacterium strain ATCC 39723 de- tinuous-flow, fixed-film methanogenic column, and vinyl chlo- graded PCP in a continuous-culture bioreactor (376). In the ride was formed (536). However, de Bruin et al. (82) achieved course of a U.S. Environmental Protection Agency, Superfund complete reductive dechlorination of tetrachloroethylene to Innovative Technology Evaluation project, a pilot-scale fixed- ethylene in a laboratory-scale, continuous-flow, fixed-bed col- film biological system was used to remove PCP from ground- umn filled with a mixture of anaerobic sediment from the water. This "BioTrol" aqueous treatment system is one of the Rhine river and anaerobic granular sludge in the presence of few examples working on a pilot scale. It involved indigenous lactate as electron donor. Ethylene was further reduced to microorganisms taken from the local soil of the contaminated ethane (82). Tetrachloroethylene, trichloromethane, and hexa- site and amended with a PCP-degrading Flavobacterium strain chlorobenzene were reductively dechlorinated to the levels of (466). Frick et al. (139) also designed a large bioreactor 670 FETZNER AND LINGENS MICROBIOL. REV. containing PCP-degrading microorganisms immobilized in a Simultaneous removal of 1,2-dichloroethane and vinyl chlo- biofilm to decontaminate groundwater at a site where an ride from waste gases may be possible by establishing mixed aquifer had been contaminated with PCP. cultures of the vinyl chloride-utilizing strain Mycobacterium After adsorptive immobilization on granular clay, Alcali- aurum Li and the dichloroethane utilizer X autotrophicus genes sp. strain A7-2 was used in a percolator to degrade GJ10 (190). Vinyl chloride, which occurs in the exhaust gas of 4-chlorophenol (27). Technical chlorophenol, consisting of a polyvinyl chloride production factories, was removed from an mixture of 2,3,4,6-tetrachlorophenol, 2,4,6-trichlorophenol, artificial waste gas by using both a column with immobilized and PCP, was degraded to carbon dioxide and chloride by cells and a fermentor with a growing population of Mycobac- Rhodococcus cells immobilized on a polyurethane carrier. This terium aurum Li (188). Vapor-phase TCE was degraded aerobic (bench-scale) trickling filter remained active for more cometabolically in a multiple-segment trickling filter with than 4 months (513). Polyurethane entrapment was also used isoprene-grown cells of Rhodococcus erythropolis JE77 (510). to immobilize Streptomyces cells. Immobilized S. chromofuscus Another strategy of cometabolic TCE removal involved P. and S. rochei strains in an air-lift reactor dechlorinated high- cepacia G4 or P. mendocina KR-1, which oxidized TCE in a molecular-mass compounds in spent sulfite bleach effluents bench-scale gas lift bioreactor in the presence of the cosub- from a cellulose pulp mill. The bleach effluent compounds strate phenol or toluene (114). Cosubstrate feed apparently were dechlorinated but not depolymerized (583). did not cause competitive inhibition of TCE degradation A mixture of Hyphomicrobium strain DM2, Methylobacte- (however, see above and references 133 and 134). Especially rium strain DM4, and Pseudomonas strain DMSR was used for the removal of contaminants with a relatively low water with a degradation efficiency above 99.99% for the treatment solubility, membrane bioreactors with hydrophobic micro- of synthetic dichloromethane-containing waste water in a porous membranes seem to be well suited for waste gas fluidized-bed bioreactor (142). Hyphomicrobium strain DM2 treatment. Because of the high mass transfer rates of a was immobilized on sand particles in a fluidized-bed reactor to polypropylene membrane bioreactor constructed by Hartmans treat dichloromethane-containing chemical process effluents. et al. (191), dichloromethane was removed from waste gas with However, the degradation of other wastewater components high efficiency. was favored in the process effluent, thus making the process Most bioremediation reports are bench-scale studies dealing less attractive for waste streams containing large amounts of with biotreatments of single-component model waste systems dissolved organic carbon other than dichloromethane (473). or relatively simple model effluents or waste gases. In addition Two different bioreactors, a fixed-bed reactor with sintered to the scaling-up problem, a major problem in developing a glass beads run in a flowthrough mode and a fixed-bed reactor continuous bioreactor is the adaptation of the system from with granular activated carbon as the carrier material run with "model" to "real" waste. Since biotechnological methods are a recycle, were inoculated with a mixture of the dichloroeth- largely unproven at the process level so far, many companies ane-degrading strains X autotrophicus GJ10 and a gram- involved in environmental cleanup prefer physicochemical negative bacterium designated strain DEL. Dichloroethane is waste treatment methods, which, although expensive, guaran- an important groundwater contaminant. Low levels of dichlo- tee high efficiency. However, the use of natural, adapted, or roethane were removed at low temperatures and low salinity, genetically engineered organisms is now receiving more and i.e., under groundwater conditions. On the basis of these more attention as the difficulties and costs of conventional results, a full-scale groundwater purification plant (20 m3 h-1) physicochemical cleanup methods become explosive. In the was designed (475). future, biotechnological approaches should help to prevent Bioreactors can also be used for the biological elimination of further contamination of the environment and to cope with the volatile haloorganic compounds, when the gases to be removed legacy of contaminated sites. Further basic research on the solubilize in the water film over the humidified biomass (379). physiology, biochemistry, and genetics of the degradative ca- The purification of waste gases containing volatile halogenated pacities of microorganisms will be a prerequisite in the design organic compounds by means of biofilters or trickling filters of future technologies for waste management. was studied with various model compounds. Biological trickling filters inoculated with dichloromethane utilizers such as Hyphomicrobium sp. strain DM20 (192) were ACKNOWVLEDGMENTS used to eliminate dichloromethane from industrial waste gases. Our work was supported by the Fonds der Chemischen Industrie However, a trickling filter inoculated with X autotrophicus and by grant 0319416AO from the Bundesministerium fur Forschung GJ10 designed to remove 1,2-dichloroethane was not usable, und Technologie. because clogging and decreased removal efficiencies occurred We thank Barbara Tshisuaka for critical reading of the manuscript. within some months of operation. The causes of such an instability could be the accumulation of toxic compounds, the REFERENCES (stress-induced) formation of slime, or the formation of sheets 1. Abramowicz, D. A. 1990. Aerobic and anaerobic biodegradation of excessive biomass (521). This study showed that the use of of PCBs: a review. Crit. Rev. Biotechnol. 10:241-251. specific strains in continuous biological processes for waste gas 2. Acea, M. J., and M. Alexander. 1988. Growth and survival of treatment requires knowledge about the competitive behavior bacteria introduced into carbon-amended soil. Soil Biol. Bio- of the strain and knowledge of ways to stimulate the growth of chem. 20:703-709. the desirable organisms over that of less favorable ones. This 3. Acea, M. J., C. R. Moore, and M. Alexander. 1988. Survival and also includes insight into the kinetics of substrate removal and growth of bacteria introduced into soil. Soil Biol. Biochem. the requirement for organic cosubstrates. For instance, a 20:509-515. mixed culture of X autotrophicus and Pseudomonas sp. 4. Adams, R. H., C.-M. Huang, F. K. Higson, V. Brenner, and D. D. GJ10 Focht. 1992. Construction of a 3-chlorobiphenyl-utilizing recom- strain GJi grew more stably on 1,2-dichloroethane than did binant from an intergeneric mating. Appl. Environ. Microbiol. strain GJ10 alone, since GJi fulfilled the requirement of strain 58:647-654. GJ10 for biotin and GJ1 degraded the toxic compounds that 5. Adriaens, P., and D. D. Focht. 1991. Cometabolism of 3,4- accumulated from the dichloroethane-utilizing strain GJ10 dichlorobenzoate by Acinetobacter sp. strain 4-CB1. Appl. Envi- under suboptimal conditions (521). ron. Microbiol. 57:173-179. VOL. 58, 1994 BACTERIAL DEHALOGENASES 671

6. Adriaens, P., H.-P. E. Kohler, D. Kohler-Staub, and D. D. Focht. under different redox conditions. Appl. Environ. Microbiol. 54: 1989. Bacterial dehalogenation of chlorobenzoates and coculture 143-149. biodegradation of 4,4'-dichlorobiphenyl. Appl. Environ. Micro- 25. Bakac, A., and J. H. Espenson. 1986. Kinetics and mechanism of biol. 55:887-892. the alkylnickel formation in one-electron reductions of alkyl 7. Adrian, N.R, and J. M. Suflita. 1990. Reductive dehalogenation halides and hydroperoxides by a macrocyclic nickel(I) complex. J. of a nitrogen heterocyclic herbicide in anoxic aquifer slurries. Am. Chem. Soc. 108:713-719. Appl. Environ. Microbiol. 56:292-294. 26. Balajee, S., and A. Mahadevan. 1990. Dissimilation of 2,4- 8. Ahmad, D., M. Sylvestre, and M. Sondossi. 1991. Subcloning of dichlorophenoxyacetic acid by Azotobacter chroococcum. Xeno- bph genes from Pseudomonas testosteroni B-356 in Pseudomonas biotica 20:607-617. putida and Escherichia coli: evidence for dehalogenation during 27. Balfanz, J., and H.-J. Rehm. 1991. Biodegradation of 4-chloro- initial attack on chlorobiphenyls. Appl. Environ. Microbiol. 57: phenol by adsorptive immobilized Alcaligenes sp. A 7-2 in soil. 2880-2887. Appl. Microbiol. Biotechnol. 35:662-668. 9. Ahmed, A. E., and M. W. Anders. 1978. Metabolism of diha- 28. Bamo-Lage, G., F. Z. Parsons,R S. Nassar, and P. A. Lorenzo. lomethanes to formaldehyde and inorganic halide-IT. Studies on 1986. Sequential dehalogenation of chlorinated ethenes. Environ. the mechanism of the reaction. Biochem. Pharmacol. 27:2021- Sci. Technol. 20:96-99. 2025. 29. Bartels, I., H.-J. Knackmuss, and W. Reineke. 1984. Suicide 10. Ahr, H. J., L. J. King, W. Nastainczyk, and V. Ullrich. 1980. The inactivation of catechol 2,3-dioxygenase from Pseudomonas mechanism of chloroform and carbon monoxide formation from putida mt-2 by 3-halocatechols. Appl. Environ. Microbiol. 47: carbon tetrachloride by microsomal cytochrome P-450. Biochem. 500-505. Pharmacol. 29:2855-2861. 30. Barth, P. T., L. Bolton, and J. C. Thomson. 1992. Cloning and 11. Allison, N., A. J. Skinner, andR A. Cooper. 1983. The dehalo- partial sequencing of an operon encoding two Pseudomonas genases of a 2,2-dichloropropionate-degrading bacterium. J. putida haloalkanoate dehalogenases of opposite stereospecificity. Gen. Microbiol. 129:1283-1293. J. Bacteriol. 174:2612-2619. 12. Altenschmidt, U., and G. Fuchs. 1992. Novel aerobic 2-amino- 31. Bartnicki, E. W., and C. E. Castro. 1969. Biodehalogenation. The benzoate metabolism. Purification and characterization of 2-ami- pathway for transhalogenation and the stereochemistry of ep- nobenzoate-CoA ligase, localisation of the gene on a 8-kbp oxide formation from halohydrins. Biochemistry 8:4677-4680. plasmid, and cloning and sequencing of the gene from a denitri- 32. Baxter, R. M. 1990. Reductive dechlorination of certain chlori- fying Pseudomonas sp. Eur. J. Biochem. 205:721-727. nated organic compounds by reduced hematin compared with 13. Altenschmidt, U., B. Oswald, and G. Fuchs. 1991. Purification their behaviour in the environment. Chemosphere 21:451-458. and characterization of benzoate-coenzyme A ligase and 2-ami- 33. Beeching, J. R, A. J. Weightman, and J. H. Slater. 1983. The nobenzoate-coenzyme A ligases from a denitrifying Pseudomonas formation of an R-prime carrying the fraction I dehalogenase sp. J. Bacteriol. 173:5494-5501. gene from Pseudomonas putida PP3 using the IncP plasmid 14. Altenschmidt, U., B. Oswald, E. Steiner, H. Herrmann, and G. R68.44. J. Gen. Microbiol. 129:2071-2078. Fuchs. 1993. New aerobic benzoate oxidation pathway via ben- 34. Belay, N., and L. Daniels. 1987. Production of ethane, ethylene, zoyl-coenzyme A and 3-hydroxybenzoyl-coenzyme A in a deni- and acetylene from halogenated hydrocarbons by methanogenic trifying Pseudomonas sp. J. Bacteriol. 175:4851-4858. bacteria. Appl. Environ. Microbiol. 53:1604-1610. 15. Apajalahti, J. H. A., and M. S. Salkinoja-Salonen. 1986. Degra- 35. Biegert, T., U. Altenschmidt, C. Eckerskorn, and G. Fuchs. 1993. dation of polychlorinated phenols by Rhodococcus chlorophenoli- Enzymes of anaerobic metabolism of phenolic compounds. 4-Hy- cus. Appl. Microbiol. Biotechnol. 25:62-67. droxybenzoate-CoA ligase from a denitrifying Pseudomonas spe- 16. Apajalahti, J. H. A., and M. S. Salkinoja-Salonen. 1987. Dechlo- cies. Eur. J. Biochem. 213:555-561. rination and para-hydroxylation of polychlorinated phenols by 36. Blow, D. M., J. J. Birktoft, and B. S. Hartley. 1969. Role of a Rhodococcus chlorophenolicus. J. Bacteriol. 169:675-681. buried acid group in the mechanism of action of chymotrypsin. 17. Apajalahti, J. H. A., and M. S. Salkinoja-Salonen. 1987. Com- Nature (London) 221:337-340. plete dechlorination of tetrachlorohydroquinone by cell extracts 37. Bordeleau, L. M., and R Bartha. 1972. Biochemical transforma- of pentachlorophenol-induced Rhodococcus chlorophenolicus. J. tions of herbicide-derived anilines in culture medium and in soil. Bacteriol. 169:5125-5130. Can. J. Microbiol. 18:1857-1864. 18. Arciero, D., T. Vannelli, M. Logan, and A. B. Hooper. 1989. 38. Bosma, T. N. P., J. R van der Meer, G. Schraa, M. E. Tros, and Degradation of trichloroethylene by the ammonia-oxidizing bac- A. J. B. Zehnder. 1988. Reductive dechlorination of all trichloro- terium Nitrosomonas europaea. Biochem. Biophys. Res. Com- and dichlorobenzene isomers. FEMS Microbiol. Ecol. 53:223- mun. 159:640-643. 229. 19. Assaf-Anid, N., L. Nies, and T. M. Vogel. 1992. Reductive 39. Bouwer, E. J., and P. L. McCarty. 1983. Transformations of 1- dechlorination of a polychlorinated biphenyl congener and hexa- and 2-carbon halogenated aliphatic organic compounds under chlorobenzene by vitamin B12. Appl. Environ. Microbiol. 58: methanogenic conditions. Appl. Environ. Microbiol. 45:1286- 1057-1060. 1294. 20. Attaway, H. H., N. D. Camper, and M. J. B. Paynter. 1982. 40. Bouwer, E. J., and P. L. McCarty. 1983. Transformations of Anaerobic microbial degradation of diuron by pond sediment. halogenated organic compounds under denitrification conditions. Pestic. Biochem. Physiol. 17:96-101. Appl. Environ. Microbiol. 45:1295-1299. 21. Au, K. G., and C. T. Walsh. 1984. Stereochemical studies on a 41. Bouwer, E. J., and P. L. McCarty. 1985. Ethylene dibromide plasmid-coded fluoroacetate halidohydrolase. Bioorg. Chem. 12: transformation under methanogenic conditions. Appl. Environ. 197-205. Microbiol. 50:527-528. 22. Babbitt, P. C., G. L. Kenyon, B. M. Martin, H. Charest, M. 42. Bouwer, E. J., B. E. Rittmann, and P. L. McCarty. 1981. Sylvestre, J. D. Scholten, K.-H. Chang, P.-H. Liang, and D. Anaerobic degradation of halogenated 1- and 2-carbon organic Dunaway-Mariano. 1992. Ancestry of the 4-chlorobenzoate de- compounds. Environ. Sci. Technol. 15:596-599. halogenase: analysis of amino acid sequence identities among 43. Boyd, S. A., and D. R Shelton. 1984. Anaerobic biodegradation families of acyl:adenyl ligases, enoyl-CoA hydratases/isomerases, of chlorophenols in fresh and acclimated sludge. Appl. Environ. and acyl-CoA thioesterases. Biochemistry 31:5594-5604. Microbiol. 47:272-277. 23. Bachmann, A., W. de Bruin, J. C. Jumelet, H. H. N. Rijnaarts, 44. Boyd, S. A., D. R Shelton, D. Berry, and J. M. Tiedje. 1983. and A. J. B. Zehnder. 1988. Aerobic biomineralization of alpha- Anaerobic biodegradation of phenolic compounds in digested hexachlorocyclohexane in contaminated soil. Appl. Environ. Mi- sludge. Appl. Environ. Microbiol. 46:50-54. crobiol. 54:548-554. 45. Boyle, M. 1989. The environmental microbiology of chlorinated 24. Bachmann, A., P. Walet, P. Winen, W. de Bruin, J. L. M. aromatic decomposition. J. Environ. Qual. 18:395-402. Huntjens, W. Roelofsen, and A. J. B. Zehnder. 1988. Biodegra- 46. Brady, L., A. M. Brzozowski, Z. S. Derewenda, E. Dodson, G. dation of alpha- and beta-hexachlorocyclohexane in a slurry Dodson, S. Tolley, J. P. Turkenburg, L. Christiansen, B. Huge- 672 FETZNER AND LINGENS MICROBIOL. REV.

