degradation of 1,2,4-trichloro- and 1,2,4,5-tetrachlorobenzene

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1991, p. 1430-1440 Vol. 57, No. 50099-2240/91/051430-11$02.00/0Copyright © 1991, American Society for Microbiology

Degradation of 1,2,4-Trichloro- and 1,2,4,5-Tetrachlorobenzeneby Pseudomonas Strains

PETER SANDER,' ROLF-MICHAEL WITTICH,1 PETER FORTNAGEL,1* HEINZ WILKES,2AND WITTKO FRANCKE2

Institut fur Allgemeine Botanik, Abteilung Mikrobiologie, Ohnhorststrasse 18,1 and Institut fur Organische Chemie,Martin-Luther-King-Platz 6,2 Universitat Hamburg, D-2000 Hamburg, Federal Republic of Germany

Received 11 October 1990/Accepted 5 March 1991

Two Pseudomonas sp. strains, capable of growth on chlorinated benzenes as the sole source of carbon andenergy, were isolated by selective enrichment from soil samples of an industrial waste deposit. Strain PS12 grewon monochlorobenzene, all three isomeric dichlorobenzenes, and 1,2,4-trichlorobenzene (1,2,4-TCB). StrainPS14 additionally used 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB). During growth on these compounds bothstrains released stoichiometric amounts of chloride ions. The first steps of the catabolism of 1,2,4-TCB and1,2,4,5-TeCB proceeded via dioxygenation of the aromatic nuclei and furnished 3,4,6-trichlorocatechol. Theintermediary cis-3,4,6-trichloro-1,2-dihydroxycyclohexa-3,5-diene (TCB dihydrodiol) formed from 1,2,4-TCBwas rearomatized by an NAD+-dependent dihydrodiol dehydrogenase activity, while in the case of 1,2,4,5-TeCB oxidation the catechol was obviously produced by spontaneous elimination of hydrogen chloride from theinitially formed 1,3,4,6-tetrachloro-1,2-dihydroxycyclohexa-3,5-diene. Subsequent ortho cleavage was cata-lyzed by a type II catechol 1,2-dioxygenase producing the corresponding 2,3,5-trichloromuconate which waschanneled into the tricarboxylic acid pathway via an ordinary degradation sequence, which in the present caseincluded 2-chloro-3-oxoadipate. From the structure-related compound 2,4,5-trichloronitrobenzene the nitrogroup was released as nitrite, leaving the above metabolite as 3,4,6-trichlorocatechol. Enzyme activities for theoxidation of chlorobenzenes and halogenated metabolites were induced by both strains during growth on thesehaloaromatics and, to a considerable extent, during growth of strain PS12 on acetate.

The extensive use of haloaromatics as solvents, odorizers,fire retardants, and pesticides has led to considerable releaseof these compounds into the environment. Additionally, thispollution was and is still accompanied with toxic side effectsto the biosphere (51, 59). Polychlorinated benzenes, evengenerated biologically in minute amounts (62), enter theenvironment from industrial sources and effluents of hazard-ous waste disposal sites, consequently becoming detectablein soil, air, water, and living organisms (59). Although theabove and many other persistent compounds resist microbialmineralization because of their recalcitrant haloaromaticstructure (26, 46), microbial enzyme systems were subjectedto rapid evolution (17), now allowing the utilization of suchxenobiotic compounds as polychlorinated biphenyls (7, 29),pentachlorophenol (16, 61, 71), and many other compoundsof environmental concern.The first information on the aerobic biodegradation of

halogenated benzenes was by Shelat and Patel (69), whoreported that bromobenzene should be utilized by a Bacilluspolymyxa. Mineralization of monochlorobenzene by a Pseu-domonas strain was demonstrated by Reineke and Knack-muss (63). The three isomeric dichlorobenzenes and themore xenobiotic 1,2,4-trichlorobenzene (1,2,4-TCB) wereutilized by enriched and constructed strains as well asundefined communities in natural and laboratory environ-ments (1, 9, 10, 19, 24, 38, 47, 48, 56, 66, 70, 74). Theevolution of radiolabeled CO2 even from 1,2,3-TCB wasreported by Marinucci and Bartha (48), and Haider et al. (35)and Ballschmiter and Scholz (6) described the cometabolicturnover of numerous halobenzenes by benzene-growingbacteria forming chlorocatechols and -phenols. These chlo-

* Corresponding author.

rophenols obviously were artifacts from workup procedures,since the formation of benzene dihydrodiols has been de-scribed to proceed by dioxygenation (32) and the nonenzy-matic dehydration of dihydrodiols is known to occur evenunder mild conditions (34).Whereas 1,3,5-TCB and other highly halogenated benzene

isomeres still seem to resist aerobic biodegradation (55), thereductive dehalogenation of tri- and dichlorobenzenes tomonochlorobenzene and even that of hexachlorobenzene,resulting in a mixture of tri- and dichlorobenzenes, has beenwell demonstrated to occur under obviously rather unspe-cific anaerobic conditions (8, 25, 72, 73).To our knowledge, detailed investigations on the biodeg-

radation of 1,2,4-TCB and 1,2,4,5-tetrachlorobenzene(1,2,4,5-TeCB) have not been reported, with the only excep-tion being that plasmid-encoded genes coding for 1,2,4-TCBdegradation have been cloned and expressed recently (75).In the present study, we demonstrate the enrichment, isola-tion, and characterization of two bacterial strains capable ofusing these compounds as the sole source of carbon andenergy and to release nitrite from 2,4,5-trichloronitroben-zene. In addition, we describe biochemical properties andprovide information on the isolation and structure elucida-tion of degradation products, most of which are reportedhere for the first time. We also suggest a converging pathwayfor the biodegradation of both chlorobenzenes.

(This paper is based in part on a doctoral study by PeterSander in the Faculty of Biology and by Heinz Wilkes in theFaculty of Chemistry, The University of Hamburg.)

MATERIALS AND METHODS

Enrichment, isolation, and growth of bacteria. The bacte-rial strains PS12 and PS14 were enriched from soil samples

1430

DEGRADATION OF 1,2,4-TCB AND 1,2,4,5-TeCB 1431

collected from the Hamburg-Georgswerder industrial wastedeposit. Serum bottles (100 ml) sealed with rubber stopperswere filled with 30 ml of a mineral salts medium containing(per liter of distilled water) 7.0 g of Na2HPO4 2H20, 2.0 gof KH2PO4, 500 mg of (NH4)2S04, 100 mg of MgCl2 6H20,50 mg of Ca(NO3)2 4H20, 1 ml of a trace-element solution(as described in reference 60 but without EDTA), and 5 mgof yeast extract. About 1 g of soil was added to each bottle.Corresponding to a final concentration of 5 mM, liquid1,2,4-TCB as the halogenated carbon source was fed overthe gas phase by evaporation from a test tube placed in thebottles. The same amount of fine mortar-ground crystalline1,2,4,5-TeCB was directly added to the freshly autoclavedhot medium. The bottles were incubated at 28°C on a rotaryshaker at 100 rpm, and subcultures were made at weeklyintervals. After growth of bacteria became visible by in-creasing turbidity and decreasing pH, aliquots of the cellsuspensions were plated on solid media consisting of theabove mineral salts medium and 12 g of Agar No. 1 (OxoidLtd., Basingstoke, Hampshire, United Kingdom) per liter.Plates were incubated in desiccators at 28°C in the presenceof 1,2,4-TCB vapor as the substrate. Crystals of 1,2,4,5-TeCB were dispersed in the hot, freshly autoclaved liquidagar solution by rapid stirring before plates were poured.Colonies grown on plates were isolated and reinoculated intothe above mineral salts medium; yeast extract was omitted inall further procedures and experiments. Pure cultures wereobtained by serial dilutions on nutrient agar and retransfer ofsingle colonies onto mineral salts agar; the chlorobenzeneswere supplied as described above. This solid medium wasalso used for stock cultures of strains. Mass cultures weregrown in magnetically stirred 5-liter Erlenmeyer flasks filledwith 3 liters of mineral salts medium containing the substrateas described above, corresponding to a 5 mM concentration.Growth parameters (protein and optical density) were deter-mined as described previously (27).

Identification of bacterial strains. Strains obtained from theenrichment procedures were identified on the basis of clas-sification schemes published in Bergey's Manual of System-atic Bacteriology (57), using tests described therein, and bystandard laboratory procedures. The guanine-plus-cytosine(G+C) content of bacterial DNA was determined as de-scribed by Frank-Kamenetskii (28).

Chloride and nitrite release. Chloride concentrations weredetermined at 25°C with the pMX 2000 Ion Meter (WTWGmbH, Weilheim, Federal Republic of Germany) equippedwith a chloride-sensitive electrode (Ingold AG, Urdorf,Switzerland). For end point determinations, cells weregrown in a mineral salts medium in which chloride salts hadbeen replaced by sulfate. Standards for calibration measure-ments were made up in this medium. Specific releasing rateswere determined by using washed cells resuspended in 50mM sodium phosphate buffer. Releasing rates were cor-rected for electrode drift by using data obtained from exper-iments performed in the absence of chlorinated substratesand, additionally, using benzene for blank runs. The quan-titation of nitrite ions released by washed cell suspensionsfrom nitroaromatic compounds was performed as describedby Montgomery and Dymock (52) using sodium nitrite forcalibration.Oxygen uptake. The determination of oxygen uptake rates

of extensively washed, resting cell suspensions was per-formed polarographically as described previously (27). Con-centrated stock solutions of the substrates (100 mM) weremade up in dimethyl sulfoxide.

Preparation of cell extracts. Residual chlorobenzenes were

removed from growing cultures by decantation and filtrationfollowed by further incubation for at least 1 h to allowcomplete consumption of still-dissolved substrates. Bacteriawere harvested by centrifugation at 14,000 x g for 20 min at4°C and washed with 33 mM Tris-HCl buffer (pH 8.0).Pelleted cells were suspended in about a fivefold volume ofthis buffer and broken by two passages through a chilledFrench pressure cell (Aminco, Silver Spring, Md.) at 10,000lb/in2. Cell debris were removed by centrifugation at 100,000x g for 1 h at 4°C. The resulting supernatant was stored onice until use for enzyme assays. Soluble protein was deter-mined by the method of Bradford (11) with bovine serumalbumin as the standard.Enzyme assays. Catechol 2,3-dioxygenase (C230, EC

1.13.11.2) activity was assayed as described by Nozaki (54).Catechol 1,2-dioxygenase (C120, EC 1.13.11.1) activities forcatechol and its chlorinated and methylated derivatives weredetermined by the procedures of Dorn and Knackmuss (20,21). The conversion of 3,6-dichlorocatechol into 2,5-dichlo-romuconate was determined by the use of spectral data givenby Oltmanns et al. (56) and Spain and Nishino (70); for theestimation of 2,3,5-trichloromuconate produced from 3,4,6-trichlorocatechol, the molar A260 coefficient was determinedto be 9,500 cm-1 mol-1 in the assay buffer. Simultaneouslypresent and interfering C230 activity was first destroyed byincubating the enzyme preparation in the presence of 0.01%(vol/vol) H202 for 10 min as described by Nakazawa andYokota (53). cis-1,2-Dihydroxycyclohexa-3,5-diene (dihy-drodiol) dehydrogenase activity was determined by monitor-ing NAD+ reduction as described by Reineke and Knack-muss (63).

Isolation and derivatization of metabolites. For isolation ofneutral metabolites, the culture broth, freed from cells bycentrifugation, was extracted twice with equal volumes ofethyl acetate. Acidic metabolites were extracted at pH 2.5 asdescribed above. The organic layers were dried with anhy-drous magnesium or sodium sulfate, and after evaporation ofthe solvent, residues were taken up in small volumes ofmethanol or ethyl acetate and stored at -20°C before theywere further investigated. Column chromatography wasperformed on silica gel (230/400 mesh, according to ASTM;E. Merck, Darmstadt, Federal Republic of Germany) withhexane/ethyl acetate (10/1 by volume). Methylation ofmetabolites was carried out with diazomethane as describedpreviously (78).