Jensen, L. Norskov, L. Thim, and U. Menge. 1990. A serine 65. Chaudhry, G. R, and S. Chapalamadugu. 1991. Biodegradation protease triad forms the catalytic centre of a triacylglycerol of halogenated organic compounds. Microbiol. Rev. 55:59-79. lipase. Nature (London) 343:767-770. 66. Chu, J., and E. J. Kirsch. 1973. Utilization of halophenols by a 47. Braus-Stromeyer, S. A., R. Hermann, A. M. Cook, and T. pentachlorophenol metabolizing bacterium. Dev. Ind. Microbiol. Leisinger. 1993. Dichloromethane as the sole carbon source for 14:264-273. an acetogenic mixed culture and isolation of a fermentative, 67. Chu, J. P., and E. J. Kirsch. 1972. Metabolism of pentachloro- dichloromethane-degrading bacterium. Appl. Environ. Micro- phenol by an axenic bacterial culture. Appl. Microbiol. 23:1033- biol. 59:3790-3797. 1035. 48. Brenner, V., B. S. Hernandez, and D. D. Focht. 1993. Variation in 68. Claus, H., and Z. Filip. 1990. Enzymatic oxidation of some chlorobenzoate catabolism by Pseudomonas putida Plll as a substituted phenols and aromatic amines, and the behaviour of consequence of genetic alterations. Appl. Environ. Microbiol. 59: some phenoloxidases in the presence of soil related adsorbents. 2790-2794. Water Sci. Technol. 22:69-77. 49. Briglia, M., E.-L. Nurmiaho-Lassila, G. Vallini, and M. Salki- 69. Colby, J., D. I. Stirling, and H. Dalton. 1977. The soluble noja-Salonen. 1990. The survival of the pentachlorophenol- methane mono-oxygenase of Methylococcus capsulatus (Bath). degrading Rhodococcus chlorophenolicus PCP-1 and Flavobacte- Biochem. J. 165:395-402. rium sp. in natural soil. Biodegradation 1:273-281. 70. Cook, A. M. 1987. Biodegradation of s-triazine xenobiotics. 50. Brokamp, A., and F. R J. Schmidt. 1991. Survival ofAlcaligenes FEMS Microbiol. Rev. 46:93-116. xylosoxidans degrading 2,2-dichloropropionate and horizontal 71. Cook, A. M., H. Grossenbacher, and R Huitter. 1984. Bacterial transfer of its halidohydrolase gene in a soil microcosm. Curr. degradation of N-cyclopropylmelamine. The steps to ring cleav- Microbiol. 22:299-306. age. Biochem. J. 222:315-320. 51. Brunner, W., D. Staub, and T. Leisinger. 1980. Bacterial degra- 72. Cook, A. M., and R Hutter. 1984. Deethylsimazine: bacterial dation of dichloromethane. Appl. Environ. Microbiol. 40:950- dechlorination, deamination, and complete degradation. J. Agric. 958. Food Chem. 32:581-585. 52. Brunner, W., F. H. Sutherland, and D. D. Focht. 1985. Enhanced 73. Cook, A. M., and R Hutter. 1986. Ring dechlorination of biodegradation of polychlorinated biphenyls in soil by analog deethylsimazine by hydrolases from Rhodococcus corallinus. enrichment and bacterial inoculation. J. Environ. Qual. 14:324- FEMS Microbiol. Lett. 34:335-338. 328. 74. Copley, S. D., and G. P. Crooks. 1992. Enzymic dehalogenation of 53. Bryant, F. O., D. D. Hale, and J. E. Rogers. 1991. Regiospecific 4-chlorobenzoyl coenzyme A in Acinetobacter sp. strain 4-CB1. dechlorination of pentachlorophenol by dichlorophenol-adapted Appl. Environ. Microbiol. 58:1385-1387. microorganisms in freshwater, anaerobic sediment slurries. Appl. 75. Cozza, C. L., and S. L. Woods. 1992. Reductive dechlorination Environ. Microbiol. 57:2293-2301. pathways for substituted benzenes: a correlation with electronic 54. Bull, A. T., G. Holt, and D. J. Hardman. 1988. Environmental properties. Biodegradation 2:265-278. pollution policies in light of biotechnological assessment: Organi- 76. Crawford, K L, and W. W. Mohn. 1985. Microbiological removal sation for Economic Cooperation, United Kingdom, and Euro- of pentachlorophenol from soil using a Flavobacterium. Enzyme pean Economic Council Perspectives, p. 351-371. In G. S. Microb. Technol. 7:617-620. Omenn (ed.), Environmental biotechnology. Plenum Press, New 77. Criddle, C. S., J. T. DeWitt, D. Grbic-Gali, and P. L. McCarty. York. 1990. Transformation of carbon tetrachloride by Pseudomonas 55. Buswell, J. A., and E. Odier. 1987. Lignin biodegradation. Crit. sp. strain KC under denitrification conditions. Appl. Environ. Rev. Biotechnol. 6:1-60. Microbiol. 56:3240-3246. 56. Castro, C. E., and E. W. Bartnicki. 1968. Biodehalogenation. 78. Criddle, C. S., J. T. DeWitt, and P. L. McCarty. 1990. Reductive Epoxidation of halohydrins, epoxide opening, and transhaloge- dehalogenation of carbon tetrachloride by Escherichia coli K-12. nation by a Flavobactenum sp. Biochemistry 7:3213-3218. Appl. Environ. Microbiol. 56:3247-3254. 57. Castro, C. E., and E. W. Bartnicki. 1975. Conformational iso- 79. Crooks, G. P., and S. D. Copley. 1993. A surprising effect of merism and effective redox geometry in the oxidation of heme leaving group on the nucleophilic aromatic substitution reaction proteins by alkyl halides, cytochrome c, and cytochrome oxidase. catalyzed by 4-chlorobenzoyl-CoA dehalogenase. J. Am. Chem. Biochemistry 14:498-503. Soc. 115:6422-6423. 58. Castro, C. E., and N. 0. Belser. 1990. Biodehalogenation: 80. Davies, J. I., and W. C. Evans. 1962. The elimination of halide oxidative and reductive metabolism of 1,1,2-trichloroethane by ions from aliphatic halogen-substituted organic acids by an Pseudomonas putida-biogeneration of vinyl chloride. Environ. enzyme preparation from Pseudomonas dehalogenans. Biochem. Toxicol. Chem. 9:707-714. J. 82:50P-S1P. 59. Castro, C. E., and W. C. Kray, Jr. 1963. The cleavage of bonds by 81. de Bont, J. A. M., M. J. A. W. Vorage, S. Hartmans, and W. J. J. low valent transition metal ions. The homogeneous reduction of van den Tweel. 1986. Microbial degradation of 1,3-dichloroben- alkyl halides by chromous sulfate. J. Am. Chem. Soc. 85:2768-2773. zene. Appl. Environ. Microbiol. 52:677-680. 60. Castro, C. E., R S. Wade, and N. 0. Belser. 1985. Biodehaloge- 82. de Bruin, W. P., M. J. J. Kotterman, M. A. Posthumus, G. nation: reactions of cytochrome P-450 with polyhalomethanes. Schraa, and A. J. B. Zehnder. 1992. Complete biological reduc- Biochemistry 24:204-210. tive transformation of tetrachloroethene to ethane. Appl. Envi- 61. Castro, C. E., W. H. Yokoyama, and N. 0. Belser. 1988. Biode- ron. Microbiol. 58:1996-2000. halogenation. Reductive reactivities of microbial and mammalian 83. de Jong, E., A. E. Cazemier, J. A. Field, and J. A. M. de Bont. cytochromes P-450 compared with heme and whole-cell models. 1994. Physiological role of chlorinated aryl alcohols biosynthe- J. Agric. Food Chem. 36:915-919. sized de novo by the white rot fungus Bjerkandera sp. strain 62. Chang, K.-H., P.-H. Liang, W. Beck, J. D. Scholten, and D. BOS55. Appl. Environ. Microbiol. 60:271-277. Dunaway-Mariano. 1992. Isolation and characterization of the 84. de Jong, E., J. A. Field, H.-E. Spinnler, J. B. P. A. Winberg, and three polypeptide components of 4-chlorobenzoate dehalogenase J. A. M. de Bont. 1994. Significant biogenesis of chlorinated from Pseudomonas sp. strain CBS-3. Biochemistry 31:5605-5610. aromatics by fungi in natural environments. Appl. Environ. 63. Chapman, P. J. 1979. Degradation mechanisms, p. 28-66. In Microbiol. 60:264-270. A. W. Bourquin and P. H. Pritchard (ed.), Proceedings of the 85. DeWeerd, K. A., F. Concannon, and J. M. Suflita. 1991. Rela- workshop: microbial degradation of pollutants in marine envi- tionship between hydrogen consumption, dehalogenation, and ronments. U.S. Environmental Protection Agency, Gulf Breeze, the reduction of sulfur oxyanions by Desulfomonile tiedjei. Appl. Fla. Environ. Microbiol. 57:1929-1934. 64. Chapman, P. J. 1988. Constructing microbial strains for degra- 86. DeWeerd, K. A., L. Mandelco, R S. Tanner, C. R Woese, and dation of halogenated aromatic hydrocarbons, p. 81-95. In G. S. J. M. Suflita. 1990. Desulfomonile tiedjei gen. nov. and sp. nov., a Omenn (ed.), Environmental biotechnology. Plenum Press, New novel anaerobic, dehalogenating, sulfate-reducing bacterium. York. Arch. Microbiol. 154:23-30. VOL. 58, 1994 BACTERIAL DEHALOGENASES 673

87. DeWeerd, K. A., and J. M. Suflita. 1990. Anaerobic aryl reductive 107. Egli, C., T. Tschan, R. Scholtz, A. M. Cook, and T. Leisinger. dehalogenation of halobenzoates by cell extracts of "Desulfomo- 1988. Transformation of tetrachloromethane to dichloromethane nile tiedjei." Appl. Environ. Microbiol. 56:2999-3005. and carbon dioxide by Acetobacterium woodii. Appl. Environ. 88. DeWeerd, K A., J. M. Suflita, T. Linkfield, J. M. Tiedje, and P. H. Microbiol. 54:2819-2824. Pritchard. 1986. The relationship between reductive dehaloge- 108. Elsner, A., F. LbIller, K. Miyashita, R. Muller, and F. Lingens. nation and other aryl substituent removal reactions catalyzed by 1991. Resolution of 4-chlorobenzoate dehalogenase from Pseu- anaerobes. FEMS Microbiol. Ecol. 38:331-339. domonas sp. strain CBS3 into three components. Appl. Environ. 89. DiIlio, C., A. Aceto, R. Piccolomini, N. Allocati, A. Faraone, L. Microbiol. 57:324-326. Cellini, G. Ravagnan, and G. Federici. 1988. Purification and 109. Elsner, A., R. Muller, and F. Lingens. 1991. Separate cloning and characterization of three forms of glutathione transferase from expression analysis of two protein components of 4-chlorobenzo- Proteus mirabilis. Biochem. J. 255:971-975. ate dehalogenase from Pseudomonas sp. CBS3. J. Gen. Micro- 90. Dijkstra, B. W., J. Drenth, and K. H. Kalk. 1981. Active site and biol. 137:477-481. catalytic mechanism of phospholipase A2. Nature (London) 289: 110. Engesser, K.-H., G. Auling, J. Busse, and H.-J. Knackmuss. 1990. 604-606. 3-Fluorobenzoate enriched bacterial strain FLB 300 degrades 91. DiSpirito, A. A., J. Gulledge, A. K. Shiemke, J. C. Murrell, M. E. benzoate and all three isomeric monofluorobenzoates. Arch. Lidstrom, and C. L. Krema. 1992. Trichloroethylene oxidation by Microbiol. 153:193-199. the membrane-associated methane monooxygenase in type I, 111. Engesser, K.-H., E. Schmidt, and H.-J. Knackmuss. 1980. Adap- type II and type X methanotrophs. Biodegradation 2:151-164. tation of Alcaligenes eutrophus B9 and Pseudomonas sp. B13 to 92. DiStefano, T. D., J. M. Gossett, and S. H. Zinder. 1991. Reduc- 2-fluorobenzoate as growth substrate. Appl. Environ. Microbiol. tive dechlorination of high concentrations of tetrachloroethene 39:68-73. to ethene by an anaerobic enrichment culture in the absence of 112. Engesser, K.-H., and P. Schulte. 1989. Degradation of 2-bromo-, methanogenesis. Appl. Environ. Microbiol. 57:2287-2292. 2-chloro- and 2-fluorobenzoate by Pseudomonas putida CLB250. 93. Dolfing, J. 1990. Reductive dechlorination of 3-chlorobenzoate is FEMS Microbiol. Lett. 60:143-148. coupled to ATP production and growth in an anaerobic bacte- 113. Engvild, K. C. 1986. Chlorine-containing natural compounds in rium, strain DCB-1. Arch. Microbiol. 153:264-266. higher plants. Phytochemistry 25:781-791. 94. Dolfing, J. 1990. Desulfomonile tiedjei and other anaerobic bac- 114. Ensley, B. D., and P. R. Kurisko. 1994. A gas lift bioreactor for teria with a taste for halogenated aromatic compounds, p. 47-64. removal of contaminants from the vapor phase. Appl. Environ. In B. N. Jacobsen, J. Zeyer, B. Jensen, P. Westermann, and B. Microbiol. 60:285-290. Ahring (ed.), Anaerobic biodegradation of xenobiotic com- 115. Erickson, B. D., and F. J. Mondello. 1993. Enhanced biodegra- pounds. Water pollution research report 25. Commission of the dation of polychlorinated biphenyls after site-directed mutagen- European Communities. esis of a biphenyl dioxygenase gene. Appl. Environ. Microbiol. 95. Dolfing, J., and B. K. Harrison. 1992. Gibbs free energy of 59:3858-3862. formation of halogenated aromatic compounds and their poten- 116. Evans, W. C., and G. Fuchs. 1988. Anaerobic degradation of tial role as electron acceptors in anaerobic environments. Envi- aromatic compounds. Annu. Rev. Microbiol. 42:289-317. ron. Sci. Technol. 26:2213-2218. 117. Evans, W. C., B. S. W. Smith, H. N. Fernley, and J. I. Davies. 96. Dolfing, J., and J. M. Tiedje. 1986. Hydrogen cycling in a 1971. Bacterial metabolism of 2,4-dichlorophenoxyacetate. Bio- three-tiered food web growing on the methanogenic conversion chem. J. 122:543-551. of 3-chlorobenzoate. FEMS Microbiol. Ecol. 38:293-298. 118. Fairbanks, B. C., G. A. O'Connor, and S. E. Smith. 1987. 97. Dolfing, J., and J. M. Tiedje. 1987. Growth yield increase linked Mineralization and volatilization of polychlorinated biphenyls in to reductive dechlorination in a defined 3-chlorobenzoate de- sludge-amended soils. J. Environ. Qual. 16:18-25. grading methanogenic coculture. Arch. Microbiol. 149:102-105. 119. Fathepure, B. Z., and S. A. Boyd. 1988. Reductive dechlorination 98. Dolfing, J., and J. M. Tiedje. 1991. Influence of substituents on of perchloroethylene and the role of methanogens. FEMS Mi- reductive dehalogenation of 3-chlorobenzoate analogs. Appl. crobiol. Lett. 49:149-156. Environ. Microbiol. 57:820-824. 120. Fathepure, B. Z., and S. A. Boyd. 1988. Dependence of tetrachlo- 99. Dorn, E., and H.-J. Knackmuss. 1978. Chemical structure and roethylene dechlorination on methanogenic substrate consump- biodegradability of halogenated aromatic compounds. Two cat- tion by Methanosarcina sp. strain DCM. Appl. Environ. Micro- echol 1,2-dioxygenases from a 3-chlorobenzoate-grown pseudo- biol. 54:2976-2980. monad. Biochem. J. 174:73-84. 121. Fathepure, B. Z., J. P. Nengu, and S. A. Boyd. 1987. Anaerobic 100. Dorn, E., and H.-J. Knackmuss. 1978. Chemical structure and bacteria that dechlorinate perchloroethene. Appl. Environ. Mi- biodegradability of halogenated aromatic compounds. Substitu- crobiol. 53:2671-2674. ent effects on 1,2-dioxygenation of catechol. Biochem. J. 174:85- 122. Fathepure, B. Z., J. M. Tiedje, and S. A. Boyd. 1988. Reductive 94. dechlorination of hexachlorobenzene to tri- and dichloroben- 101. Duxbury, J. M., J. M. Tiedje, M. Alexander, and J. E. Dawson. zenes in anaerobic sewage sludge. Appl. Environ. Microbiol. 54: 1970. 2,4-D metabolism: enzymatic conversion of chloromaleyl- 327-330. acetic acid to succinic acid. J. Agric. Food Chem. 18:199-201. 123. Fathepure, B. Z., and T. M. Vogel. 1991. Complete degradation 102. Edgehill, R. U., and R. K. Finn. 1983. Microbial treatment of soil of polychlorinated hydrocarbons by a two-stage biofilm reactor. to remove pentachlorophenol. Appl. Environ. Microbiol. 45: Appl. Environ. Microbiol. 57:3418-3422. 1122-1125. 124. Fernandez-Valverde, M., A. Reglero, H. Martinez-Blanco, and 103. Edgehill, R. U., and R. K. Finn. 1983. Activated sludge treatment J. M. Luengo. 1993. Purification of Pseudomonas putida acyl of synthetic wastewater containing pentachlorophenol. Biotech- coenzyme A ligase active with a range of aliphatic and aromatic nol. Bioeng. 15:2165-2176. substrates. Appl. Environ. Microbiol. 59:1149-1154. 104. Egli, C., R. Scholtz, A. M. Cook, and T. Leisinger. 1987. Anaer- 125. Fetzner, S., R. Muller, and F. Lingens. 1989. Degradation of obic dechlorination of tetrachloromethane and 1,2-dichloroeth- 2-chlorobenzoate by Pseudomonas cepacia 2CBS. Biol. Chem. ane to degradable products by pure cultures of Desulfobactenum Hoppe-Seyler 370:1173-1182. sp. and Methanobacterium sp. FEMS Microbiol. Lett. 43:257-261. 126. Fetzner, S., R. Muller, and F. Lingens. 1992. Purification and 105. Egli, C., S. Stromeyer, A. M. Cook, and T. Leisinger. 1990. some properties of 2-halobenzoate 1,2-dioxygenase, a two-com- Transformation of tetra- and trichloromethane to CO2 by anaer- ponent enzyme system from Pseudomonas cepacia 2CBS. J. obic bacteria is a non-enzymic process. FEMS Microbiol. Lett. Bacteriol. 174:279-290. 68:207-212. 127. Finn, R. K. 1983. Use of specialized microbial strains in the 106. Egli, C., M. Thuer, D. Suter, A. M. Cook, and T. Leisinger. 1989. treatment of industrial waste and in soil decontamination. Expe- Monochloro- and dichloroacetic acids as carbon and energy rientia 39:1231-1236. sources for a stable, methanogenic mixed culture. Arch. Micro- 128. Focht, D. D., and M. Alexander. 1970. Bacterial degradation of biol. 152:218-223. diphenylmethane, a DDT model substrate. Appl. Microbiol. 674 FETZNER AND LINGENS MICROBIOL. REV.