Analytical methods. Water-soluble compounds and metab-olites produced by bacterial cultures were analyzed byreverse-phase high-performance liquid chromatography(HPLC) as described previously (27). HPLC was also usedfor the isolation of metabolites for nuclear magnetic reso-nance (NMR) spectroscopic investigations. Routine gaschromatography was performed on a Carlo Erba Fractovap6130 model (Erba Science, Hofheim, Federal Republic ofGermany) on a 25-m fused silica column OV1 (ChrompackGmbH, Frankfurt, Federal Republic of Germany) under atemperature regimen from 80 to 300°C at a rate of 5°C/min.Gas chromatography-mass spectrometry (GC-MS, El at 70eV) studies were carried out on a HP 5890 gas chromato-graph (Hewlett-Packard GmbH, Bad Homburg, FederalRepublic of Germany) coupled to a VG 70-250S mass spec-trometer (Vacuum Generators, Manchester, United King-dom). A 70-eV mass spectrum of 3,4,6-trichloro-1,2-dihy-droxycyclohexa-3,5-diene was obtained upon direct inlet ofthe isolated compound by using the above instrument. NMRspectra were recorded on a Bruker WM 400 and an AM 250P instrument (Bruker GmbH, Karlsruhe, Federal Republic

VOL. 57, 1991

APPL. ENVIRON. MICROBIOL.

of Germany). Optical rotation of chiral compounds wasmeasured on the Perkin-Elmer 243 polarimeter (Perkin-Elmer & Co. GmbH, Uberlingen, Federal Republic of Ger-many) at 549 nm.

Chemicals. 3-Chloro-2-hydroxybenzaldehyde and 4,5-dichloro-2-hydroxybenzaldehyde were synthesized from2-chlorophenol and 3,4-dichlorophenol, respectively, by themethods of Reimer and Thiemann (76) and Duff (22). Fol-lowing the procedures of Baker et al. (5) and Dakin (18), thealdehydes served as starting material for the syntheses of3-chlorocatechol, 4-chlorocatechol, 3,5-dichlorocatechol,and 4,5-dichlorocatechol. 3-Methylcatechol and 4-methyl-catechol were synthesized by the same methods from 2-meth-ylphenol and 3-methylphenol, respectively. Some of thecompounds used in this study were prepared biologicallywith the aid of low-level-induced, peptone-grown cells of thetwo strains described in this paper: 3,4- and 3,6-dichloro-catechol were obtained from cooxidation of 1,2- and 1,4-dichlorobenzene, while 1,2,3- and 1,2,4-trichlorobenzeneserved as starting material for 3,4,5- and 3,4,6-trichlorocate-chol. Structural proof of all samples was carried out byGC-MS and NMR spectroscopy. Chemicals used for synthe-ses and experiments were purchased from Aldrich-ChemieGmbH & Co. KG (Steinheim, Federal Republic of Germa-ny). D- and L-chlorosuccinic acid and biochemicals werefrom Sigma Chemie GmbH (Deisenhofen, Federal Republicof Germany). Mineral salts were of the highest commerciallyavailable purity.

RESULTS

Isolation and characterization of bacterial strains. Follow-ing the described enrichment and purification methods,1,2,4-TCB- and 1,2,4,5-TeCB-utilizing mixed cultures wereobtained. From these mixed cultures the 1,2,4-TCB-utilizingstrain PS12 was obtained in pure culture about 10 weeksafter starting the enrichment. About 2 months later, the1,2,4,5-TeCB-mineralizing strain PS14 could be isolated.Both strains were strictly aerobic, gram negative, and oxi-dase and catalase positive. Motility was imparted by a singlepolar flagellum, and the rod-shaped organisms, about 1.0 by1.5 ,um, formed turbid, creamy colonies on plates. Estima-tion of the guanine-plus-cytosine (G+C) content resulted in68 mol% for strain PS12 and 64 mol% for strain PS14. Nitratecould be used as a nitrogen source. Both organisms couldnot grow at 37°C, and only strain PS14 grew at 4°C. Apartfrom the utilization of (halogenated) aromatic compoundsdescribed later, strain PS12 could use acetate, fumarate,succinate, 2-oxoglutarate, malonate, glutamate, and glucoseas sole sources of carbon and energy. Strain PS14 could notutilize any of these compounds but solely grew on peptone (5g/liter) containing mineral salts medium. However, bothstrains could grow on D- and L-chlorosuccinate, whereaschloroacetate did not serve as a carbon source. On the basisof the above characteristics, both strains were tentativelyclassified as Pseudomonas species.Growth with 1,2,4-TCB, 1,2,4,5-TeCB, and other aromatic

compounds. The ability of Pseudomonas sp. strain PS12 togrow with 1,2,4-TCB and that of Pseudomonas sp. strainPS14 to grow with 1,2,4,5-TeCB is shown in Fig. 1A and B.Doubling times during the early growth phase were about 12h for strain PS12 when it was fed with 1,2,4-TCB over thevapor phase and 8 h for strain PS14 when the substrate1,2,4,5-TeCB was present as a fine crystal suspension in themineral salts medium. Endpoint determinations were per-formed 8 days after inoculation of the cultures for turbidity,

0150 \15LA1-0.4- 6.6

0 2 6 10

E

0.2. -S '07501

0 0 -- 04 0 6.20 2 4 6 8 10

Trichlorobenzene (mM)

0.7 ,120 - 30 74

0.6 - 100 25 7.