20:608-611. Characterization of anaerobic dechlorinating consortia derived 129. Focht, D. D., and M. Alexander. 1970. DDT metabolites and from aquatic sediments. Appl. Environ. Microbiol. 55:1472-1476. analogs: ring fission by Hydrogenomonas. Science 170:91-92. 151. Gibson, S. A., and J. M. Suflita. 1986. Extrapolation of biodeg- 130. Focht, D. D., and W. Brunner. 1985. Kinetics of biphenyl and radation results to groundwater aquifers: reductive dehalogena- polychlorinated biphenyl metabolism in soil. Appl. Environ. tion of aromatic compounds. Appl. Environ. Microbiol. 52:681- Microbiol. 50:1058-1063. 688. 131. Focht, D. D., and D. Shelton. 1987. Growth kinetics of Pseudo- 152. Gibson, S. A., and J. M. Suflita. 1990. Anaerobic biodegradation monas alcaligenes C-0 relative to inoculation and 3-chlorobenzo- of 2,4,5-trichlorophenoxyacetic acid in samples from a methano- ate metabolism in soil. Appl. Environ. Microbiol. 53:1846-1849. genic aquifer: stimulation by short-chain organic acids and alco- 132. Fogel, M. M., A. R. Taddeo, and S. Fogel. 1986. Biodegradation hols. Appl. Environ. Microbiol. 56:1825-1832. of chlorinated ethenes by a methane-utilizing mixed culture. 153. Goldman, P. 1965. The enzymatic cleavage of the carbon-fluorine Appl. Environ. Microbiol. 51:720-724. bond in fluoroacetate. J. Biol. Chem. 240:3434-3438. 133. Folsom, B. R., and P. J. Chapman. 1991. Performance charac- 154. Goldman, P., G. W. A. Milne, and D. B. Keister. 1968. Carbon- terization of a model bioreactor for the biodegradation of halogen bond cleavage. III. Studies on bacterial halidohydro- trichloroethylene by Pseudomonas cepacia G4. Appl. Environ. lases. J. Biol. Chem. 243:428-434. Microbiol. 57:1602-1608. 155. Goldstein, R. M., L. M. Mallory, and M. Alexander. 1985. 134. Folsom, B. R., P. J. Chapman, and P. H. Pritchard. 1990. Phenol Reasons for possible failure of inoculation to enhance biodegra- and trichloroethylene degradation by Pseudomonas cepacia G4: dation. Appl. Environ. Microbiol. 50:977-983. kinetics and interactions between substrates. Appl. Environ. 156. Golovleva, L. A., R. N. Pertsova, L. L. Evtushenko, and B. P. Microbiol. 56:1279-1285. Baskunov. 1990. Degradation of 2,4,5-trichlorophenoxyacetic 135. Fowden, L. 1968. The occurrence and metabolism of carbon- acid by a Nocardioides simplex culture. Biodegradation 1:263- halogen compounds. Proc. R. Soc. London Ser. B 171:5-18. 271. 136. Fox, B. G., J. G. Borneman, L. P. Wackett, and J. D. Lipscomb. 157. Golovleva, L. A., 0. Zaborina, R. Pertsova, B. Baskunov, Y. 1990. Haloalkene oxidation by the soluble methane monooxygen- Schurukhin, and S. Kuzmin. 1992. Degradation of polychlori- ase from Methylosinus trichosporium OB3b: mechanistic and nated phenols by Streptomyces rochei 303. Biodegradation 2:201- environmental implications. Biochemistry 29:6419-6427. 208. 137. Franken, S. M., H. J. Rozeboom, K. H. Kalk, and B. W. Dijkstra. 158. Green, J., and H. Dalton. 1989. Substrate specificity of soluble 1991. Crystal structure of haloalkane dehalogenase: an enzyme to methane monooxygenase. Mechanistic implications. J. Biol. detoxify halogenated alkanes. EMBO J. 10:1297-1302. Chem. 264:17698-17703. 138. Freedman, D. L., and J. M. Gossett. 1989. Biological reductive 159. Griffith, G. D., J. R. Cole, J. F. Quensen Ill, and J. M. Tiedje. dechlorination of tetrachloroethylene and trichloroethylene to 1992. Specific deuteration of dichlorobenzoate during reductive ethylene under methanogenic conditions. Appl. Environ. Micro- dehalogenation by Desulfomonile tiedjei in D20. Appl. Environ. biol. 55:2144-2151. Microbiol. 58:409-411. 139. Frick, T. D., R. L. Crawford, M. Martinson, T. Chresand, and G. 160. Groenewegen, P. E. J., W. J. J. van den Tweel, and J. A. M. de Bateson. 1988. Microbiological cleanup of groundwater contam- Bont. 1992. Anaerobic bioformation of 4-hydroxybenzoate from inated by pentachlorophenol, p. 173-191. In G. S. Omenn (ed.), 4-chlorobenzoate by the coryneform bacterium NTB-1. Appl. Environmental biotechnology. Plenum Press, New York. Microbiol. Biotechnol. 36:541-547. 140. Fulthorpe, R. R., and R. C. Wyndham. 1991. Transfer and 161. Grossenbacher, H., C. Horn, A. M. Cook, and R. Hutter. 1984. expression of the catabolic plasmid pBRC60 in wild bacterial 2-Chloro-4-amino-1,3,5-triazine-6(5H)-one: a new intermediate recipients in a freshwater ecosystem. Appl. Environ. Microbiol. in the biodegradation of chlorinated s-triazines. Appl. Environ. 57:1546-1553. Microbiol. 48:451-453. 141. Fulthorpe, R. R., and R. C. Wyndham. 1992. Involvement of a 162. Grove, G., R. P. Zarlengo, K. P. Timmerman, N.-Q. Li, M. F. chlorobenzoate-catabolic transposon, Tn5271, in community ad- Tam, and C.-P. D. Tu. 1988. Characterization and heterospecific aptation to chlorobiphenyl, chloroaniline, and 2,4-dichlorophe- expression of cDNA clones of genes in the maize GSH S- noxyacetic acid in a freshwater ecosystem. Appl. Environ. Micro- transferase multigene family. Nucleic Acids Res. 16:425-438. biol. 58:314-325. 163. Gschwend, P. M., J. K MacFarlane, and K A. Newman. 1985. 142. Galli, R. 1987. Biodegradation of dichloromethane in waste water Volatile halogenated organic compounds released to seawater using a fluidized bed bioreactor. Appl. Microbiol. Biotechnol. from temperate marine macroalgae. Science 227:1033-1035. 27:206-213. 164. Guthrie, M. A., E. J. Kirsch, R. F. Wukasch, and C. P. L. Grady, 143. Galli, R., and T. Leisinger. 1985. Specialized bacterial strains for Jr. 1984. Pentachlorophenol biodegradation. II. Anaerobic. Wa- the removal of dichloromethane from industrial waste. Conserv. ter Res. 18:451-461. Recycl. 8:91-100. 165. Haak, B., and S. Fetzner. Unpublished data. 144. Galli, R., and T. Leisinger. 1988. Plasmid analysis and cloning of 166. Haak, B., S. Fetzner, and F. Lingens. Cloning, nucleotide se- the dichloromethane-utilization genes of Methylobacterium sp. quence, and expression of the plasmid-encoded genes for the DM4. J. Gen. Microbiol. 134:943-952. two-component 2-halobenzoate 1,2-dioxygenase from Pseudomo- 145. Galli, R., and P. L. McCarty. 1989. Biotransformation of 1,1,1- nas cepacia 2CBS. Submitted for publication. trichloroethane, trichloromethane, and tetrachloromethane by a 167. Haggblom, M. M. 1992. Microbial breakdown of halogenated Clostridium sp. Appl. Environ. Microbiol. 55:837-844. aromatic pesticides and related compounds. FEMS Microbiol. 146. Galli, R., G. Stucki, and T. Leisinger. 1982. Mechanism of Rev. 103:29-72. dehalogenation of dichloromethane by cell extracts of Hyphomi- 168. Haggblom, M. M., J. H. A. Apajalahti, and M. S. Salkinoja- crobium DM2. Experientia 38:1378. Salonen. 1988. Hydroxylation and dechlorination of chlorinated 147. Gantzer, C. J., and L. P. Wackett. 1991. Reductive dechlorination guaiacols and syringols by Rhodococcus chlorophenolicus. Appl. catalyzed by bacterial transition-metal coenzymes. Environ. Sci. Environ. Microbiol. 54:683-687. Technol. 25:715-722. 169. Haggblom, M. M., D. Janke, and M. S. Salkinoja-Salonen. 1989. 148. Geissler, J. F., C. S. Harwood, and J. Gibson. 1988. Purification Hydroxylation and dechlorination of tetrachlorohydroquinone by and properties of benzoate-coenzyme A ligase, a Rhodopseudo- Rhodococcus sp. strain CP-2 cell extracts. Appl. Environ. Micro- monas palustris enzyme involved in the anaerobic degradation of biol. 55:516-519. benzoate. J. Bacteriol. 170:1709-1714. 170. Haggblom, M. M., L. J. Nohynek, and M. S. Salkinoja-Salonen. 149. Genthner, B. R. S., W. A. Price II, and P. H. Pritchard. 1989. 1988. Degradation and 0-methylation of chlorinated phenolic Anaerobic degradation of chloroaromatic compounds in aquatic compounds by Rhodococcus and Mycobacterium strains. Appl. sediments under a variety of enrichment conditions. Appl. Envi- Environ. Microbiol. 54:3043-3052. ron. Microbiol. 55:1466-1471. 171. Haggblom, M. M., M. D. Rivera, and L. Y. Young. 1993. Influence 150. Genthner, B. R. S., W. A. Price II, and P. H. Pritchard. 1989. of alternative electron acceptors on the anaerobic biodegradabil- VOL. 58, 1994 BACTERIAL DEHALOGENASES 675

ity of chlorinated phenols and benzoic acids. Appl. Environ. 194. Hasan, A. K. M. Q., K. Motosugi, N. Esaki, and K. Soda. 1991. Microbiol. 59:1162-1167. Total conversion of racemic 2-chloropropionic acid into D-lactate 172. Haggblom, M. M., and L. Y. Young. 1990. Chlorophenol degra- by combination of enzymatic and chemical dehalogenations. J. dation coupled to sulfate reduction. Appl. Environ. Microbiol. Ferment. Bioeng. 72:481-482. 56:3255-3260. 195. Haugland, R. A., D. J. Schlemm, R. P. Lyons III, P. R. Sferra, and 173. Haider, K. 1979. Degradation and metabolization of lindane and A. M. Chakrabarty. 1990. Degradation of the chlorinated phe- other hexachlorocyclohexane isomers by anaerobic and aerobic noxyacetate herbicides 2,4-dichlorophenoxyacetic acid and 2,4,5- soil microorganisms. Z. Naturforsch. Sect. C 34:1066-1069. trichlorophenoxyacetic acid by pure and mixed bacterial cultures. 174. Haider, K., G. Jagnow, R. Kohnen, and S. U. Lim. 1974. Appl. Environ. Microbiol. 56:1357-1362. Degradation of chlorinated benzenes, phenols and cyclohexane 196. Heitkamp, M. A., and C. E. Cerniglia. 1989. Polycyclic aromatic derivatives by benzene and phenol utilizing soil bacteria under hydrocarbon degradation by a Mycobactenum sp. in microcosms aerobic conditions. Arch. Microbiol. 96:183-200. containing sediment and water from a pristine ecosystem. Appl. 175. Haigler, B. E., S. F. Nishino, and J. C. Spain. 1988. Degradation Environ. Microbiol. 55:1968-1973. of 1,2-dichlorobenzene by a Pseudomonas sp. Appl. Environ. 197. Hendriksen, H. V., and B. K. Ahring. 1993. Anaerobic dechlori- Microbiol. 54:294-301. nation of pentachlorophenol in fixed-film and upflow anaerobic 176. Hakulinen, R., and M. Salkinoja-Salonen. 1982. Treatment of sludge blanket reactors using different inocula. Biodegradation pulp and paper industry wastewaters in an anaerobic fluidised 3:399-408. bed reactor. Process Biochem. 17:18-22. 198. Hendriksen, H. V., S. Larsen, and B. K. Ahring. 1991. Anaerobic 177. Hale, D. D., J. E. Rogers, and J. Wiegel. 1990. Reductive degradation of PCP and phenol in fixed-film reactors: the influ- dechlorination of dichlorophenols by nonadapted and adapted ence of an additional substrate. Water Sci. Technol. 24:431-436. microbial communities in pond sediments. Microb. Ecol. 20:185- 199. Hendriksen, H. V., S. Larsen, and B. K. Ahring. 1992. Influence 196. of a supplemental carbon source on anaerobic dechlorination of 178. Halpern, J., and J. P. Maher. 1965. Kinetics of the reactions of pentachlorophenol in granular sludge. Appl. Environ. Microbiol. pentacyanocobaltate(II) with organic halides. J. Am. Chem. Soc. 58:365-370. 87:5361-5366. 200. Henson, J. M., M. V. Yates, J. W. Cochran, and D. L. Shackle- 179. Hammel, K. E., and P. J. Tardone. 1988. The oxidative 4-dechlo- ford. 1988. Microbial removal of halogenated methanes, ethanes, rination of polychlorinated phenols is catalyzed by extracellular and ethylenes in an aerobic soil exposed to methane. FEMS fungal lignin peroxidases. Biochemistry 27:6563-6568. Microbiol. Ecol. 53:193-201. 180. Harayama, S., M. Rekik, and K. N. Timmis. 1986. Genetic 201. Heritage, A. D., and I. C. MacRae. 1977. Degradation of lindane analysis of a relaxed substrate specificity aromatic ring dioxyge- by cell-free preparations of Clostridium sphenoides. Appl. Envi- nase, toluate 1,2-dioxygenase, encoded by TOL plasmid pWWO ron. Microbiol. 34:222-224. of Pseudomonas putida. Mol. Gen. Genet. 202:226-234. 202. Hernandez, B. S., F. K Higson, R. Kondrat, and D. D. Focht. 181. Hardman, D. J. 1991. Biotransformation of halogenated com- 1991. Metabolism of and inhibition by chlorobenzoates in pounds. Crit. Rev. Biotechnol. 11:1-40. Pseudomonas putida P1ll. Appl. Environ. Microbiol. 57:3361- 182. Hardman, D. J., P. C. Gowland, and J. H. Slater. 1986. Large 3366. plasmids from soil bacteria enriched on halogenated alkanoic 203. Hickey, W. J., V. Brenner, and D. D. Focht. 1992. Mineralization acids. Appl. Environ. Microbiol. 51:44-51. of 2-chloro- and 2,5-dichlorobiphenyl by Pseudomonas sp. strain 183. Hardman, D. J., and J. H. Slater. 1981. Dehalogenases in soil UCR2. FEMS Microbiol. Lett. 98:175-180. bacteria. J. Gen. Microbiol. 123:117-128. 204. Hickey, W. J., and D. D. Focht. 1990. Degradation of mono-, di-, 184. Hardman, D. J., and J. H. Slater. 1981. The dehalogenase and trihalogenated benzoic acids by Pseudomonas aeruginosa complement of a soil pseudomonad grown in closed and open JB2. Appl. Environ. Microbiol. 56:3842-3850. cultures on haloalkanoic acids. J. Gen. Microbiol. 127:399-405. 205. Hickey, W. J., D. B. Searles, and D. D. Focht. 1993. Enhanced 185. Harker, A. R., and Y. Kim. 1990. Trichloroethylene degradation mineralization of polychlorinated biphenyls in soil inoculated by two independent aromatic-degrading pathways in Alcaligenes with chlorobenzoate-degrading bacteria. Appl. Environ. Micro- eutrophus JMP134. Appl. Environ. Microbiol. 56:1179-1181. biol. 59:1194-1200. 186. Hartmann, J., W. Reineke, and H.-J. Knackmuss. 1979. Metab- 206. Higgins, I. J., R. C. Hammond, F. S. Sariaslani, D. Best, M. M. olism of 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate by a Davies, S. E. Tryhorn, and F. Taylor. 1979. Biotransformation of pseudomonad. Appl. Environ. Microbiol. 37:421-428. hydrocarbons and related compounds by whole organism suspen- 187. Hartmans, S., J. A. M. de Bont, and W. Harder. 1989. Microbial sions of methane-grown Methylosinus trichosporium OB3b. Bio- metabolism of short-chain unsaturated hydrocarbons. FEMS chem. Biophys. Res. Commun. 89:671-677. Microbiol. Rev. 63:235-264. 207. Higson, F. K., and D. D. Focht. 1990. Degradation of 2-bromo- 188. Hartmans, S., J. A. M. de Bont, J. Tramper, and K. C. A. M. benzoic acid by a strain of Pseudomonas aeruginosa. Appl. Luyben. 1985. Bacterial degradation of vinyl chloride. Biotech- Environ. Microbiol. 56:1615-1619. nol. Lett. 7:383-388. 208. Hill, H. A. O., J. M. Pratt, M. P. O'Riordan, F. R. Williams, and 189. Hartmans, S., M. W. Jansen, M. J. van der Werf, and J. A. M. de R. J. P. Williams. 1971. The chemistry of vitamin B12. XV. Bont. 1991. Bacterial metabolism of 3-chloroacrylic acid. J. Gen. Catalysis of alkyl halide reduction by vitamin B12a: studies using Microbiol. 137:2025-2032. controlled potential reduction. J. Chem. Soc. A 1971:1859-1862. 190. Hartmans, S., A. Kaptein, J. Tramper, and J. A. M. de Bont. 209. Holliger, C., S. W. M. Kengen, G. Schraa, A. J. M. Stams, and 1992. Characterization of a Mycobacterium sp. and a Xan- A. J. B. Zehnder. 1992. Methyl-coenzyme M reductase of Meth- thobacter sp. for the removal of vinyl chloride and 1,2-dichloro- anobacterium thermoautotrophicum AH catalyzes the reductive ethane from waste gases. Appl. Microbiol. Biotechnol. 37:796- dechlorination of 1,2-dichloroethane to ethylene and chloroeth- 801. ane. J. Bacteriol. 174:4435-4443. 191. Hartmans, S., E. J. T. M. Leenen, and G. T. H. Voskuilen. 1992. 210. Holliger, C., G. Schraa, A. J. M. Stams, and A. J. B. Zehnder. Membrane bioreactor with porous hydrophobic membranes for 1990. Reductive dechlorination of 1,2-dichloroethane and chlo- waste-gas treatment, p. 103-106. In A. J. Dragt and J. van Ham roethane by cell suspensions of methanogenic bacteria. Biodeg- (ed.), Biotechniques for air pollution abatement and odour radation 1:253-261. control policies. Elsevier Science Publishers BV, Amsterdam. 211. Holliger, C., G. Schraa, A. J. M. Stams, and A. J. B. Zehnder. 192. Hartmans, S., and J. Tramper. 1991. Dichloromethane removal 1992. Enrichment and properties of an anaerobic mixed culture from waste gases with a trickle-bed bioreactor. Bioprocess Eng. reductively dechlorinating 1,2,3-trichlorobenzene to 1,3-dichloro- 6:83-92. benzene. Appl. Environ. Microbiol. 58:1636-1644. 193. Harwood, C. S., and J. Gibson. 1986. Uptake of benzoate by 212. Holliger, C., G. Schraa, A. J. M. Stams, and A. J. B. Zehnder. Rhodopseudomonas palustris grown anaerobically in light. J. 1993. A highly purified enrichment culture couples the reductive Bacteriol. 165:504-509. dechlorination of tetrachloroethene to growth. Appl. Environ. 676 FETZNER AND LINGENS MICROBIOL. REV.