0.580 0 20

g 0.4- T.OP 7.0'60-15~

r-0.3-protein content,amount f chlordeionsreleasefromt6.8

cab nsouces an deraei H hsebthscnan

0.2-c-~~~~~~~~~~1..0 0.2 00 ~20 66a.Li 0.

parameters.Adecreaseinp to below 6.34whichouldb6.40 2 4 6 8 10

Tetrachlorobenzene (mM)FIG. 1. End point determinations of culture turbidity (0), pro-

tein content (0), chloride release (t), and pH change (0) aftergrowth of Pseudomonas sp. PS12 on 1,2,4-TCB (A) and of Pseudo-monas sp. PS14 on 1,2,4,5-TeCB (B). Data were determined fromparallel batches of increasing amounts of substrate 8 days afterinoculation.

protein content, amount of chloride ions released from thecarbon sources, and decrease in pH. Those batches contain-ing substrate concentrations up to 3 mM showed an almostlinear response with respect to the above-described growthparameters. A decrease in pH to below 6.3, which could beprevented by neutralization of the hydrochloric acid pro-duced during dehalogenation, inhibited further growth ofcultures. Both strains did not tolerate buffer concentrationshigher than 60 mM with respect to phosphate and a pHbeyond 7.8. Yields per mole of substrate utilized by strainPS12 grown on 1,2,4-TCB and P814 grown on 1,2,4,5-TeCBwere 38 and 36 g of protein, respectively. Almost identicalyields were also obtained by monochlorobenzene- and tolu-ene-grown P812 cells.Both strains failed to grow with benzene but could metab-

olize a wide spectrum of halogenated aromatic compoundssuch as monochlorobenzene, the three dichlorobenzenes,1,2,4-TCB, and the corresponding brominated analogs. Ad-ditionally, strain P814 utilized 1,2,4,5-TeCB but not thebromoderivative. Other isomers of the tri- and tetrahaloge-nated benzenes, as well as penta- and hexachlorobenzene,were no substrate for bacterial growth; however, mixedhalogenated aromatics like 2-chloro-1-iodobenzene and 2,5-dichloro-1-iodobenzene were used by both strains, whereas1-bromo-4-chlorobenzene and 2,4-dichloro-1-iodobenzene

1432 SANDER ET AL.


TABLE 1. Specific rates of oxygen uptake with aromatic compounds by washed-cell suspensions ofPseudomonas sp. strains PS12 and PS14

Specific oxygen uptake ratesa after growth of:

Assay substrate Strain PS12 Strain PS14

CB 1,2-DCB 1,3-DCB 1,4-DCB 1,2,4-TCB 3-CBzt Bzt Ac 1,2,4,5-TeCB Pept

Catechol 352 163 162 382 216 467 283 85 154 93-Chlorocatechol 572 294 309 506 357 438 131 110 201 54-Chlorocatechol 473 289 316 633 354 431 121 108 205 83,4-Dichlorocatechol 196 98 134 281 157 199 40 49 91 103,5-Dichlorocatechol 475 244 306 524 305 460 113 107 172 113,6-Dichlorocatechol 226 122 155 264 182 210 55 54 108 134,5-Dichlorocatechol 25 8 12 37 21 44 <5 24 15 53,4,5-Trichlorocatechol 50 13 13 33 ND ND ND ND 17 103,4,6-Trichlorocatechol 171 90 115 235 129 170 36 37 87 16Tetrachlorocatechol 20 11 11 59 36 50 <5 <5 19 <53-Methylcatechol 869 292 432 984 357 891 217 178 286 54-Methylcatechol 792 310 453 675 443 934 234 180 251 9Benzene <5 <5 <5 <5 <5 <5 <5 <5 <5 <5Monochlorobezene 91 60 102 31 70 92 <5 24 107 81,2-DCB 106 45 79 44 60 132 <5 20 131 161,3-DCB 76 40 75 20 58 89 6 17 79 141,4-DCB 74 41 77 34 60 95 <5 22 103 121,2,3-TCB 31 26 27 18 31 59 <5 28 6 141,2,4-TCB 79 38 46 39 49 151 5 34 72 301,3,5-TCB <5 <5 <5 <5 <5 <5 <5 5 <5 <51,2,3,4-TeCB 8 18 13 <5 6 10 <5 8 11 71,2,3,5-TeCB <5 <5 <5 <5 <5 <5 <5 <5 <5 <51,2,4,5-TeCB 13 13 13 11 9 10 <5 <5 65 5Pentachlorobenzene <5 <5 <5 <5 <5 <5 <5 <5 <5 <5Hexachlorobenzene <5 <5 <5 <5 <5 <5 <5 <5 <5 <5

a Results represent means of at least two independently performed experiments. Oxygen uptake rates are expressed as specific activities (nanomoles of 02consumption per minute per milligram of protein) and are corrected for endogenous respiration. CB, chlorobenzene; DCB, dichlorobenzene; TCB,trichlorobenzene; Bzt, benzoate; CBzt, chlorobenzoate, Ac, acetate; Pept, peptone; TeCB, tetrachlorobenzene; ND, not determined.

were only used by strain PS12. Fluorinated benzenes andhalogenated phenols were not mineralized at all. Toluene;4-chlorotoluene; 2,4-, 2,5- and 3,4-dichlorotoluene (but not2- and 3-chlorotoluene or 2,3- and 2,6-dichlorotoluene);benzoate; 3-chlorobenzoate; 3-methylbenzoate; the threemonohydroxybenzoates; gentisate; and protocatechuatewere utilized by strain PS12 only.Growth of both strains on acetate and peptone, respec-

tively, for more than 100 generations did not result in the lossof their capability to utilize the chlorobenzenes.Oxygen uptake rates and enzyme activities. Washed cell

suspensions of Pseudomonas sp. PS12 were tested for theiroxidative potential for a number of compounds after growthon chlorinated benzenes, 3-chlorobenzoate, benzoate, andacetate as shown in Table 1. Since cells of Pseudomonas sp.PS14 grown on chlorobenzenes gave similar results, for thisstrain only activities from cells pregrown on 1,2,4,5-TeCBand peptone are given because neither acetate nor benzoateand 3-chlorobenzoate were utilized.Monochlorobenzene-grown cells of strain PS12 oxidized

this carbon source, some higher chlorinated benzenes, andcatechols. Benzoate-grown cells of strain PS12, however,were not induced for the oxidation of chlorobenzenes, butthey oxidized catechol and, to a lesser extent, chloro- andmethylcatechols with the exception of 4,5-dichlorocatecholand tetrachlorocatechol, as was found for the acetate-growncells; the acetate-grown cells, on the other hand, showedminute potential for the oxidation of some chlorinated ben-zenes. In contrast to cells grown on benzoate or acetate,3-chlorobenzoate-grown cells were highly induced for theoxidation of chlorinated catechols and benzenes, often pro-

ducing significantly higher uptake rates with these com-pounds than the chlorobenzene-grown cells. Halogenatedbenzenes like 1,3,5-TCB and 1,2,3,5-TeCB which allowed nogrowth were not oxidized; however, the ability to oxidize1,2,3-TCB and, to a lesser extent, 1,2,3,4-TeCB was signif-icant.

Parallel experiments performed with strain PS14 show that1,2,4,5-TeCB-grown cells very well oxidized their haloaro-matic carbon source and, in analogy to induced PS12 cells,most of the other compounds tested for this organism. Aftergrowth with peptone, however, no significant induction ofenzymes oxidizing aromatic compounds was detectable.Only 1,2,4-TCB was slowly oxidized as had been found foracetate-grown PS12 cells. Chlorophenols, including 2,4,5-trichlorophenol, were not oxidized by both strains.Enzyme activities for the oxidation of the dihydrodiols of

benzene and 1,2,4-TCB and for catechol and its halogenatedas well as methylated derivatives were estimated by usingcell extracts (Table 2). The benzene dihydrodiol dehydroge-nases of both strains were significantly induced even whencells were grown with acetate, benzoate, 3-chlorobenzoate,or peptone as the only carbon source. Highest rates werefound for the 1,2,4,5-TeCB-grown strain PS14. Comparisonof specific activities revealed that those for the chloroderiv-atives were always about 70% of those for the nonchlori-nated compounds.While tetrachlorocatechol was considerably converted by

cells of both strains grown with an aromatic compound, onlynegligible activities for the degradation of 4,5-di- and 3,4,5-trichlorocatechol were detected. Cells grown with chloroar-omatics exhibited significantly higher activities for the oxi-

VOL. 57, 1991

1434 SANDER ET AL.

TABLE 2. Specific catabolic enzyme activities in cell extracts of Pseudomonas sp. strains PS12 and PS14

Enzyme activity' for growth substrate:Enzyme and Strain PS12 Strain PS14test substrate

CB 1,2-DCB 1,3-DCB 1,4-DCB 1,2,4-TCB Bzt 3-CBzt Ac 1,2,4,5-TeCB Pept

Dihydrodiol dehydrogenaseBenzene dihydrodiol 220 153 107 207 97 27 62 88 380 551,2,4-TCB dihydrodiol 151 97 84 137 77 19 37 81 261 42

Catechol 1,2-dioxygenaseCatechol 284 146 298 200 152 508 905 64 124 73-Chlorocatechol 417 252 646 285 321 189 737 128 190 54-Chlorocatechol 403 209 573 246 290 202 724 120 184 53,4-Dichlorocatechol 63 59 169 37 90 39 234 39 43 23,5-Dichlorocatechol 257 184 461 145 235 129 546 107 121 43,6-Dichlorocatechol 76 54 158 58 91 37 234 42 46 24,5-Dichlorocatechol (<0.01) (<0.01) (<0.01) (<0.01) (<0.01) (<0.01) (<0.01) (<0.01) (<0.01) (<0.01)3,4,5-Trichlorocatechol (<0.01) ND (0.015) ND (<0.01) ND ND (<0.01) (<0.01) (<0.01)3,4,6-Trichlorocatechol 79 81 167 50 85 38 186 37 47 2Tetrachlorocatechol (0.130) (0.092) (0.112) (0.048) (0.031) (0.090) (0.140) (<0.01) (0.072) (<0.01)3-Methylcatechol 732 511 950 440 558 347 1352 202 278 84-Methylcatechol 703 427 833 469 412 419 986 136 300 7a Absolute specific activities are expressed in nanomoles per minute per milligram of protein. Activities in parentheses (AE per minute per milligram of protein)

are given for the formation of those chlorinated muconates of unknown e. Abbreviations are defined in Table 1, footnote a.

dation of monohalogenated catechols and often for 3,5-dichlorocatechol than for catechol. Benzoate-grown cells ofstrain PS12 exhibited high activities for the conversion ofcatechol, whereas chlorocatechols were oxidized moreslowly in contrast to acetate-grown cells, and only negligibleactivities were detected from peptone-grown PS14 cells.Methylcatechols were all subjected to the ortho-cleavagepathway. Significantly high activities for the conversion ofthese compounds were produced by cells pregrown on

chlorobenzenes, benzoate, 3-chlorobenzoate, and acetate,and again there was no induction after growth on peptone.However, toluene-grown cells of strain PS12 induced cate-chol 2,3-dioxygenase activities (16.4 nmol/min/mg for cate-chol, 21.8 nmol/min/mg for 3-methylcatechol, and 21.5 nmol/min/mg for 4-methylcatechol) together with catechol 1,2-dioxygenase activities (147 nmol/min/mg for catechol, 250nmol/min/mg for 3-methylcatechol, and 192 nmol/min/mg for4-methylcatechol).

Determination of chloride release. Chloride ions were re-leased very rapidly by strain PS14 from various chloroben-zenes (Table 3). For all chlorobenzenes used as carbonsources, and even for 1,2,3-TCB and 1,2,3,4-TeCB, highinitial dechlorination rates were obtained. Dehalogenation inall these cases was quantitative with respect to endpointdeterminations (Fig. 1A and B). 1,2,3,4-TeCB lost only one

of its four chlorine substituents. 1,3,5-TCB and 1,2,3,5-TeCB were found to be inefficient substrates for the dehalo-genation, confirming the above-described oxygen uptakeexperiments. Peptone-grown cells showed high activitiesonly for those chlorobenzenes which could be utilized forgrowth; rates found for dechlorination of penta- andhexachlorobenzene might be considered artifacts, althoughthey were reproducible.