Microbiol. 59:2991-2997. 231. Johnston, H. W., G. G. Briggs, and M. Alexander. 1972. Metab- 213. Holliger, C., G. Schraa, E. Stupperich, A. J. M. Stams, and olism of 3-chlorobenzoic acid by a pseudomonad. Soil Biol. A. J. B. Zehnder. 1992. Evidence for the involvement of corri- Biochem. 4:187-190. noids and factor F430 in the reductive dechlorination of 1,2- 232. Johri, S., G. N. Qazi, and C. L Chopra. 1991. Evidence of dichloroethane byMethanosarcina barkeri. J. Bacteriol. 174:4427- plasmid mediated dechlorinase activity in Pseudomonas sps. J. 4434. Biotechnol. 20:73-82. 214. Holmstead, R. L. 1976. Studies of the degradation of mirex with 233. Jones, D. H. A., P. T. Barth, D. Byrom, and C. M. Thomas. 1992. an iron(II)porphyrin model system. J. Agric. Food Chem. 24: Nucleotide sequence of the structural gene encoding a 2-haloal- 620-624. kanoic acid dehalogenase of Pseudomona'sputida strain AJ1 and 215. Hopkins, G. D., L. Semprini, and P. L. McCarty. 1993. Micro- purification of the encoded protein. J. Gen. Microbiol. 138:675- cosm and in situ field studies of enhanced biotransformation of 683. trichloroethylene by phenol-utilizing microorganisms. Appl. En- 234. Joshi, D. K., and M. H. Gold. 1993. Degradation of 2,4,5- viron. Microbiol. 59:2277-2285. trichlorophenol by the lignin-degrading basidiomycete Phanero- 216. Horowitz, A., D. R. Shelton, C. P. Cornell, and J. M. Tiedje. 1982. chaete chrysosponum. Appl. Environ. Microbiol. 59:1779-1785. Anaerobic degradation of aromatic compounds in sediments and 235. Jutzi, K., A. M. Cook, and R. Hutter. 1982. The degradative digested sludge. Dev. Ind. Microbiol. 23:435-444. pathway of the s-triazine melamine. The steps to ring cleavage. 217. Husain, M., B. Entsch, D. P. Ballou, V. Massey, and P. J. Biochem. J. 208:679-684. Chapman. 1980. Fluoride elimination from substrates in hydroxy- 236. Kafkewitz, D., P. M. Armenante, G. Lewandowski, and C.-M. lation reactions catalyzed by p-hydroxybenzoate hydroxylase. J. Kung. 1992. Dehalogenation and mineralization of 2,4,6-trichlo- Biol. Chem. 255:4189-4197. rophenol by the sequential activity of anaerobic and aerobic 218. Ide, A., Y. Niki, F. Sakamoto, I. Watanabe, and H. Watanabe. microbial populations. Biotechnol. Lett. 14:143-148. 1972. Decomposition of pentachlorophenol in paddy soil. Agric. 237. Karns, J. S., J. J. Kilbane, S. Duttagupta, and A. M. Biol. Chem. 36:1937-1944. Chakrabarty. 1983. Metabolism of halophenols by 2,4,5-trichlo- 219. Imai, R., Y. Nagata, M. Fukuda, M. Takagi, and K. Yano. 1991. rophenoxyacetic acid-degrading Pseudomonas cepacia. Appl. En- Molecular cloning of a Pseudomonas paucimobilis gene encoding viron. Microbiol. 46:1176-1181. a 17-kilodalton polypeptide that eliminates HCI molecules from 238. Kasai, N., K. Tsujimura, K. Unoura, and T. Suzuki. 1990. -y-hexachlorocyclohexane. J. Bacteriol. 173:6811-6819. Degradation of 2,3-dichloro-1-propanol by a Pseudomonas sp. 220. Imai, R., Y. Nagata, K. Senoo, H. Wada, M. Fukuda, M. Takagi, Agric. Biol. Chem. 54:3185-3190. and K. Yano. 1989. Dehydrochlorination of y-hexachlorocyclo- 239. Kasai, N., K. Tsujimura, K. Unoura, and T. Suzuki. 1992. hexane (-y-BHC) by y-BHC assimilating Pseudomonas paucimo- Preparation of (S)-2,3-dichloro-1-propanol by Pseudomonas sp. bilis. Agric. Biol. Chem. 53:2015-2017. and its use in the synthesis of (R)-epichlorohydrin. J. Ind. 221. Ishida, M. 1968. Comparative studies on BHC metabolizing Microbiol. 9:97-101. enzymes, DDT dehydrochlorinase and glutathione S-trans- 240. Kasai, N., K. Tsujimura, K. Unoura, and T. Suzuki. 1992. ferases. Agric. Biol. Chem. 32:947-955. Isolation of (S)-2,3-dichloro-1-propanol assimilating bacterium, 222. Jacobsen, C. S., and J. C. Pedersen. 1992. Mineralization of its characterization, and its use in preparation of (R)-2,3-di- 2,4-dichlorophenoxyacetic acid (2,4-D) in soil inoculated with chloro-1-propanol and (S)-epichlorohydrin. J. Ind. Microbiol. Pseudomonas cepacia DBOI(pRO101), Alcaligenes eutrophus 10:37-43. AEO106(pRO101) and Alcaligenes eutrophus JMP134(pJP4): ef- 241. Kaschabek, S. R., and W. Reineke. 1992. Maleylacetate reductase fects of inoculation level and substrate concentration. Biodegra- of Pseudomonas sp. strain B13: dechlorination of chloromaleyl- dation 2:253-263. acetates, metabolites in the degradation of chloroaromatic com- 223. Jagnow, G., K. Haider, and P.-C. Ellwardt. 1977. Anaerobic pounds. Arch. Microbiol. 158:412-417. dechlorination and degradation of hexachlorocyclohexane iso- 242. Kastner, M. 1991. Reductive dechlorination of tri- and tetrachlo- mers by anaerobic and facultative anaerobic bacteria. Arch. roethylenes depends on transition from aerobic to anaerobic Microbiol. 115:285-292. conditions. Appl. Environ. Microbiol. 57:2039-2046. 224. Janssen, D. B., J. Gerritse, J. Brackman, C. Kalk, D. Jager, and 243. Kawasaki, H., S. Hayashi, H. Yahara, F. Minami, and K. B. Witholt. 1988. Purification and characterization of a bacterial Tonomura. 1982. Plasmid pUO2 determining haloacetate deha- dehalogenase with activity toward halogenated alkanes, alcohols logenase and mercury resistance in Pseudomonas sp. J. Ferment. and ethers. Eur. J. Biochem. 171:67-72. Technol. 60:5-11. 225. Janssen, D. B., G. Grobben, R. Hoekstra, R. Oldenhuis, and B. 244. Kawasaki, H., K. Miyoshi, and K. Tonomura. 1981. Purification, Witholt. 1988. Degradation of trans-1,2-dichloroethene by mixed crystallization and properties of haloacetate halidohydrolase and pure cultures of methanotrophic bacteria. Appl. Microbiol. from Pseudomonas species. Agric. Biol. Chem. 45:543-544. Biotechnol. 29:392-399. 245. Kawasaki, H., M. Takao, A. Koiso, and K. Tonomura. 1985. 226. Janssen, D. B., D. Jager, and B. Witholt. 1987. Degradation of Genetic rearrangement of plasmids: in vivo recombination be- n-haloalkanes and a,w-dihaloalkanes by wild-type and mutants of tween a dehalogenation plasmid and multiple-resistance plasmid Acinetobacter sp. strain GJ70. Appl. Environ. Microbiol. 53:561- RP4 in Pseudomonas sp. Appl. Environ. Microbiol. 49:1544- 566. 1546. 227. Janssen, D. B., R. Oldenhuis, and A. J. van den WQngaard. 1990. 246. Kawasaki, H., N. Tone, and K. Tonomura. 1981. Plasmid- Hydrolytic and oxidative degradation of chlorinated aliphatic determined dehalogenation of haloacetates in Moraxella species. compounds by aerobic microorganisms. Adv. Appl. Biotechnol. Agric. Biol. Chem. 45:29-34. Ser. Biotechnol. Biodegrad. 4:105-125. 247. Kawasaki, H., N. Tone, and K. Tonomura. 1981. Purification and 228. Janssen, D. B., F. Pries, J. van der Ploeg, B. Kazemier, P. properties of haloacetate halidohydrolase specified by plasmid Terpstra, and B. Witholt. 1989. Cloning of 1,2-dichloroethane from Moraxella sp. strain B. Agric. Biol. Chem. 45:35-42. degradation genes of Xanthobacter autotrophicus GJ10 and ex- 247a.Kawasaki, H., T. Toyama, T. Maeda, H. Nishino, and K. Tono- pression and sequencing of the dhlA gene. J. Bacteriol. 171:6791- mura. 1994. Cloning and sequence analysis of a plasmid-encoded 6799. 2-haloacid dehalogenase gene from Pseudomonasputida no. 109. 229. Janssen, D. B., A. Scheper, L. DUkhuizen, and B. Witholt. 1985. Biosci. Biotechnol. Biochem. 58:160-163. Degradation of halogenated aliphatic compounds by Xantho- 248. Kawasaki, H., K. Tsuda, L. Matsushita, and K. Tonomura. 1992. bacterautotrophicus GJ10. Appl. Environ. Microbiol. 49:673-677. Lack of homology between two haloacetate dehalogenase genes 230. Janssen, D. B., and B. Witholt. 1992. Aerobic and anaerobic encoded on a plasmid from Moraxella sp. strain B. J. Gen. degradation of halogenated aliphatics, p. 299-327. In H. Sigel Microbiol. 138:1317-1323. and A. Sigel (ed.), Metal ions in biological systems, vol. 28. 249. Kawasaki, H., H. Yahara, and K. Tonomura. 1981. Isolation and Degradation of environmental pollutants by microorganisms and characterization of plasmid pUO1 mediating dehalogenation of their metalloenzymes. Marcel Dekker Inc., New York. haloacetate and mercury resistance in Moraxella sp. B. Agric. VOL. 58, 1994 BACTERIAL DEHALOGENASES 677

Biol. Chem. 45:1477-1481. dechlorination of 2,4-dichlorophenol in freshwater sediments in 250. Kersten, P. J., P. J. Chapman, and S. Dagley. 1985. Enzymatic the presence of sulfate. Appl. Environ. Microbiol. 55:2735-2737. release of halogens or methanol from some substituted protocat- 273. Krone, U. E., K. Laufer,R K. Thauer, and H. P. C. Hogenkamp. echuic acids. J. Bacteriol. 162:693-697. 1989. Coenzyme F430 as a possible catalyst for the reductive 251. Keuning, S., D. B. Janssen, and B. Witholt. 1985. Purification and dehalogenation of chlorinated C, hydrocarbons in methanogenic characterization of hydrolytic haloalkane dehalogenase from bacteria. Biochemistry 28:10061-10065. Xanthobacter autotrophicus GJ10. J. Bacteriol. 163:635-639. 274. Krone, U. E., and R. K. Thauer. 1992. Dehalogenation of 252. Khalifa, S., R. L. Holmstead, and J. E. Casida. 1976. Toxaphene trichlorofluoromethane (CFC-11) by Methanosarcina barkeri. degradation by iron(II) protoporphyrin systems. J. Agric. Food FEMS Microbiol. Lett. 90:201-204. Chem. 24:277-282. 275. Krone, U. E., R K. Thauer, and H. P. C. Hogenkamp. 1989. 253. Kilbane, J. J., D. K. Chatterjee, and A. M. Chakrabarty. 1983. Reductive dehalogenation of chlorinated C,-hydrocarbons medi- Detoxification of 2,4,5-trichlorophenoxyacetic acid from contam- ated by corrinoids. Biochemistry 28:4908-4914. inated soil by Pseudomonas cepacia. Appi. Environ. Microbiol. 276. Krone, U. E., R. K. Thauer, H. P. C. Hogenkamp, and K. 45:1697-1700. Steinbach. 1991. Reductive formation of carbon monoxide from 254. Kilbane, J. J., D. K. Chatterjee, J. S. Karns, S. T. Kellogg, and CCd4 and FREONs 11, 12, and 13 catalyzed by corrinoids. A. M. Chakrabarty. 1982. Biodegradation of 2,4,5-trichlorophe- Biochemistry 30:2713-2719. noxyacetic acid by a pure culture of Pseudomonas cepacia. Appl. 277. Krumme, M. L., and S. A. Boyd. 1988. Reductive dechlorination Environ. Microbiol. 44:72-78. of chlorinated phenols in anaerobic upflow bioreactors. Water 255. King, G. M. 1988. Dehalogenation in marine sediments contain- Res. 22:171-177. ing natural sources of halophenols. Appl. Environ. Microbiol. 54: 278. Kuhm, A. E., M. Schlomann, H.-J. Knackmuss, and D. H. Pieper. 3079-3085. 1990. Purification and characterization of dichloromuconate cy- 256. Kingsbury, D. T. 1988. Balancing regulatory control, scientific cloisomerase from Alcaligenes eutrophus JMP134. Biochem. J. knowledge, and public understanding, p. 341-350. In G. S. Omenn 266:877-883. (ed.), Environmental biotechnology. Plenum Press, New York. 279. Kuhn, E. P., and J. M. Suflita. 1989. Sequential reductive 257. Kirk, T. K., and R. L. Farrell. 1987. Enzymatic "combustion". dehalogenation of chloroanilines by microorganisms from a The microbial degradation of lignin. Annu. Rev. Microbiol. methanogenic aquifer. Environ. Sci. Technol. 23:848-852. 41:465-505. 280. Kuhn, E. P., G. T. Townsend, and J. M. Suflita. 1990. Effect of 258. Kirkpatrick, D., S. R. Biggs, B. Conway, C. M. Finn, D. R. sulfate and organic carbon supplements on reductive dehaloge- Hawkins, T. Honda, M. Ishida, and G. P. Powell. 1981. Metab- nation of chloroanilines in anaerobic aquifer slurries. Appl. olism of N-(2,3-dichlorophenyl)-3,4,5,6-tetrachlorophthalamic Environ. Microbiol. 56:2630-2637. acid (techlofthalam) in paddy soil and rice. J. Agric. Food Chem. 281. LaRoche, S. D., and T. Leisinger. 1990. Sequence analysis and 29:1149-1153. expression of the bacterial dichloromethane dehalogenase struc- 259. Kiyohara, H., T. Hatta, Y. Ogawa, T. Kakuda, H. Yokoyama, and tural gene, a member of the glutathione S-transferase supergene N. Takizawa. 1992. Isolation of Pseudomonaspickettii strains that family. J. Bacteriol. 172:164-171. degrade 2,4,6-trichlorophenol and their dechlorination of chlo- 282. LaRoche, S. D., and T. Leisinger. 1991. Identification of dcmR, rophenols. Appl. Environ. Microbiol. 58:1276-1283. the regulatory gene governing expression of dichloromethane 260. Kiages, U., S. Krauss, and F. Lingens. 1983. 2-Haloacid dehalo- dehalogenase in Methylobacterium sp. strain DM4. J. Bacteriol. genase from a 4-chlorobenzoate-degrading Pseudomonas species 173:6714-6721. CBS3. Hoppe-Seyler's Z. Physiol. Chem. 364:529-535. 283. Larsen, S., H. V. Hendriksen, and B. K. Ahring. 1991. Potential 261. Kiages, U., and F. Lingens. 1979. Degradation of 4-chlorobenzoic for thermophilic (50°C) anaerobic dechlorination of pentachlo- acid by a Nocardia species. FEMS Microbiol. Lett. 6:201-203. rophenol in different ecosystems. Appl. Environ. Microbiol. 57: 262. Kiages, U., and F. Lingens. 1980. Degradation of 4-chlorobenzoic 2085-2090. acid by a Pseudomonas sp. Zentralbl. Bakteriol. Mikrobiol. Hyg. 284. Layton, A. C., J. Sanseverino, W. Wallace, C. Corcoran, and G. S. 1 Abt Orig. C 1:215-223. Sayler. 1992. Evidence for 4-chlorobenzoic acid dehalogenation 263. Kiecka, G. M., and D. T. Gibson. 1981. Inhibition of catechol mediated by plasmids related to pSS50. Appl. Environ. Micro- 2,3-dioxygenase from Pseudomonas putida by 3-chlorocatechol. biol. 58:399-402. Appl. Environ. Microbiol. 41:1159-1165. 285. Lee, M. D., J. M. Thomas, R. C. Borden, P. B. Bedient, C. H. 264. Kleka, G. M., and S. J. Gonsior. 1984. Reductive dechlorination Ward, and J. T. Wilson. 1988. Biorestoration of aquifers contam- of chlorinated methanes and ethanes by reduced iron(II) porphy- inated with organic compounds. Crit. Rev. Environ. Control rins. Chemosphere 13:391-402. 18:29-89. 265. Knackmuss, H.-J. 1981. Degradation of halogenated and sulfon- 286. Leigh, J. A., A. J. Skinner, and R. A. Cooper. 1988. Partial ated hydrocarbons. FEMS Symp. 12:189-212. purification, stereospecificity and stoichiometry of three dehalo- 266. Kobayashi, H., and B. E. Rittmann. 1982. Microbial removal of genases from a Rhizobium species. FEMS Microbiol. Lett. 49: hazardous organic compounds. Environ. Sci. Technol. 16:170A- 353-356. 183A. 287. Leisinger, T., and R Bader. 1993. Microbial dehalogenation of 267. Koh, S.-C., J. P. Bowman, and G. S. Sayler. 1993. Soluble synthetic organohalogen compounds: hydrolytic dehalogenases. methane monooxygenase production and trichloroethylene deg- Chimia 47:116-121. radation by a type I methanotroph, Methylomonas methanica 288. Leisinger, T., and D. Kohler-Staub. 1990. Dichloromethane 68-1. Appl. Environ. Microbiol. 59:960-967. dehalogenase from Hyphomicrobium DM2. Methods Enzymol. 268. Kohler-Staub, D., S. Hartmans, R. Galli, F. Suter, and T. 188:355-361. Leisinger. 1986. Evidence for identical dichloromethane dehalo- 289. Lewis, T. A., and R. L. Crawford. 1993. Physiological factors genases in different methylotrophic bacteria. J. Gen. Microbiol. affecting carbon tetrachloride dehalogenation by the denitrifying 132:2837-2843. bacterium Pseudomonas sp. strain KC. Appl. Environ. Microbiol. 269. Kohler-Staub, D., and H.-P. E. Kohler. 1989. Microbial degrada- 59:1635-1641. tion of ,B-chlorinated four-carbon aliphatic acids. J. Bacteriol. 290. Li, D.-Y., J. Eberspacher, B. Wagner, J. Kuntzer, and F. Lingens. 171:1428-1434. 1991. Degradation of 2,4,6-trichlorophenol by Azotobacter sp. 270. Kohler-Staub, D., and T. Leisinger. 1985. Dichloromethane strain GP1. Appl. Environ. Microbiol. 57:1920-1928. dehalogenase of Hyphomicrobium sp. strain DM2. J. Bacteriol. 291. Li, S., and L. P. Wackett. 1992. Trichloroethylene oxidation by 162:676-681. toluene dioxygenase. Biochem. Biophys. Res. Commun. 185:443- 271. Kohring, G.-W., J. E. Rogers, and J. Wiegel. 1989. Anaerobic 451. biodegradation of 2,4-dichlorophenol in freshwater lake sediments 292. Liao, D.-I., and S. J. Remington. 1990. Structure of wheat serine at different temperatures. Appl. Environ. Microbiol. 55:348-353. carboxypeptidase II at 3.5-A resolution. J. Biol. Chem. 265:6528- 272. Kohring, G.-W., X. Zhang, and J. Wiegel. 1989. Anaerobic 6531. 678 FETZNER AND LINGENS MICROBIOL. REV.