Preparation and characterization of metabolites. Duringgrowth on chlorobenzenes as sole carbon sources, screeningfor excreted metabolites by HPLC analysis of the culturesupernatant was always negative. Peptone-grown low-level-induced resting cells of strain PS14, however, accumulatedmetabolites in the presence of 1,2,4-TCB, 1,2,4,5-TeCB, andthe analogous 2,4,5-trichloronitrobenzene.

For the oxidation of 1,2,4-TCB a washed cell suspensionof strain PS14 pregrown in peptone medium was used. To 55ml of cells suspended in 54 mM phosphate buffer of pH 7.4(adjusted to a protein concentration of 0.65 mg/ml), 1,2,4-TCB was added corresponding to a final concentration of 10mM. Accumulation of metabolites became visible by render-ing the medium a brownish-green color, and samples ana-lyzed by HPLC showed the formation of two distinct com-pounds (I and II). The time-dependent formation of thesemetabolites was quantified by using UV spectral data ofisolated samples of both metabolites (UV maxima at 282 andat 221 nm, respectively), obtained from a 20-fold scaled-up

TABLE 3. Specific rates and stoichiometry of chloride release byresting cells of Pseudomonas sp. strain PS14'

Rate of chloriderelease (pmol/h/mg % of chlorideof protein) after released after

Assay substrate growth on: growth on

1,2,4,5- 1,2,4,5-TeCBPeptone TeCB

Chlorobenzene 0.693 0.763 1051,2-Dichlorobenzene 1.290 1.188 1031,3-Dichlorobenzene 0.603 0.818 1001,4-Dichlorobenzene 0.559 1.178 931,2,3-Trichlorobenzene 0.052 0.623 1021,2,4-Trichlorobenzene 0.611 1.521 1031,3,5-Trichlorobenzene 0.067 0.112 81,2,3,4-Tetrachlorobenzene 0.075 1.392 251,2,3,5-Tetrachlorobenzene 0.049 0.031 31,2,4,5-Tetrachlorobenzene 0.661 0.802 106Pentachlorobenzene 0.039 0.002 2Hexachlorobenzene 0.058 0.013 1

a Cells of a peptone-grown and a 1,2,4,5-TeCB-grown culture were exten-sively washed with 55 mM phosphate buffer and resuspended therein at anoptical density (A578) of 0.43 corresponding to 140 ,ug of protein per ml.Chlorobenzenes were added, corresponding to a concentration of 0.5 mM,and the release of chloride was monitored. Initial rates and end point values(6 to 50 h) are given.



90(

70C

x

:zI-

zLLIz0

50s

300

100

1 2 3 4 5 20HOURS

FIG. 2. Formation of cis-3,4,6-trichloro-1,2-dihydroxycyclo-hexa-3,5-diene (0) and 3,4,6-trichlorocatechol (LI) from 1,2,4-TCBby peptone-grown cells of Pseudomonas sp. PS14.

experiment after extraction and purification by preparativeHPLC (Fig. 2). Structure elucidation of these metaboliteswas achieved by MS and NMR spectrometry (1H NMR, 13CNMR).

Since cis-3,4,6-trichloro-1,2-dihydroxycyclohexa-3,5-di-ene (compound I) proved to be labile under GC conditions,the mass spectrum in Fig. 3A was recorded upon direct inletof a purified sample. The molecular ion at m/z = 214 isaccompanied by an ion of low intensity at mlz = 212, whichindicates loss of two hydrogen equivalents. Significant frag-ments are formed upon loss of water (mlz = 196) andhydrogen peroxide (mlz = 180). The recorded intensities ofthe molecular ion signals (100.0:93.5:31.7:3.1) are in goodaccord with the theoretical pattern (100.0:96.5:31.3:3.5 forC6H5C1302); the same is true for the ions at M-H20 andM-H202. The base peak at mlz = 143 results from loss ofchlorine (m/z = 179) followed by elimination of hydrogenchloride. Data obtained by 'H-NMR spectroscopy (250.13MHz, CD30D; tetramethylsilane [TMS] as internal stan-dard) were as follows: 8 = 4.36 (H-2), 4.40 (H-1, J1,2 = 6.0Hz, J1,5 = 1.2 Hz), and 6.16 (H-5) ppm. According toC,H-COSY (42) and COLOC (77) 2-D-experiments, 13C-NMR data (62.89 MHz, CD30D; TMS as internal standard)are attributed as follows: C-1 = 72.43, C-2 = 73.86, C-5 =124.21, C-4 = 126.02, C-3 = 131.14, and C-6 = 138.50 ppm.The specific rotation of the compound proved to be [a]2 =+22.40 in methanol. The structure of 3,4,6-trichlorocatechol

1I(-

50-

A

i' iAikiL JLLI

'OH

.OHGI

40 60 80 100 120 140 160 180 200

100 -

50 -

B

1. 60

40} 60

Cl

COOMeCOOMe

Cl

M+ = 272 (0.1 %)

I L..I,L.h fI- 1.1". .ILiwu-..,

220 240m/z

1.0

80 100 120 140 160 180 200 220 240mlZ

FIG. 3. 70-eV mass spectra of cis-3,4,6-trichloro-1,2-dihydroxy-cyclohexa-3,5-diene (A) and dimethyl 2,3,5-trichloromuconate (B).