293. Linkfield, T. G., and J. M. Tiedje. 1990. Characterization of the 3,4-dioxygenase from Pseudomonas species strain CBS3. J. Biol. requirements and substrates for reductive dehalogenation by Chem. 261:12883-12888. strain DCB-1. J. Ind. Microbiol. 5:9-16. 315. Marietta, M. A., Y.-F. Cheung, and C. Walsh. 1982. Stereochem- 294. Lipke, H., and C. W. Kearns. 1959. DDT dehydrochlorinase. I. ical studies on the hydration of monofluorofumarate and 2,3- Isolation, chemical properties, and spectrophotometric assay. J. difluorofumarate by fumarase. Biochemistry 21:2637-2644. Biol. Chem. 234:2123-2128. 316. Martinez-Blanco, H., A. Reglero, and J. M. Luengo. 1990. Carbon 295. Lipke, H., and C. W. Kearns. 1959. DDT dehydrochlorinase. II. catabolite regulation of phenylacetyl-CoA ligase from Pseudomo- Substrate and cofactor specificity. J. Biol. Chem. 234:2129-2132. nas putida. Biochem. Biophys. Res. Commun. 167:891-897. 296. Little, C. D., A. V. Palumbo, S. E. Herbes, M. E. Lidstrom, R. L. 317. Martinez-Blanco, H., A. Reglero, L B. Rodriguez-Aparicio, and Tyndall, and P. J. Gilmer. 1988. Trichloroethylene biodegrada- J. M. Luengo. 1990. Purification and biochemical characterization by a methane-oxidizing bacterium. Appl. Environ. Microbiol. tion of phenylacetyl-CoA ligase from Pseudomonas putida. A 54:951-956. specific enzyme for the catabolism of phenylacetic acid. J. Biol. 297. Little, M., and P. A. Williams. 1971. A bacterial halidohydrolase: Chem. 265:7084-7090. its purification, some properties and its modification by specific 318. Merkel, S. M., A. E. Eberhard, J. Gibson, and C. S. Harwood. amino acid reagents. Eur. J. Biochem. 21:99-109. 1989. Involvement of coenzyme A thioesters in anaerobic metab- 298. L4ffler, F., F. Lingens, and R. Muller. Dehalogenation of 4-chlo- olism of 4-hydroxybenzoate by Rhodopseudomonas palustris. J. robenzoate. Characterisation of 4-chlorobenzoyl-coenzyme A Bacteriol. 171:1-7. dehalogenase from Pseudomonas sp. CBS3. Submitted for pub- 319. Meyer, D. J., B. Coles, S. E. Pemble, K. S. Gilmore, G. M. Fraser, lication. and B. Ketterer. 1991. Theta, a new class of glutathione trans- 299. LfiIler, F., and R. Muller. 1991. Identification of 4-chloroben- ferases purified from rat and man. Biochem. J. 274:409-414. zoyl-coenzyme A as intermediate in the dehalogenation catalyzed 320. Middeldorp, P. J. M., M. Briglia, and M. S. Salkinoja-Salonen. by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3. 1990. Biodegradation of pentachlorophenol in natural soil by FEBS Lett. 290:224-226. inoculated Rhodococcus chlorophenolicus. Microb. Ecol. 20:123- 300. Liffler, F., R. Muller, and F. Lingens. 1991. Dehalogenation of 139. 4-chlorobenzoate by 4-chlorobenzoate dehalogenase from 321. Miguez, C. B., C. W. Greer, and J. M. Ingram. 1990. Degradation Pseudomonas sp. CBS3: an ATP/coenzyme A dependent reac- of mono- and dichlorobenzoic acid isomers by two natural tion. Biochem. Biophys. Res. Commun. 176:1106-1111. isolates ofAlcaligenes denitrficans. Arch. Microbiol. 154:139-143. 301. Lfifer, F., R. Muller, and F. Lingens. 1992. Purification and 322. Miguez, C. B., C. W. Greer, and J. M. Ingram. 1993. Purification properties of 4-halobenzoate-coenzyme A ligase from Pseudomo- and properties of chlorocatechol 1,2-dioxygenase from Alcali- nas sp. CBS3. Biol. Chem. Hoppe-Seyler 373:1001-1007. genes denitrificans BRI 6011. Can. J. Microbiol. 39:1-5. 302. Logan, M. S. P., F. A. Blocki, K. J. Stimpfl, and L. P. Wackett. 323. Mikesell, M. D., and S. A. Boyd. 1985. Reductive dechlorination 1993. Haloacetonitriles are low K, inhibitors of bacterial dichlo- of the pesticides 2,4-D, 2,4,5-T, and pentachlorophenol in anaer- romethane dehalogenases. Biochem. Biophys. Res. Commun. obic sludges. J. Environ. Qual. 14:337-340. 197:853-858. 324. Mikesell, M. D., and S. A. Boyd. 1986. Complete reductive 303. Lovelock, J. E. 1975. Natural halocarbons in the air and in the dechlorination and mineralization of pentachlorophenol by sea. Nature (London) 256:193-194. anaerobic microorganisms. Appl. Environ. Microbiol. 52:861- 304. Lowe, S. E., M. K. Jain, and J. G. Zeikus. 1993. Biology, ecology, 865. and biotechnological applications of anaerobic bacteria adapted 325. Mikesell, M. D., and S. A. Boyd. 1988. Enhancement of penta- to environmental stresses in temperature, pH, salinity, or sub- chlorophenol degradation in soil through induced anaerobiosis strates. Microbiol. Rev. 57:451-509. and bioaugmentation with anaerobic sewage sludge. Environ. Sci. 305. MacRae, L. C., K. Raghu, and E. M. Bautista. 1969. Anaerobic Technol. 22:1411-1414. degradation of the insecticide lindane by Clostridium sp. Nature 326. Mikesell, M. D., and S. A. Boyd. 1990. Dechlorination of chloro- (London) 221:859-860. form by Methanosarcina strains. Appl. Environ. Microbiol. 56: 306. Madsen, T., and J. Aamand. 1991. Effects of sulfuroxy anions on 1198-1201. degradation of pentachlorophenol by a methanogenic enrich- 327. Milne, G. W. A., P. Goldman, and J. L. Holtzman. 1968. The ment culture. Appl. Environ. Microbiol. 57:2453-2458. metabolism of 2-fluorobenzoic acid. II. Studies with 802Q J. Biol. 307. Madsen, T., and J. Aamand. 1992. Anaerobic transformation and Chem. 243:5374-5376. toxicity of trichlorophenols in a stable enrichment culture. Appl. 328. Miskus, R. P., D. P. Blair, and J. E. Casida. 1965. Conversion of Environ. Microbiol. 58:557-561. DDT to DDD by bovine rumen fluid, lake water, and reduced 308. Madsen, T., and D. Licht. 1992. Isolation and characterization of porphyrins. J. Agric. Food Chem. 13:481-483. an anaerobic chlorophenol-transforming bacterium. Appl. Envi- 329. Mohamed, M. E.-S., and G. Fuchs. 1993. Purification and char- ron. Microbiol. 58:2874-2878. acterization of phenylacetate-coenzyme A ligase from a denitri- 309. Mannervik, B., P. Alin, C. Guthenberg, H. Jensson, M. K. Tahir, fying Pseudomonas sp., an enzyme involved in the anaerobic M. Warholm, and H. Jornvall. 1985. Identification of three degradation of phenylacetate. Arch. Microbiol. 159:554-562. classes of cytosolic glutathione transferase common to several 330. Mohamed, M. E.-S., B. Seyfried, A. Tschech, and G. Fuchs. 1993. mammalian species: correlation between structural data and Anaerobic oxidation of phenylacetate and 4-hydroxyphenylac- enzymatic properties. Proc. Natl. Acad. Sci. USA 82:7202-7206. etate to benzoyl-coenzyme A and CO2 in denitrifying Pseudomo- 310. Marks, T. S., J. D. Allpress, and A. Maule. 1989. Dehalogenation nas sp. Arch. Microbiol. 159:563-573. of lindane by a variety of porphyrins and corrins. Appl. Environ. 331. Mohn, W. W., and K. J. Kennedy. 1992. Reductive dehalogena- Microbiol. 55:1258-1261. tion of chlorophenols by Desulfomonile tiedjei DCB-1. Appl. 311. Marks, T. S., and A. Maule. 1992. The use of immobilized Environ. Microbiol. 58:1367-1370. porphyrins and corrins to dehalogenate organochlorine pollut- 332. Mohn, W. W., and K. J. Kennedy. 1992. Limited degradation of ants. Appl. Microbiol. Biotechnol. 38:413-416. chlorophenols by anaerobic sludge granules. Appl. Environ. 312. Marks, T. S., R. Wait, A. R. W. Smith, and A. V. Quirk. 1984. The Microbiol. 58:2131-2136. origin of the oxygen incorporated during the dehalogenation/ 333. Mohn, W. W., and J. M. Tiedje. 1990. Strain DCB-1 conserves hydroxylation of 4-chlorobenzoate by an Arthrobacter sp. Bio- energy for growth from reductive dechlorination coupled to chem. Biophys. Res. Commun. 124:669-674. formate oxidation. Arch. Microbiol. 153:267-271. 313. Markus, A., U. Kiages, S. Krauss, and F. Lingens. 1984. Oxida- 334. Mohn, W. W., and J. M. Tiedje. 1991. Evidence for chemiosmotic tion and dehalogenation of 4-chlorophenylacetate by a two- coupling of reductive dechlorination and ATP synthesis in De- component enzyme system from Pseudomonas sp. strain CBS3. J. sulfomonile tiedjei. Arch. Microbiol. 157:1-6. Bacteriol. 160:618-621. 335. Mohn, W. W., and J. M. Tiedje. 1992. Microbial reductive 314. Markus, A., D. Krekel, and F. Lingens. 1986. Purification and dehalogenation. Microbiol. Rev. 56:482-507. some properties of component A of the 4-chlorophenylacetate 336. Mokross, H., E. Schmidt, and W. Reineke. 1990. Degradation of VOL. 58, 1994 BACTERIAL DEHALOGENASES 679

3-chlorobiphenyl by in vivo constructed hybrid pseudomonads. N-1074. J. Bacteriol. 174:7613-7619. FEMS Microbiol. Lett. 71:179-186. 356. Nakamura, T., F. Yu, W. Mizunashi, andI. Watanabe. 1991. 337. Moon, Y. H., and S. Kuwatsuka. 1984. Properties and conditions Microbial transformation of prochiral 1,3-dichloro-2-propanol of soils causing the dechlorination of the herbicide benthiocarb into optically active 3-chloro-1,2-propanediol. Agric. Biol. Chem. (thiobencarb) in flooded soils. J. Pestic. Sci. 9:745-754. 55:1931-1933. 338. Moon, Y. H., and S. Kuwatsuka. 1985. Factors influencing 357. Nakamura, T., F. Yu, W. Mizunashi, and L. Watanabe. 1993. microbial dechlorination of benthiocarb (thiobencarb) in the soil Production of (R)-3-chloro-1,2-propanediol from prochiral 1,3- suspension. J. Pestic. Sci. 10:523-528. dichloro-2-propanol by Corynebacterium sp. strain N-1074. Appl. 339. Moon, Y. H., and S. Kuwatsuka. 1985. Characterization of Environ. Microbiol. 59:227-230. microbes causing dechlorination of benthiocarb (thiobencarb) in 358. Nakatsu, C., J. Ng,R Singh, N. Straus, and C. Wyndham. 1991. diluted soil suspension. J. Pestic. Sci. 10:541-547. Chlorobenzoate catabolic transposon Tn5271 is a composite class 340. Morgan, P., and R. J. Watkinson. 1989. Microbiological methods I element with flanking class II insertion sequences. Proc. Natl. for the cleanup of soil and ground water contaminated with Acad. Sci. USA 88:8312-8316. halogenated organic compounds. FEMS Microbiol. Rev. 63:277- 359. Nakatsu, C. H., andR C. Wyndham. 1993. Cloning and expres- 300. sion of the transposable chlorobenzoate 3,4-dioxygenase genes of 341. Morsberger, F.-M., R. Muller, M. K. Otto, F. Lingens, and K. D. Alcaligenes sp. strain BR60. Appl. Environ. Microbiol. 59:3625- Kulbe. 1991. Purification and characterization of 2-halocarboxy- 3633. lic acid dehalogenase II from Pseudomonas spec. CBS3. Biol. 360. Neidle, E. L., C. Hartnett, L. N. Ornston, A. Bairoch, M. Rekik, Chem. Hoppe-Seyler 372:915-922. and S. Harayama. 1991. Nucleotide sequences of the Acineto- 342. Motosugi, K., N. Esaki, and K. Soda. 1982. Bacterial assimilation bacter calcoaceticus benABC genes for benzoate 1,2-dioxygenase of D- and L-2-chloropropionates and occurrence of a new deha- reveal evolutionary relationships among multicomponent oxyge- logenase. Arch. Microbiol. 131:179-183. nases. J. Bacteriol. 173:5385-5395. 343. Motosugi, K., N. Esaki, and K. Soda. 1982. Purification and 361. Neidleman, S. L., and J. Geigert. 1986. Biohalogenation: princi- properties of a new enzyme, DL-2-haloacid dehalogenase, from ples, basic roles and applications, p. 156-171. Ellis Horwood Ltd., Pseudomonas sp. J. Bacteriol. 150:522-527. Chichester, United Kingdom. 344. Motosugi, K., N. Esaki, and K. Soda. 1982. Purification and 362. Neilson, A. H. 1990. The biodegradation of halogenated organic properties of 2-haloacid dehalogenase from Pseudomonasputida. compounds. J. Appl. Bacteriol. 69:445-470. Agric. Biol. Chem. 46:837-838. 363. Neilson, A. H., A.-S. Allard, C. Lindgren, and M. Remberger. 345. Motosugi, K., N. Esaki, and K. Soda. 1983. Determinationof L- 1987. Transformations of chloroguaiacols, chloroveratroles, and and D-2-halopropanoic acids and 2-halobutanoic acids with bac- chlorocatechols by stable consortia of anaerobic bacteria. Appl. terial dehalogenases. Anal. Lett. 16(B7):509-514. Environ. Microbiol. 53:2511-2519. 346. Motosugi, K., N. Esaki, and K. Soda. 1984. Enzymatic prepara- 364. Nelson, M. J.K.,S. 0. Montgomery, W. R Mahaffey, and P. H. tionof D- and L-lactic acid from racemic 2-chloropropionic acid. Pritchard. 1987. Biodegradation of trichloroethylene and in- Biotechnol. Bioeng. 26:805-806. volvement of an aromatic biodegradative pathway. Appl. Envi- 347. Muller, R., R. H. Oltmanns, and F. Lingens. 1988. Enzymatic ron. Microbiol. 53:949-954. dehalogenation of 4-chlorobenzoate by extracts from Ar- 365. Nelson, M. J. K., S. 0. Montgomery, and P. H. Pritchard. 1988. throbacter sp. SU DSM 20407. Biol. Chem. Hoppe-Seyler 369: Trichloroethylene metabolism by microorganisms that degrade 567-571. aromatic compounds. Appl. Environ. Microbiol. 54:604-606. 348. Muller, R., J. Thiele, U. Klages, and F. Lingens. 1984. Incorpo- 366. Nelson, M. J. K., P. H. Pritchard, and A. W. Bourquin. 1988. ration of [180]water into 4-hydroxybenzoic acid in the reaction of Preliminary development of a bench-scale treatment system for 4-chlorobenzoate dehalogenase from Pseudomonas spec. CBS3. aerobic degradation of trichloroethylene, p. 203-209. In G. S. Biochem. Biophys. Res. Commun. 124:178-182. Omenn (ed.), Environmental biotechnology. Plenum Press, New 349. Murdiyatmo, U., W. Asmara, J. S. H. Tsang, A. J. Baines, A. T. York. Bull, and D. J. Hardman. 1992. Molecular biology of the 367. Newman, L. M., and L. P. Wackett. 1991. Fate of 2,2,2-trichlo- 2-haloacid halidohydrolase IVa from Pseudomonas cepacia roacetaldehyde (chloral hydrate) produced during trichloroeth- MBA4. Biochem. J. 284:87-93. ylene oxidation by methanotrophs. Appl. Environ. Microbiol. 57: 350. Nagasawa, T., T. Nakamura, F. Yu, L Watanabe, and H. Yamada. 2399-2402. 1992. Purification and characterization of halohydrin hydrogen- 368. Nies, L., and T. M. Vogel. 1991. Identification of the proton halide lyase from a recombinant Escherichia coli containing the source for the microbial reductive dechlorination of 2,3,4,5,6- gene from a Corynebacterium sp. Appl. Microbiol. Biotechnol. pentachlorobiphenyl. Appl. Environ. Microbiol. 57:2771-2774. 36:478-482. 369. Ohisa, N., N. Kurihara, and M. Nakajima. 1982. ATP synthesis 351. Nagata, Y., T. Hatta, R. Imai, K. Kimbara, M. Fukuda, K. associated with the conversion of hexachlorocyclohexane related Yano, and M. Takagi. 1993. Purification and characterization of compounds. Arch. Microbiol. 131:330-333. -y-hexachlorocyclohexane (y-HCH) dehydrochlorinase (LinA) 370. Ohisa, N., and M. Yamaguchi. 1978. Gamma BHC degradation from Pseudomonas paucimobilis. Biosci. Biotechnol. Biochem. accompanied by the growth of Clostridium rectum isolated from 57:1582-1583. paddy field soil. Agric. Biol. Chem. 42:1819-1823. 352. Nagata, Y., T. Nariya, R Ohtomo, M. Fukuda, K. Yano, and M. 371. Ohisa, N., M. Yamaguchi, and N. Kurihara. 1980. Lindane Takagi. 1993. Cloning and sequencing of a dehalogenase gene degradation by cell-free extracts of Clostridium rectum. Arch. encoding an enzyme with hydrolase activity involved in the Microbiol. 125:221-225. degradation of y-hexachlorocyclohexane in Pseudomonas pauci- 372. Oldenhuis, R, and D. B. Janssen. 1992. Degradation of trichlo- mobilis. J. Bacteriol. 175:6403-6410. roethylene by methanotrophic bacteria, p. 121-133. In J. C. 353. Nakajima, H., M. Onda, R Tsurutani, and K. Motosugi. (Unitika Murrell and D. P. Kelly (ed.), Microbial growth on Cl com- Ltd.). 1990. Optically active p-halolactic acid or glycidic acid. pounds. Intercept Ltd., Andover, United Kingdom. European patent application EP 365,029 (Cl.C12P41/00), 25 April 373. Oldenhuis, R, R. L. J. M. Vink, D. B. Janssen, and B. Witholt. 1990. Japanese patent application 88/265,838, 20 October 1988. 1989. Degradation of chlorinated aliphatic hydrocarbons by 354. Nakamura, T., T. Nagasawa, F. Yu, I. Watanabe, and H. Yamada. Methylosinus trichosporium OB3b expressing soluble methane 1991. A new catalytic function of halohydrin hydrogen-halide- monooxygenase. Appl. Environ. Microbiol. 55:2819-2826. lyase, synthesis of P-hydroxynitriles from epoxides and cyanide. 374. Omori, T., and M. Alexander. 1978. Bacterial and spontaneous Biochem. Biophys. Res. Commun. 180:124-130. dehalogenation of organic compounds. Appl. Environ. Microbiol. 355. Nakamura, T., T. Nagasawa, F. Yu, I. Watanabe, and H. Yamada. 35:512-516. 1992. Resolution and some properties of enzymes involved in 375. Omori, T., and M. Alexander. 1978. Bacterial dehalogenation of enantioselective transformation of 1,3-dichloro-2-propanol to halogenated alkanes and fatty acids. Appl. Environ. Microbiol. (R)-3-chloro-1,2-propanediol by Corynebactenum sp. strain 35:867-871. 680 FETZNER AND LINGENS MICROBIOL. REV.

376. O'Reilly, K. T., and R L. Crawford. 1989. Degradation of pathway for catabolism of alkylbenzoates. Science 235:593-596. pentachlorophenol by polyurethane-immobilized Flavobacterium 396. Rasche, M. E., R E. Hicks, M. R Hyman, and D. J. Arp. 1990. cells. Appl. Environ. Microbiol. 55:2113-2118. Oxidation of monohalogenated ethanes and n-chlorinated al- 377. Orser, C. S., J. Dutton, C. Lange, P. Jablonski, L. Xun, and M. kanes by whole cells of Nitrosomonas europaea. J. Bacteriol. Hargis. 1993. Characterization of a Flavobacterium glutathione 172:5368-5373. S-transferase gene involved in reductive dechlorination. J. Bac- 397. Read, R J., and M. N. G. James. 1988. Refined crystal structure teriol. 175:2640-2644. of Streptomyces griseus trypsin at 1.7 A resolution. J. Mol. Biol. 378. Orser, C. S., C. C. Lange, L Xun, T. C. Zahrt, and B. J. 200:523-551. Schneider. 1993. Cloning, sequence analysis, and expression of 398. Rees, D. C., M. Lewis, and W. N. Lipscomb. 1983. Refined crystal the Flavobacterium pentachlorophenol-4-monooxygenase gene in structure of carboxypeptidase A at 1.54 A resolution. J. Mol. Biol. Escherichia coli. J. Bacteriol. 175:411-416. 168:367-387. 379. Ottengraf, S. P. P. 1987. Biological systems for waste gas elimi- 399. Reineke, W. 1989. The degradation of chlorinated aromatic nation. Trends Biotechnol. 5:132-136. compounds by bacteria: biochemistry, strain development and 380. Pasti, M. B., A. L. Pometto III, M. P. Nuti, and D. L. Crawford. decontamination of soil and waste water. Forum Mikrobiol. 1990. Lignin-solubilizing ability of actinomycetes isolated from 12:402-411. termite (Termitidae) gut. Appl. Environ. Microbiol. 56:2213- 400. Reineke, W., and H.-J. Knackmuss. 1984. Microbial metabolism 2218. of haloaromatics: isolation and properties of a chlorobenzene- 381. Patel, R. N., C. T. Hou, A. L. Laskin, and A. Felix. 1982. Microbial degrading bacterium. Appl. Environ. Microbiol. 47:395-402. oxidation of hydrocarbons: properties of a soluble methane 401. Reineke, W., and H.-J. Knackmuss. 1988. Microbial degradation monooxygenase from a facultative methane-utilizing organism, of haloaromatics. Annu. Rev. Microbiol. 42:263-287. Methylobacterium sp. strain CRL-26. Appl. Environ. Microbiol. 402. Renganathan, V. 1989. Possible involvement of toluene 2,3- 44:1130-1137. dioxygenase in defluorination of 3-fluoro-substituted benzenes by 382. Pathak, D., K. L. Ngai, and D. Ollis. 1988. X-ray crystallographic toluene-degrading Pseudomonas sp. strain T-12. Appl. Environ. structure of dienelactone hydrolase at 2.8 A. J. Mol. Biol. Microbiol. 55:330-334. 204:435-445. 403. Rojo, F., D. H. Pieper, K.-H. Engesser, H.-J. Knackmuss, and 383. Pathak, D., and D. Ollis. 1990. Refined structure of dienelactone K. N. Timmis. 1987. Assemblage of ortho cleavage route for hydrolase at 1.8 A. J. Mol. Biol. 214:497-525. simultaneous degradation of chloro- and methylaromatics. Sci- 384. Pavlostathis, S. G., and P. Zhuang. 1991. Transformation of ence 238:1395-1398. trichloroethylene by sulfate-reducing cultures enriched from a 403a.Romanov, V., and R P. Hausinger. 1994. Pseudomonas aerugi- contaminated subsurface soil. Appl. Microbiol. Biotechnol. 36: nosa 142 uses a three-component ortho-halobenzoate 1,2-dioxy- 416-420. genase for metabolism of 2,4-dichloro- and 2-chlorobenzoate. J. 385. Petty, M. A. 1961. An introduction to the origin and biochemistry Bacteriol. 176:3368-3374. of microbial halometabolites. Bacteriol. Rev. 25:111-130. 404. Rosenberg, A., and M. Alexander. 1980. Microbial metabolism of 386. Pflug, A. D., and M. B. Burton. 1988. Remediation of multimedia 2,4,5-trichlorophenoxyacetic acid in soil, soil suspensions, and contamination from the wood-preserving industry, p. 193-201. In axenic culture. J. Agric. Food Chem. 28:297-302. G. S. Omenn (ed.), Environmental biotechnology. Plenum Press, 405. Rott, B., S. Nitz, and F. Korte. 1979. Microbial decomposition of New York. sodium pentachlorophenolate. J. Agric. Food Chem. 27:306-310. 387. Phelps, T. J., J. J. Niedzielski, R. M. Schram, S. E. Herbes, and 406. Roy-Arcand, L., and F. S. Archibald. 1991. Direct dechlorination D. C. White. 1990. Biodegradation of trichloroethylene in con- of chlorophenolic compounds by laccases from Trametes (Corio- tinuous-recycle expanded-bed bioreactors. Appl. Environ. Micro- lus) versicolor. Enzyme Microb. Technol. 13:194-203. biol. 56:1702-1709. 407. Rozeboom, H. J., J. Kingma, D. B. Janssen, and B. W. DUkstra. 388. Picardal, F. W., R G. Arnold, H. Couch, A. M. Little, and M. E. 1988. Crystallization of haloalkane dehalogenase from Xan- Smith. 1993. Involvement of cytochromes in the anaerobic bio- thobacter autotrophicus GJ10. J. Mol. Biol. 200:611-612. transformation of tetrachloromethane by Shewanella putrefaciens 408. Ruisinger, S., U. Klages, and F. Lingens. 1976. Abbau der 200. Appl. Environ. Microbiol. 59:3763-3770. 4-Chlorbenzoesaure durch eine Arthrobacter-Species. Arch. Mi- 389. Pipke, R., I. Wagner-Dobler, K. N. Timmis, and D. F. Dwyer. crobiol. 110:253-256. 1992. Survival and function of a genetically engineered pseudo- 409. Saber, D. L, and R L Crawford. 1985. Isolation and character- monad in aquatic sediment microcosms. Appl. Environ. Micro- ization of Flavobacterium strains that degrade pentachlorophe- biol. 58:1259-1265. nol. Appl. Environ. Microbiol. 50:1512-1518. 389a.Pries, F., J. Kingma, M. Pentenga, G. van Pouderoyen, C. M. 410. Sahasrabudhe, A. V., and V. V. Modi. 1991. Degradation of Jeronimus-Stratingh, A. P. Bruins, and D. B. Janssen. 1994. isomeric monochlorobenzoates and 2,4-dichlorophenoxyacetic Site-directed mutagenesis and oxygen isotope incorporation stud- acid by a constructed Pseudomonas sp. Appl. Microbiol. Biotech- ies of the nucleophilic aspartate of haloalkane dehalogenase. nol. 34:556-557. Biochemistry 33:1242-1247. 411. Sahasrabudhe, A., A. Pande, and V. Modi. 1991. Dehalogenation 390. Quensen, J. F., Ill, J. M. Tiedje, and S. A. Boyd. 1988. Reductive of a mixture of chloroaromatics by immobilized Pseudomonas sp. dechlorination of polychlorinated biphenyls by anaerobic micro- USI ex cells. Appl. Microbiol. Biotechnol. 35:830-832. organisms from sediments. Science 242:752-754. 412. Sahu, S. K., K. K. Patnaik, M. Sharmila, and N. Sethunathan. 391. Radehaus, P. M., and S. K. Schmidt. 1992. Characterization of a 1990. Degradation of alpha-, beta-, and gamma-hexachlorocyclo- novel Pseudomonas sp. that mineralizes high concentrations of hexane by a soil bacterium under aerobic conditions. Appl. pentachlorophenol. Appl. Environ. Microbiol. 58:2879-2885. Environ. Microbiol. 56:3620-3622. 392. Ramachandra, M., D. L. Crawford, and G. Hertel. 1988. Char- 413. Sallis, P. J., S. J. Armfield, A. T. Bull, and D. J. Hardman. 1990. acterization of an extracellular lignin peroxidase of the lignocel- Isolation and characterization of a haloalkane halidohydrolase lulolytic actinomycete Streptomyces viridosporus. Appl. Environ. from Rhodococcus erythropolis Y2. J. Gen. Microbiol. 136:115- Microbiol. 54:3057-3063. 120. 393. Ramanand, K., M. T. Balba, and J. Duffy. 1993. Reductive 414. Sander, P., R-M. Wittich, P. Fortnagel, H. Wilkes, and W. dehalogenation of chlorinated benzenes and toluenes under Francke. 1991. Degradation of 1,2,4-trichloro- and 1,2,4,5-tetra- methanogenic conditions. Appl. Environ. Microbiol. 59:3266- chlorobenzene by Pseudomonas strains. Appl. Environ. Micro- 3272. biol. 57:1430-1440. 394. Ramanand, K, A. Nagarajan, and J. M. Suflita. 1993. Reductive 415. Sandmann, E. R I. C., and M. A. Loos. 1988. Aromatic metab- dechlorination of the nitrogen heterocyclic herbicide picloram. olism by a 2,4-D degrading Arthrobacter sp. Can. J. Microbiol. Appl. Environ. Microbiol. 59:2251-2256. 34:125-130. 395. Ramos, J. L., A. Wasserfallen, K. Rose, and K. N. Timmis. 1987. 416. Sangodkar, U. M. X., P. J. Chapman, and A. M. Chakrabarty. Redesigning metabolic routes: manipulation of TOL plasmid 1988. Cloning, physical mapping and expression of chromosomal VOL. 58, 1994 BACTERIAL DEHALOGENASES 681