(compound II) was assigned on the basis of MS investiga-tions (M = C6H302C13), and its 'H-NMR spectrum (250.13MHz, CDCl3; TMS as internal standard) was as follows: 8 =7.1 (s, 1H) ppm, 2 exchangeable OH protons. Besides thesemetabolites, traces of 2,4,5-trichlorophenol, obviouslyformed from the dihydrodiol during extraction of the acidi-fied medium, could also be identified upon GC-MS bycomparison with an authentic sample.The oxidation of the 1,2,4,5-TeCB analog, 2,4,5-trichlo-

ronitrobenzene, by induced resting cells of strains PS12 orPS14 revealed the transient formation of minute amounts of3,4,6-trichlorocatechol. Neither the corresponding dihy-drodiols nor any other chloroorganic compounds were de-tected in the spent medium. In the course of the oxidation of2,4,5-trichloronitrobenzene, the stoichiometric formation ofnitrite, but not of the above trichlorocatechol, was evidentand the releasing rate, which was linear for several hours,was determined with 0.74 nmol/h/mg of protein for bothstrains. The trichloronitrobenzene, however, did not servefor growth. 1,2,4-TCB and 1,2,4,5-TeCB conversion, hereused as controls, did not result in the accumulation ofdetectable amounts of nitrite.An upscaled catechol 1,2-dioxygenase assay was per-

formed with extracts of strain PS12 and 3,4,6-trichlorocate-chol as the substrate. After acidic extraction, derivatizationwith diazomethane and subsequent purification by columnchromatography, dimethyl 2,3,5-trichloromuconate was ob-tained. The mass spectrum (GC-MS) of the diester is given inFig. 3B. The signal of the molecular ion (mlz = 272) is ofvery low intensity; however, loss of chlorine forms the

)

o_.

01*1 ci

o 0,...o".CI OH

I,0I0/. OHI 9-- I ~ Ji

/II\I

\

t

c

VOL. 57, 1991

-

-L- I 1

%.# I

I III-

1436 SANDER ET AL.

'2E

44

A.j

-

2 .x

D-E

4.4cc

a.

100-

50-

2p0

1,5 2

1.0 iJ

0,5

0

40 80 120 160TIME 6nin)

FIG. 4. Conversion of 3,4,6-trichlorocatechol (A, left peak areascale) by cell extracts of Pseudomonas sp. PS12 to 2,3,5-trichloro-muconate (0), 2,5-dichloro-4-oxo-hex-2-endioic acid (0), 2-chloro-3-oxoadipate (A, right peak area scale), and chloride (U). Theconversion was followed by HPLC. The decarboxylated com-pounds, however, were detected. Samples additionally were ana-lyzed by GC/MS and NMR spectroscopy.

dominating base peak. The recorded isotopic pattern of mlz= 237 (C8H7C1204) is in full accord with the predictedpattern for the presence of two chlorines (high-resolutionMS). Further characteristic ions are observed at mlz = 241and mlz = 213, both still containing all three chlorines andformed upon loss of 31 amu (methoxy, -OCH3) and 59 amu(carbomethoxy, -COOCH3) from the molecular ion, respec-tively. The presence of the carbomethoxy group in thecompound is also indicated by an intense ion at m/z = 59.'H-NMR data (400.14 MHz, CDCl3, TMS as internal stan-dard) were as follows: 8 = 3.85 (2s, 6H); 7.21 (s, 1H) ppm;the following "3C-NMR data (100.03 MHz, CDCl3, TMS asinternal standard) were obtained: 8 = 53.35, 53.46, 124.35,128.57, 132.19, 137.48, 161.59, and 161.92 ppm. From thesedata it is impossible to determine the configuration of thedouble bounds, which, with respect to the genesis of thismetabolite, should be cis,cis. An interesting by-productformed during degradation of 3,4,6-trichlorocatechol, how-ever, could not be isolated; but GC-MS data (M =C7H4C1204) suggest the compound to be methyl 5-carboxy-2,5-dichloropenta-2,4-dien-4-olide.The lower pathway was investigated with respect to the

elimination of residual chlorine by using cell extracts ofstrain PS12 as described above. However, in this caseEDTA was omitted to allow further conversion of thetrichloromuconate formed from trichlorocatechol. The ex-periment was performed in 30 ml of 10 mM Tris-acetatebuffer of pH 8.0. 3,4,6-Trichlorocatechol (initial concentra-tion, 1 mM) was oxidized by cell extract (13.5 mg of protein)prepared from induced cells. The time-dependent formationof metabolites was monitored by HPLC and is demonstratedin Fig. 4. Quantitation of all new metabolites was notpossible since sufficient amounts of isolated compoundsneeded for calibration were not available. Therefore, peakareas obtained from HPLC analysis are given. Figure 4shows that the rapid turnover of 3,4,6-trichlorocatecholcoincides with the early and transient formation of 2,3,5-trichloromuconate. Under stoichiometric chloride release,2,3,5-trichloromuconate is converted to a new metaboliteaccumulating in the medium. Upon the addition ofNADH (1

100-

50-

A 119 Ci 13777**-. -.. 59

CIt1.65

49, 147 165 .

JLi II i. .1I . I I I I I.

40 60 80 100 120 140 160

B

,l I1,... All L 1.

40 60 80 100

M+= 196

(0.1 %)

id. I180 m/z

87 105 ..59

77 --..-OMe

I::49j115 133,

M+ = 164

(0.1%)

120 140 160 180 m/z

FIG. 5. 70-eV mass spectra of methyl 2,5-dichloro-4-oxopent-2-enoate (A) and methyl 5-chloro-4-oxopentanoate (B).

mM) the decrease of the concentration of this new com-pound accompanies a second step of chloride release underformation of a further product.From analogous experiments, 2,5-dichloro-4-oxopent-2-

enoate was isolated by extraction of the acidified mediumbefore the addition of NADH. 1H-NMR data [250.13 MHz,(CD3)2CO, TMS as internal standard] were as follows: 8 =4.04 (s, 2H), 7.58 (s, 1H) ppm; one exchangeable COOH-proton. The mass spectrum of the corresponding methylester is given in Fig. 5A. After the addition of NADH, theculture medium was acidified and extracted; methylation ofthe extract and subsequent purification by column chroma-tography yielded methyl 5-chloro-4-oxopentanoate. Themass spectrum of this compound is given in Fig. SB; thestructure was confirmed by the following 'H-NMR data[250.13 MHz, (CD3)2CO, TMS as internal standard]: 8 =2.60 (t, J = 6.4 Hz, 2H), 2.89 (t, J = 6.4 Hz, 2H), 3.61 (s,3H), and 4.42 (s, 2H) ppm. The mass spectra of both methylesters show similar fragmentation patterns, as illustrated inFig. 5. Base peaks in both spectra result from loss of achloromethyl group due to an a cleavage of the molecularion. The complementary signal is found at m/z = 49 (CH2Cl).The low intensity of several ions in the spectrum of Fig. 5Ais due to the stability of the conjugated double-bond system.Peaks at mlz = 97 and mlz = 55 in the spectrum of Fig. SBcan be rationalized as loss of water and the carbomethoxygroup from m/z = 115.