genes specifying degradation of the herbicide 2,4,5-T by Pseudo- 437. Scholtz, R, F. Messi, T. Leisinger, and A. M. Coolk 1988. Three monas cepacia AC1100. Gene 71:267-277. dehalogenases and physiological restraints in the biodegradation 417. Sariaslani, F. S. 1989. Microbial enzymes for oxidation of organic of haloalkanes by Arthrobacter sp. strain HAL. Appl. Environ. molecules. Crit. Rev. Biotechnol. 9:171-257. Microbiol. 54:3034-3038. 418. Sato, K., and H. Tanaka. 1987. Degradation and metabolism of a 438. Scholtz, R, A. Schmuckle, A. M. Cook, and T. Leisinger. 1987. fungicide, 2,4,5,6-tetrachloroisophthalonitrile (TPN) in soil. Biol. Degradation of eighteen 1-monohaloalkanes by Arthrobacter sp. Fertil. Soils 3:205-209. strain HAl. J. Gen. Microbiol. 133:267-274. 419. Savard, P., H. Charest, M. Sylvestre, F. Shareck, J. D. Scholten, 439. Scholtz, R, L. P. Wackett, C. Egli, A. M. Cook, and T. Leisinger. and D. Dunaway.Mariano. 1992. Expression of the 4-chloroben- 1988. Dichloromethane dehalogenase with improved catalytic zoate dehalogenase genes from Pseudomonas sp. CBS3 in Esch- activity isolated from a fast-growing dichloromethane-utilizing erichia coli and identification of the gene translation products. bacterium. J. Bacteriol. 170:5698-5704. Can. J. Microbiol. 38:1074-1083. 440. Scholz-Muramatsu, H., R Szewzyk, U. Szewzyk, and S. Gaiser. 420. Savard, P., L. Peloquin, and M. Sylvestre. 1986. Cloning of 1990. Tetrachloroethylene as electron acceptor for the anaerobic Pseudomonas sp. strain CBS3 genes specifying dehalogenation of degradation of benzoate. FEMS Microbiol. Lett. 66:81-86. 4-chlorobenzoate. J. Bacteriol. 168:81-85. 441. Schraa, G., M. L. Boone, M. S. M. Jetten, A. R. W. van Neerven, 421. Schanke, C. A., and L. P. Wackett. 1992. Environmental reductive P. J. Colberg, and A. J. B. Zehnder. 1986. Degradation of elimination reactions of polychlorinated ethanes mimicked by 1,4-dichlorobenzene by Alcaligenes sp. strain A175. Appl. Envi- transition-metal coenzymes. Environ. Sci. Technol. 26:830-833. ron. Microbiol. 52:1374-1381. 422. Schenk, T., R. Muller, and F. Lingens. 1990. Mechanism of 442. Schrag, J. D., Y. Li, S. Wu, and M. Cygler. 1991. Ser-His-Glu enzymatic dehalogenation of pentachlorophenol by Arthrobacter triad forms the catalytic site of the lipase from Geotrichum sp. strain ATCC 33790. J. Bacteriol. 172:7272-7274. candidum. Nature (London) 351:761-764. 423. Schenk, T., R. Muller, F. Morsberger, M. K. Otto, and F. 443. Schweizer, D., A. Markus, M. Seez, H. H. Ruf, and F. Lingens. Lingens. 1989. Enzymatic dehalogenation of pentachlorophenol 1987. Purification and some properties of component B of the by extracts fromArthrobacter sp. strain ATCC 33790. J. Bacteriol. 4-chlorophenylacetate 3,4-dioxygenase from Pseudomonas spe- 171:5487-5491. cies strain CBS3. J. Biol. Chem. 262:9340-9346. 424. Schennen, U., K. Braun, and H.-J. Knackmuss. 1985. Anaerobic 444. Seech, A. G., J. T. Trevors, and T. L. Bulman. 1991. Biodegra- degradation of 2-fluorobenzoate by benzoate-degrading, denitri- dation of pentachlorophenol in soil: the response to physical, fying bacteria. J. Bacteriol. 161:321-325. chemical, and biological treatments. Can. J. Microbiol. 37:440- 425. Schindowski, A., R.-M. Wittich, and P. Fortnagel. 1991. Catabo- 444. lism of 3,5-dichlorosalicylate by Pseudomonas species strain JWS. 445. Seibert, V., K. Stadler-Fritzsche, and M. Schlomann. 1993. FEMS Microbiol. Lett. 84:63-70. Purification and characterization of maleylacetate reductase 426. Schlomann, M., P. Fischer, E. Schmidt, and H.-J. Knackmuss. from Alcaligenes eutrophus JMP134(pJP4). J. Bacteriol. 175: 1990. Enzymatic formation, stability, and spontaneous reactions 6745-6754. of 4-fluoromuconolactone, a metabolite of the bacterial degra- 446. Senior, E., A. T. Bull, and J. H. Slater. 1976. Enzyme evolution in dation of 4-fluorobenzoate. J. Bacteriol. 172:5119-5129. a microbial community growing on the herbicide dalapon. Nature 427. Schlomann, M., K.-L. Ngai, L. N. Ornston, and H.-J. Knack- (London) 263:476-479. muss. 1993. Dienelactone hydrolase from Pseudomonas cepacia. 447. Shelton, D. R, and J. M. Tiedje. 1984. Isolation and partial J. Bacteriol. 175:2994-3001. characterization of bacteria in an anaerobic consortium that 428. Schmidt, E., and H.-J. Knackmuss. 1980. Chemical structure and mineralizes 3-chlorobenzoic acid. Appl. Environ. Microbiol. 48: biodegradability of halogenated aromatic compounds. Conver- 840-848. sion of chlorinated muconic acids into maleoylacetic acid. Bio- 448. Shields, M. S., S. W. Hooper, and G. S. Sayler. 1985. Plasmid- chem. J. 192:339-347. mediated mineralization of 4-chlorobiphenyl. J. Bacteriol. 163: 429. Schmidt, E., and H.-J. Knackmuss. 1984. Production of cis,cis- 882-889. muconate from benzoate and 2-fluoro-cis,cis-muconate from 449. Shields, M. S., S. 0. Montgomery, P. J. Chapman, S. M. Cuskey, 3-fluorobenzoate by 3-chlorobenzoate degrading bacteria. Appl. and P. H. Pritchard. 1989. Novel pathway of toluene catabolism Microbiol. Biotechnol. 20:351-355. in the trichloroethylene-degrading bacterium G4. Appl. Environ. 430. Schmidt, E., G. Remberg, and H.-J. Knackmuss. 1980. Chemical Microbiol. 55:1624-1629. structure and biodegradability of halogenated aromatic com- 450. Shields, M. S., S. 0. Montgomery, S. M. Cuskey, P. J. Chapman, pounds. Halogenated muconic acids as intermediates. Biochem. and P. H. Pritchard. 1991. Mutants of Pseudomonas cepacia G4 J. 192:331-337. defective in catabolism of aromatic compounds and trichloroeth- 431. Schmitz, A., K.-H. Gartemann, J. Fiedler, E. Grund, and R ylene. Appl. Environ. Microbiol. 57:1935-1941. Eichenlaub. 1992. Cloning and sequence analysis of genes for 451. Shimao, M., S. Onishi, S. Mizumori, N. Kato, and C. Sakazawa. dehalogenation of 4-chlorobenzoate from Arthrobacter sp. strain 1989. Degradation of 4-chlorobenzoate by facultatively alkalo- SU. Appl. Environ. Microbiol. 58:4068-4071. philic Arthrobacter sp. strain SB8. Appl. Environ. Microbiol. 55: 432. Schneider, B., R. Muller, R Frank, and F. Lingens. 1991. 478-482. Complete nucleotide sequences and comparison of the structural 452. Shochat, E., L. Hermoni, Z. Cohen, A. Abeliovich, and S. Belkin. genes of two 2-haloalkanoic acid dehalogenases from Pseudomo- 1993. Bromoalkane-degrading Pseudomonas strains. Appl. Envi- nas sp. strain CBS3. J. Bacteriol. 173:1530-1535. ron. Microbiol. 59:1403-1409. 433. Schneider, B., R Muller, R Frank, and F. Lingens. 1993. 453. Short, K. A., J. D. Doyle, R J. King, R J. Seidler, G. Stotzky, and Site-directed mutagenesis of the 2-haloalkanoic acid dehaloge- R H. Olsen. 1991. Effects of 2,4-dichlorophenol, a metabolite of nase I gene from Pseudomonas sp. strain CBS3 and its effect on a genetically engineered bacterium, and 2,4-dichlorophenoxyac- catalytic activity. Biol. Chem. Hoppe-Seyler 374:489-496. etate on some microorganism-mediated ecological processes in 434. Schnell, S., and B. Schink 1991. Anaerobic aniline degradation soil. Appl. Environ. Microbiol. 57:412-418. via reductive deamination of 4-aminobenzoyl-CoA in Desulfobac- 454. Singh, H., and R S. Kahlon. 1989. Conjugative plasmid coding terium anilini. Arch. Microbiol. 155:183-190. for the metabolism of 2-chlorobenzoic acid by Pseudomonas 435. Scholten, J. D., K.-H. Chang, P. C. Babbitt, H. Charest, M. aeruginosa. MIRCEN J. 5:255-258. Sylvestre, and D. Dunaway-Mariano. 1991. Novel enzymic hy- 455. Siuda, J. F., and J. F. DeBernardis. 1973. Naturally occurring drolytic dehalogenation of a chlorinated aromatic. Science 253: halogenated organic compounds. Lloydia 36:107-143. 182-185. 456. Slater, J. H., D. Lovatt, A. J. Weightman, E. Senior, and A. T. 436. Scholtz, R., T. Leisinger, F. Suter, and A. M. Cook. 1987. Bull. 1979. The growth of Pseudomonas putida on chlorinated Characterization of 1-chlorohexane halidohydrolase, a dehaloge- aliphatic acids and its dehalogenase activity. J. Gen. Microbiol. nase ofwide substrate range from anArthrobacter sp. J. Bacteriol. 114:125-136. 169:5016-5021. 457. Slater, J. H., A. J. Weightman, and B. G. Hall. 1985. Dehaloge- 682 FETZNER AND LINGENS MICROBIOL. IREV.

nase genes of Pseudomonas putida PP3 on chromosomally lo- 480. Suzuki, K., T. Gomi, T. Kaidoh, and E. Itagaki. 1991. Hydroxy- cated transposable elements. Mol. Biol. Evol. 2:557-567. lation of o-halogenophenol and o-nitrophenol by salicylate hy- 458. Smith, J. M., K. Harrison, and J. Colby. 1990. Purification and droxylase. J. Biochem. 109:348-353. characterization of D-2-haloacid dehalogenase from Pseudomo- 481. Suzuki, T. 1977. Metabolism of pentachlorophenol by a soil nas putida strain AJ1/23. J. Gen. Microbiol. 136:881-886. microbe. J. Environ. Sci. Health Part B 12:113-127. 459. Smith, J. M., K. Harrison, J. Colby, and S. C. Taylor. 1989. 482. Suzuki, T. 1983. Methylation and hydroxylation of pentachloro- Determination of D-2-halopropionate dehalogenase activity from phenol by Mycobacterium sp. isolated from soil. J. Pestic. Sci. Pseudomonas putida strain AJ1/23 by ion chromatography. 8:419-428. FEMS Microbiol. Lett. 57:71-74. 483. Suzuki, T., and N. Kasai. 1991. A novel method for the genera- 460. Smith, M. R., W. J. J. van den Tweel, and J. A. M. de Bont. 1991. tion of (R)- and (S)-3-chloro-1,2-propanediol by stereospecific Degradation of 3-chloro-2-methylpropionic acid byXanthobacter dehalogenating bacteria and their use in the preparation of (R)- sp. CIMW99. Appl. Microbiol. Biotechnol. 36:246-251. and (S)-glycidol. Bioorg. Med. Chem. Lett. 1:343-346. 461. Spain, J. C., and S. F. Nishino. 1987. Degradation of 1,4- 484. Suzuki, T., N. Kasai, R Yamamoto, and N. Minamiura. 1992. dichlorobenzene by a Pseudomonas sp. Appl. Environ. Microbiol. Isolation of a bacterium assimilating (R)-3-chloro-1,2-pro- 53:1010-1019. panediol and production of (S)-3-chloro-1,2-propanediol using 462. Springael, D., L. Diels, L. Hooyberghs, S. Kreps, and M. Mer- microbial resolution. J. Ferment. Bioeng. 73:443-448. geay. 1993. Construction and characterization of heavy metal- 485. Symonds, R B., W. L Rose, and M. H. Reed. 1988. Contribution resistant haloaromatic-degrading Alcaligenes eutrophus strains. of Cl- and F-bearing gases to the atmosphere by volcanoes. Appl. Environ. Microbiol. 59:334-339. Nature (London) 334:415-418. 463. Stanlake, G. J., and R. K. Finn. 1982. Isolation and character- 486. Taha, K. M. 1990. Dissertation. Fachbereich Biologie der Tech- ization of a pentachlorophenol-degrading bacterium. Appl. En- nischen Hochschule Darmstadt, Darmstadt, Germany. viron. Microbiol. 44:1421-1427. 487. Takahashi, H., S. Tsuda, Y. Nakamura, M. Ogura, T. Shiraishi, 464. Steiert, J. G., and R. L Crawford. 1986. Catabolism of penta- Y. Shimada, and K. Watanabe. 1989. Manufacture of (R)-3- chlorophenol by a Flavobacterium sp. Biochem. Biophys. Res. chloro-1,2-propanediol with Serratia. European patent applica- Commun. 141:825-830. tion EP 286,059 (CL.C12P41/00), 12 October 1988. Japanese 465. Stevens, T. O., T. G. Linkfield, and J. M. Tiedje. 1988. Physio- patent application 87/85,248, 9 March 1987. Kanegafuchi Chem- logical characterization of strain DCB-1, a unique dehalogenat- ical Industry Co. ing sulfidogenic bacterium. Appl. Environ. Microbiol. 54:2938- 488. Takemori, S., K. Suzuki, and M. Katagiri. 1976. Variable cata- 2943. lytic functions of flavoprotein oxygenases, p. 178-183. In T. P. 466. Stinson, M. K., H. S. Skovronek, and T. J. Chresand. 1991. EPA Singer (ed.), Flavins and flavoproteins. Elsevier Scientific Pub- SITE demonstration of BioTrol aqueous treatment system. J. Air lishing Co., Amsterdam. Waste Manage. Assoc. 41:228-233. 489. Taraban, R H., D. F. Berry, D. A. Berry, and H. L Walker, Jr. 467. Stirling, D. L, and H. Dalton. 1980. Oxidation of dimethyl ether, 1993. Degradation of dicamba by an anaerobic consortium methyl formate and bromomethane by Methylococcus capsulatus enriched from wetland soil. Appl. Environ. Microbiol. 59:2332- (Bath). J. Gen. Microbiol. 116:277-283. 2334. 468. St. John, W. D., and D. J. Sikes. 1988. Complex industrial waste 490. Tardif, G., C. W. Greer, D. Labbe, and P. C. K. Lau. 1991. sites, p. 237-252. In G. S. Omenn (ed.), Environmental biotech- Involvement of a large plasmid in the degradation of 1,2- nology. Plenum Press, New York. dichloroethane by Xanthobacter autotrophicus. Appl. Environ. 469. Storm, C. B., and S. Kaufnan. 1968. The effect of variation of Microbiol. 57:1853-1857. cofactor and substrate structure on the action of phenylalanine 491. Tatara, G. M., M. J. Dybas, and C. S. Criddle. 1993. Effects of hydroxylase. Biochem. Biophys. Res. Commun. 32:788-793. medium and trace metals on kinetics of carbon tetrachloride 470. Strandberg, G. W., T. L. Donaldson, and L. L Farr. 1989. transformation by Pseudomonas sp. strain KC. Appl. Environ. Degradation of trichloroethylene and trans-1,2-dichloroethylene Microbiol. 59:2126-2131. by a methanotrophic consortium in a fixed-film, packed-bed 492. Taylor, S. C. 1987. Halidohydrolase. European patent application bioreactor. Environ. Sci. Technol. 23:1422-1425. EP 179,603 (CL.C12N9/14), 30 April 1986. Imperial Chemical 471. Strotmann, U. J., M. Pentenga, and D. B. Janssen. 1990. Degra- Industries PLC. dation of 2-chloroethanol by wild type and mutants of Pseudo- 493. Thomas, A. W., J. H. Slater, and A. J. Weightman. 1992. The monas putida US2. Arch. Microbiol. 154:294-300. dehalogenase gene dehI from Pseudomonasputida PP3 is carried 472. Struis, J., and J. E. Rogers. 1989. Reductive dehalogenation of on an unusual mobile genetic element designated DEH. J. dichloroanilines by anaerobic microorganisms in fresh and di- Bacteriol. 174:1932-1940. chlorophenol-acclimated pond sediment. Appl. Environ. Micro- 494. Thomas, A. W., A. W. Topping, J. H. Slater, and A. J. Weightman. biol. 55:2527-2531. 1992. Localization and functional analysis of structural and 473. Stucki, G. 1990. Biological decomposition of dichloromethane regulatory dehalogenase genes carried on DEH from Pseudomo- from a chemical process effluent. Biodegradation 1:221-228. nas putida PP3. J. Bacteriol. 174:1941-1947. 474. Stucki, G., R. Galli, H.-R. Ebersold, and T. Leisinger. 1981. 495. Thomas, J. M., and C. H. Ward. 1989. In situ biorestoration of Dehalogenation of dichloromethane by cell extracts of Hyphomi- organic contaminants in the subsurface. Environ. Sci. Technol. crobium DM2. Arch. Microbiol. 130:366-371. 23:760-766. 475. Stucki, G., M. Thuer, and R. Bentz. 1992. Biological degradation 496. Tiedje, J. M., S. A. Boyd, and B. Z. Fathepure. 1987. Anaerobic of 1,2-dichloroethane under groundwater conditions. Water Res. degradation of chlorinated aromatic hydrocarbons. Dev. Ind. 26:273-278. Microbiol. 27:117-127. 476. Suflita, J. M., S. A. Gibson, and R. E. Beeman. 1988. Anaerobic 497. Tiedje, J. M., J. M. Duxbury, M. Alexander, and J. E. Dawson. biotransformations of pollutant chemicals in aquifers. J. Ind. 1969. 2,4-D metabolism: pathway of degradation of chlorocat- Microbiol. 3:179-194. echols by Arthrobacter sp. J. Agric. Food Chem. 17:1021-1026. 477. Suflita, J. M., A. Horowitz, D. R Shelton, and J. M. Tiedje. 1982. 498. Timmis, K. N., F. Rojo, and J. L. Ramos. 1988. Prospects for Dehalogenation: a novel pathway for the anaerobic biodegrada- laboratory engineering of bacteria to degrade pollutants, p. tion of haloaromatic compounds. Science 218:1115-1117. 61-79. In G. S. Omenn (ed.), Environmental biotechnology. 478. Suflita, J. M., J. Stout, and J. M. Tiedje. 1984. Dechlorination of Plenum Press, New York. (2,4,5-trichlorophenoxy)acetic acid by anaerobic microorganisms. 499. Tonomura, K., F. Futai, 0. Tanabe, and T. Yamaoka. 1965. J. Agric. Food Chem. 32:218-221. Defluorination of monofluoroacetate by bacteria. I. Isolation of 479. Sussman, J. L., M. Harel, F. Frolow, C. Oefner, A. Goldman, L. bacteria and their activity of defluorination. Agric. Biol. Chem. Toker, and L Silman. 1991. Atomic structure of acetylcholines- 29:124-128. terase from Torpedo califomica: a prototypic acetylcholine-bind- 500. Topp, E., and R. S. Hanson. 1990. Degradation of pentachloro- ing protein. Science 253:872-879. phenol by a Flavobacterium species grown in continuous culture VOL. 58, 1994 BACTERIAL DEHALOGENASES 683

under various nutrient limitations. Appl. Environ. Microbiol. dehalogenase from Arthrobacter sp. strain AD2. J. Bacteriol. 56:541-544. 173:124-129. 501. Topp, E., L. Xun, and C. S. Orser. 1992. Biodegradation of the 520. van den Wijngaard, A. J., K. W. H. J. van der Kamp, J. van der herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) by Ploeg, F. Pries, B. Kazemier, and D. B. Janssen. 1992. Degrada- purified pentachlorophenol hydroxylase and whole cells of Fla- tion of 1,2-dichloroethane by Ancylobacter aquaticus and other vobacterium sp. strain ATCC 39723 is accompanied by cyanogen- facultative methylotrophs. Appl. Environ. Microbiol. 58:976-983. esis. Appl. Environ. Microbiol. 58:502-506. 521. van den Wijngaard, A. J., R. G. van der Kleij, R. E. Doornweerd, 502. Tsang, J. S. H., P. J. Sallis, A. T. Bull, and D. J. Hardman. 1988. and D. B. Janssen. 1993. Influence of organic nutrients and A monobromoacetate dehalogenase from Pseudomonas cepacia cocultures on the competitive behavior of 1,2-dichloroethane- MBA4. Arch. Microbiol. 150:441-446. degrading bacteria. Appl. Environ. Microbiol. 59:3400-3405. 503. Tsien, H.-C., G. A. Brusseau, R. S. Hanson, and L. P. Wackett. 522. van der Meer, J. R., W. M. de Vos, S. Harayama, and A. J. B. 1989. Biodegradation of trichloroethylene by Methylosinus tricho- Zehnder. 1992. Molecular mechanisms of genetic adaptation to sponumOB3b. Appl. Environ. Microbiol. 55:3155-3161. xenobiotic compounds. Microbiol. Rev. 56:677-694. 504. Tsoi, T. V., G. M. Zaitsev, E. G. Plotnikova,I. A. Kosheleva, and 523. van der Meer, J. R., R.I. L. Eggen, A. J. B. Zehnder, and W. M. A. M. Boronin. 1991. Cloning and expression of the Arthrobacter de Vos. 1991. Sequence analysis of the Pseudomonas sp. strain globiformis KZT1 fcbA gene encoding dehalogenase (4-chloro- P51 tcb gene cluster, which encodes metabolism of chlorinated benzoate-4-hydroxylase) in Escherichia coli. FEMS Microbiol. catechols: evidence for specialization of catechol 1,2-dioxygen- Lett. 81:165-170. ases for chlorinated substrates. J. Bacteriol. 173:2425-2434. 524. van der Meer, J. R., A. R. W. van Neerven, E. J. de Vries, W. M. 505. Tsuchiya, T., and T. Yamaha. 1984. Reductive dechlorination of de Vos, and A. J. B. Zehnder. 1991. Cloning and characterization 1,2,4-trichlorobenzene by Staphylococcus epidermis isolated from of plasmid-encoded genes for the degradation of 1,2-dichloro-, intestinal contents of rats. Agric. Biol. Chem. 48:1545-1550. 1,4-dichloro-, and 1,2,4-trichlorobenzene of Pseudomonas sp. 506. Tu, C. M. 1976. Utilization and degradation of lindane by soil strain P51. J. Bacteriol. 173:6-15. microorganisms. Arch. Microbiol. 108:259-263. 525. van der Meer, J. R., A. J. B. Zehnder, and W. M. de Vos. 1991. 507. Uchiyama, H., T. Nakajima, 0. Yagi, and T. Tabuchi. 1989. Identification of a novel composite transposable element, Aerobic degradation of trichloroethylene by a new type II Tn5280, carrying chlorobenzene dioxygenase genes of Pseudomo- methane-utilizing bacterium, strain M. Agric. Biol. Chem. 53: nas sp. strain P51. J. Bacteriol. 173:7077-7083. 2903-2907. 526. van der Ploeg, J., F. Pries, A. van den Wongaard, C. Kennes, and 508. Uotila, J. S., V. H. Kitunen, T. Saastamoinen, T. Coote, M. M. D. B. Janssen. 1992. Genetic adaptation of bacteria towards Haggblom, and M. S. Salkinoja-Salonen. 1992. Characterization chlorinated hydrocarbon degradation, p. 163-169. In R. Casper of aromatic dehalogenases of Mycobactenium fortuitum CG-2. J. and J. Landsmann (ed.), Proceedings of the 2nd International Bacteriol. 174:5669-5675. Symposium on the Biosafety Results of Field Tests of Genetically 509. Uotila, J. S., M. S. Salkinoja-Salonen, and J. H. A. Apajalahti. Modified Plants and Microorganisms. Biologische Bundesanstalt 1991. Dechlorination of pentachlorophenol by membrane bound fur Land- und Forstwirtschaft, Braunschweig, Germany. enzymes of Rhodococcus chlorophenolicus PCP-I. Biodegradation 527. van der Ploeg, J., G. van Hall, and D. B. Janssen. 1991. 2:25-31. Characterization of the haloacid dehalogenase from Xantho- 510. Uwers, J., W. Clemens, and H.-J. Knackmuss. 1991. Biodegra- bacter autotrophicus GJ10 and sequencing of the dhlB gene. J. dation of chloroethenes using isoprene as cosubstrate, p. 77-83. Bacteriol. 173:7925-7933. In H. Verachtert and W. Verstraete (ed.), Proceedings of the 528. van der Waarde, J. J., R. Kok, and D. B. Janssen. 1993. International Symposium on Environmental Biology, Oostende, Degradation of 2-chloroallylalcohol by a Pseudomonas sp. Appl. Belgium. Koninklijke Vlaamse Ingenieursvereiniging, Brussels. Environ. Microbiol. 59:528-535. 511. Valo, R., and M. Salkinoja-Salonen. 1986. Microbial transforma- 529. van Hylckama Vlieg, J. E. T., and D. B. Janssen. 1992. Bacterial tion of polychlorinated phenoxy phenols. J. Gen. Appl. Micro- degradation of 3-chloroacrylic acid and the characterization of biol. 32:505-517. cis- and trans-specific dehalogenases. Biodegradation 2:139-150. 512. Valo, R., and M. Salkinoja-Salonen. 1986. Bioreclamation of 530. Vannelli, T., and A. B. Hooper. 1993. Reductive dehalogenation chlorophenol-contaminated soil by composting. Appl. Microbiol. of the trichloromethyl group of nitrapyrin by the ammonia- Biotechnol. 25:68-75. oxidizing bacterium Nitrosomonas europaea. Appl. Environ. Mi- 513. Valo, R. J., M. M. Haggblom, and M. S. Salkinoja-Salonen. 1990. crobiol. 59:3597-3601. Bioremediation of chlorophenol containing simulated ground 531. Vannelli, T., M. Logan, D. M. Arciero, and A. B. Hooper. 1990. water by immobilized bacteria. Water Res. 24:253-258. Degradation of halogenated aliphatic compounds by the ammo- 514. Vandenbergh, P. A., R. H. Olsen, and J. F. Colaruotolo. 1981. nia-oxidizing bacterium Nitrosomonas europaea. Appl. Environ. Isolation and genetic characterization of bacteria that degrade Microbiol. 56:1169-1171. chloroaromatic compounds. Appl. Environ. Microbiol. 42:737- 532. Verschueren, K. H. G., S. M. Franken, H. J. Rozeboom, K. H. 739. Kalk, and B. W. Djkstra. 1993. Refined X-ray structures of 515. van den Tweel, W. J. J., J. B. Kok, and J. A. M. de Bont. 1987. haloalkane dehalogenase at pH 6.2 and pH 8.2 and implications Reductive dechlorination of 2,4-dichlorobenzoate to 4-chloro- for the reaction mechanism. J. Mol. Biol. 232:856-872. benzoate and hydrolytic dehalogenation of 4-chloro-, 4-bromo-, 533. Verschueren, K. H. G., F. Seljee, H. J. Rozeboom, K. H. Kalk, and and 4-iodobenzoate by Alcaligenes denitnificans NTB-1. Appl. B. W. Dijkstra. 1993. Crystallographic analysis of the catalytic Environ. Microbiol. 53:810-815. mechanism of haloalkane dehalogenase. Nature (London) 363: 516. van den Tweel, W. J. J., N. ter Burg, J. B. Kok, and J. A. M. de 693-698. Bont. 1986. Bioformation of 4-hydroxybenzoate from 4-chloro- 534. Vitovski, S. 1993. Phenylacetate-coenzyme A ligase is induced benzoate by Alcaligenes denitrificans NTB-1. Appl. Microbiol. during growth on phenylacetic acid in different bacteria of several Biotechnol. 25:289-294. genera. FEMS Microbiol. Lett. 108:1-6. 517. van den Wijngaard, A. J., D. B. Janssen, and B. Witholt. 1989. 535. Vogel, T. M., C. S. Criddle, and P. L. McCarty. 1987. Transfor- Degradation of epichlorohydrin and halohydrins by bacterial mations of halogenated aliphatic compounds. Environ. Sci. Tech- cultures isolated from freshwater sediment. J. Gen. Microbiol. nol. 21:722-736. 135:2199-2208. 536. Vogel, T. M., and P. L. McCarty. 1985. Biotransformation of 518. van den Wijngaard, A. J., J. Prins, A. J. A. C. Smal, and D. B. tetrachloroethylene to trichloroethylene, dichloroethylene, vinyl Janssen. 1993. Degradation of 2-chloroethylvinylether by Ancy- chloride, and carbon dioxide under methanogenic conditions. lobacter aquaticus AD25 and AD27. Appl. Environ. Microbiol. Appl. Environ. Microbiol. 49:1080-1083. 59:2777-2783. 537. Vollmer, M. D., K. Stadler-Fritzsche, and M. Schlomann. 1993. 519. van den Wijngaard, A. J., P. T. W. Reuvekamp, and D. B. Conversion of 2-chloromaleylacetate in Alcaligenes eutrophus Janssen. 1991. Purification and characterization of haloalcohol JMP134. Arch. Microbiol. 159:182-188. 684 FETZNER AND LINGENS MICROBIOL. REV.

538. Wackett, L. P. 1991. Dehalogenation of organohalide pollutants 556. Wubbolts, M. G., and K. N. Timmis. 1990. Biotransformation of by bacterial enzymes and coenzymes, p. 191-200. In J. W. Kelly substituted benzoates to the corresponding cis-diols by an engi- and T. 0. Baldwin (ed.), Applications of enzyme biotechnology. neered strain of Pseudomonas oleovorans producing the TOL Plenum Press, New York. plasmid-specified enzyme toluate 1,2-dioxygenase. Appl. Envi- 539. Wackett, L. P., G. A. Brusseau, S. R. Householder, and R. S. ron. Microbiol. 56:569-571. Hanson. 1989. Survey of microbial oxygenases: trichloroethylene 560. Wuosmaa, A. M., and L. P. Hager. 1990. Methyl chloride degradation by propane-oxidizing bacteria. Appl. Environ. Mi- transferase: a carbocation route for biosynthesis of halometabo- crobiol. 55:2960-2964. lites. Science 249:160-162. 540. Wackett, L. P., and D. T. Gibson. 1988. Degradation of trichlo- 561. Wyndham, R. C., C. Nakatsu, M. Peel, A. Cashore, J. Ng, and F. roethylene by toluene dioxygenase in whole-cell studies with Szilagyi. 1994. Distribution of the catabolic transposon TnS271 in Pseudomonasputida Fl. Appl. Environ. Microbiol. 54:1703-1708. a groundwater bioremediation system. Appl. Environ. Microbiol. 541. Wackett, L. P., and S. R. Householder. 1989. Toxicity of trichlo- 60:86-93. roethylene to Pseudomonas putida Fl is mediated by toluene 562. Wyndham, R. C., R. K. Singh, and N. A. Straus. 1988. Catabolic dioxygenase. Appl. Environ. Microbiol. 55:2723-2725. instability, plasmid gene deletion and recombination in Alcali- 542. Wackett, L. P., and C. A. Schanke. 1992. Mechanisms of reductive genes sp. BR60. Arch. Microbiol. 150:237-243. dehalogenation by transition metal cofactors found in anaerobic 563. Wyndham, R. C., and N. A. Straus. 1988. Chlorobenzoate bacteria, p. 329-356. In H. Sigel and A. Sigel (ed.), Metal ions in catabolism and interactions between Alcaligenes and Pseudomo- biological systems, vol. 28. Degradation of environmental pollut- nas species from Bloody Run Creek. Arch. Microbiol. 150:230- ants by microorganisms and their metalloenzymes. Marcel Dek- 236. ker, Inc., New York. 564. Xun, L, and C. S. Orser. 1991. Purification of a Flavobacterium 543. Wade, R. S., and C. E. Castro. 1973. Oxidation of iron(II) pentachlorophenol-induced periplasmic protein (PcpA) and nu- porphyrins by alkyl halides. J. Am. Chem. Soc. 95:226-230. cleotide sequence of the corresponding gene (pepA). J. Bacteriol. 544. Wade, R. S., and C. E. Castro. 1973. Oxidation of heme proteins 173:2920-2926. by alkyl halides. J. Am. Chem. Soc. 95:231-234. 565. Xun, L, and C. S. Orser. 1991. Purification and properties of 545. Wagner-Dobler, I., R. Pipke, K. N. Timmis, and D. F. Dwyer. pentachlorophenol hydroxylase, a flavoprotein from Flavobacte- 1992. Evaluation of aquatic sediment microcosms and their use in rium sp. strain ATCC 39723. J. Bacteriol. 173:4447-4453. assessing possible effects of introduced microorganisms on eco- 566. Xun, L., E. Topp, and C. S. Orser. 1992. Glutathione is the system parameters. Appl. Environ. Microbiol. 58:1249-1258. reducing agent for the reductive dehalogenation of tetrachloro- 546. Wang, Z., B. H. Bleakley, D. L Crawford, G. Hertel, and F. Rafii. p-hydroquinone by extracts from a Flavobacterium sp. Biochem. 1990. Cloning and expression of a lignin peroxidase gene from Biophys. Res. Commun. 182:361-366. Streptomyces viridosporus in Streptomyces lividans. J. Biotechnol. 567. Xun, L, E. Topp, and C. S. Orser. 1992. Diverse substrate range 13:131-144. of a Flavobacterium pentachlorophenol hydroxylase and reaction 547. Watt, J. C., V. S. Wroniewicz, and D. F. loli. 1988. Environmental stoichiometries. J. Bacteriol. 174:2898-2902. concerns associated with the design of genetic engineering facil- 568. Xun, L, E. Topp, and C. S. Orser. 1992. Confirmation of ities, p. 307-322. In G. S. Omenn (ed.), Environmental biotech- oxidative dehalogenation of pentachlorophenol by a Flavobacte- nology. Plenum Press, New York. num pentachlorophenol hydroxylase. J. Bacteriol. 174:5745- 548. Wedemeyer, G. 1967. Dechlorination of 1,1,1-trichloro-2,2-bis(p- 5747. chlorophenyl)ethane by Aerobacter aerogenes. Appl. Microbiol. 569. Xun, L., E. Topp, and C. S. Orser. 1992. Purification and 15:569-574. characterization of a tetrachloro-p-hydroquinone reductive deha- 549. Weightman, A. J., J. H. Slater, and A. T. Bull. 1979. The partial logenase from a Flavobactenum sp. J. Bacteriol. 174:8003-8007. purification of two dehalogenases from Pseudomonasputida PP3. 570. Yamada, T., and T. Suzuki. 1983. Occurrence of reductive FEMS Microbiol. Lett. 6:231-234. dechlorination products in the paddy field soil treated with CNP 550. Weightman, A. J., A. L. Weightman, and J. H. Slater. 1982. (chlornitrofen). J. Pestic. Sci. 8:437-443. Stereospecificity of 2-monochloropropionate dehalogenation by 571. Yamaguchi, M., and H. Fujisawa. 1978. Characterization of the two dehalogenases of Pseudomonas putida PP3: evidence for NADH-cytochrome c reductase, a component of benzoate 1,2- two different dehalogenation mechanisms. J. Gen. Microbiol. dioxygenase system from Pseudomonas arvilla C-L. J. Biol. Chem. 128:1755-1762. 253:8848-8853. 551. Weiss, U. M., L. Scheunert, W. Klein, and F. Korte. 1982. Fate of 572. Yamaguchi, M., and H. Fujisawa. 1980. Purification and charac- pentachlorophenol-14C in soil under controlled conditions. J. terization of an oxygenase component in benzoate 1,2-dioxygen- Agric. Food Chem. 30:1191-1194. ase system from Pseudomonas arvilla C-1. J. Biol. Chem. 255: 552. Wilson, J. T., and B. H. Wilson. 1985. Biotransformation of 5058-5063. trichloroethylene in soil. Appl. Environ. Microbiol. 49:242- 573. Yamaguchi, M., and H. Fujisawa. 1982. Subunit structure of 243. oxygenase component in benzoate 1,2-dioxygenase system from 553. Winkler, F. K., A. D'Arcy, and W. Hunziker. 1990. Structure of Pseudomonas arvilla C-1. J. Biol. Chem. 257:12497-12502. human pancreatic lipase. Nature (London) 343:771-774. 574. Yokota, T., H. Fuse, T. Omori, and Y. Minoda. 1986. Microbial 554. Winter, B., and W. Zimmermann. 1992. Degradation of haloge- dehalogenation of haloalkanes mediated by oxygenase or ha- nated aromatics by actinomycetes, p. 157-203. In H. Sigel and A. lidohydrolase. Agric. Biol. Chem. 50:453-460. Sigel (ed.), Metal ions in biological systems, vol. 28. Degradation 575. Yokota, T., T. Omori, and T. Kodama. 1987. Purification and of environmental pollutants by microorganisms and their met- properties of haloalkane dehalogenase from Corynebacterium sp. alloenzymes. Marcel Dekker, Inc., New York. strain mlS-3. J. Bacteriol. 169:4049-4054. 555. Winter, R. B., K.-M. Yen, and B. D. Ensley. 1989. Efficient 576. You, I.-S., and R. Bartha. 1982. Stimulation of 3,4-dichloroaniline degradation of trichloroethylene by a recombinant Escherichia mineralization by aniline. Appl. Environ. Microbiol. 44:678-681. coli. Bio/Technology 7:282-285. 577. Zaidi, B. R., Y. Murakami, and M. Alexander. 1988. Factors 556. Wood, J. M., F. S. Kennedy, and R. S. Wolfe. 1968. The reaction limiting success of inoculation to enhance biodegradation of low of multihalogenated hydrocarbons with free and bound reduced concentrations of organic chemicals. Environ. Sci. Technol. 22: vitamin B12. Biochemistry 7:1707-1713. 1419-1425. 557. Woods, S. L., J. F. Ferguson, and M. M. Benjamin. 1989. 578. Zaitsev, G. M., and Y. N. Karasevich. 1981. Preparative metab- Characterization of chlorophenol and chloromethoxybenzene olism of 4-chlorobenzoic acid inArthrobacterglobifomis. Mikro- biodegradation during anaerobic treatment. Environ. Sci. Tech- biologiya 50:423-428. nol. 23:62-68. 579. Zaitsev, G. M., and Y. N. Karasevich. 1985. Preparatory metab- 558. Wu, W.-M., L. Bhatnagar, and J. G. Zeikus. 1993. Performance olism of 4-chlorobenzoic and 2,4-dichlorobenzoic acids in of anaerobic granules for degradation of pentachlorophenol. Corynebacterium sepedonicum. Mikrobiologiya 54:356-359. Appl. Environ. Microbiol. 59:389-397. 580. Zaitsev, G. M., T. V. Tsoi, V. G. Grishenkov, E. G. Plotnikova, VOL. 58, 1994 BACTERIAL DEHALOGENASES 685

and A. M. Boronin. 1991. Genetic control of degradation of effluents by free and immobilized cells of streptomycetes. Appl. chlorinated benzoic acids in Arthrobacter globiformis, Corynebac- Microbiol. Biotechnol. 39:418-423. terium sepedonicum and Pseudomonas cepacia strains. FEMS 584. Ziegler, K., K. Braun, A. Bockler, and G. Fuchs. 1987. Studies on Microbiol. Lett. 81:171-176. the anaerobic degradation of benzoic acid and 2-aminobenzoic 581. Zehnder, A. J. B., and G. Schraa. 1988. Biologischer Abbau acid by a denitrifying Pseudomonas strain. Arch. Microbiol. 149: xenobiotischer Verbindungen bei niedrigen Konzentrationen 62-69. und das Problem der Restkonzentration. gwf Wasser/Abwasser 585. Zoro, J. A., J. M. Hunter, G. Eglinton, and G. C. Ware. 1974. 129:369-373. Degradation of p,p'-DDT in reducing environments. Nature 582. Zhang, X., and J. Wiegel. 1990. Sequential anaerobic degradation (London) 247:235-237. of 2,4-dichlorophenol in freshwater sediments. Appl. Environ. 586. Zylstra, G. J., L. P. Wackett, and D. T. Gibson. 1989. Trichloro- Microbiol. 56:1119-1127. ethylene degradation by Escherichia coli containing the cloned 583. Zhou, W., B. Winter, and W. Zimmermann. 1993. Dechlorination Pseudomonas putida Fl toluene dioxygenase genes. Appl. Envi- of high-molecular-mass compounds in spent sulphite bleach ron. Microbiol. 55:3162-3166.