DISCUSSION

The relatively short time often needed during the lastdecade for the isolation of bacteria with capabilities for the



CI

CI

I

cl

Clo2>~~C

cl

:I clst

CIHcl

O)H MOHCI

CICI,4OH

Ni OH

CI

CICX COOH

COOH

CI

ICl

HOOC0

ClIsteps

ClHOOC <

I sOOH

i steps

CI

CI

NO2

cI

HOOC >--\COOH

CI 0

FIG. 6. Proposed pathway for the degradation of 1,2,4-TCB and1,2,4,5-TeCB by Pseudomonas strains PS12 and PS14, respectively,and dioxygenolytic denitration of 2,4,5-trichloronitrobenzene byPseudomonas sp. PS14.

degradation of chlorobenzenes and many other xenobioticsgives strong evidence for permanent evolutionary processes

with respect to the development of new and productivebacterial pathways. Recruitment of such capabilities (17, 41)by microorganisms allows the filling of new functionalniches, for instance in highly contaminated ecosystems, thuspromoting natural bioremediation.The specialization of some of these enzymes for the

conversion of their new substrates apparently is concomitantwith the loss of ancestral capabilities. It is evident that our

strains, which were directly enriched on 1,2,4-TCB and1,2,4,5-TeCB, have lost the property to utilize or oxidizebenzene, and the same phenomenon has been reported forother bacterial strains also capable to grow with chlorinatedbenzenes (38, 56). The analogous specialization of initial,

xenobiotic substituents releasing dioxygenases was alsodemonstrated for bacteria mineralizing monosulfonated (12,13) and the more xenobiotic disulfonated naphthalenes (78).The bacterial degradation of unsubstituted, nonphenolic

aromatic hydrocarbons is known to proceed by well-inves-tigated, multicomponent enzyme systems requiring flavo-protein reductases and ferredoxin to achieve electron trans-port to a terminal dioxygenase component, responsible forthe conversion of the aromatic nuclei into cis-dihydrodiols(4, 30, 32, 36, 37). These compounds are rearomatized tocatechol derivatives by NAD-dependent dehydrogenases (3,33, 58).

In our studies on 1,2,4,5-TeCB mineralization, the initialoxidative attack by Pseudomonas sp. PS14 cells causes theprobably spontaneous elimination of the first chlorine as HClduring rearomatization of the dihydrodiol, yielding 3,4,6-trichlorocatechol. This compound also was identified as theproduct of enzymatic rearomatization of 3,4,6-trichloro-1,2-dihydroxycyclohexa-3,5-diene, the dihydrodiol formed from1,2,4-TCB. The above dihydrodiol recently was isolated andidentified also from 1,2,4-TCB-converting microbial cultures(61a). Two successive monooxygenase reactions, one ofthem responsible for the elimination of chlorine, cannot befully excluded since chlorine replacement by hydroxyl hasbeen reported (14, 40, 44, 45). However, strains PS12 andPS14 do not utilize or oxidize 2,4,5-trichlorophenol (in thiscase expected to be the intermediate) or other chlorophe-nols. Interestingly, 2,4,5-trichlorophenol, known as the firstcatabolite in 2,4,5-trichlorophenoxyacetate degradation (31,43, 64), seems to be further metabolized via 2,5-dichlorohy-droquinone (39), thus following the known route for themineralization of pentachlorophenol (2). Furthermore, theoxidative attack onto the benzene ring system has beenshown to proceed by incorporation of one molecule ofoxygen (32).A similar dioxygenase-mediated dehalogenation of 4-chlo-

rophenylacetate to produce 3,4-dihydroxyphenylacetate hasbeen described by Markus et al. (49), as has been the enzymesystem responsible for this reaction (50, 67). This mecha-nism of dioxygenation and dehydrohalogenation does notallow the regeneration of reduction equivalents from apostulated chlorine-containing unstable intermediate; and sothe dihydrodiol dehydrogenase activity found in our 1,2,4,5-TeCB-oxidizing and -dehalogenating PS14 cells has to beconsidered useless in the degradation of this substrate. Theregeneration of NADH, however, could be demonstrated forthe oxidation of 3,4,6-trichloro-1,2-dihydroxycyclohexa-3,5-diene to 3,4,6-trichlorocatechol. This intermediate was ring-cleaved by, in the case of strain PS12, a constitutive catechol1,2-dioxygenase of type II (20) with high activities for3-chloro-, 4-chloro-, and 3,5-dichlorocatechol.One of the three remaining halides obviously was split off

in the course of the subsequent addition-elimination process(so-called cycloisomerization [65]) of the intermediate 2,3,5-trichloromuconate obtained on ortho-cleavage of the abovetrichlorocatechol. From the trichloromuconate, under de-chlorination, the corresponding carboxymethylenebuten-olide was formed, which, after hydrolysis, yielded 2,5-dichloro-4-oxohex-2-enedioate, identified as the acid-catalyzed decarboxylation product, 2,5-dichloro-4-oxopent-2-enoate. It should be noted that ,B-keto acids are easilydecarboxylated under acidic conditions (65). Analogous de-carboxylation products of 2- and 5-chloro-4-oxohex-2-ene-dioate, which represent the biologically active intermediatesin 3,4- and 3,5-dichlorocatechol degradation, have beenreported (38, 68).

VOL. 57, 1991


For the catabolism of 2-chloro-4-oxohex-2-enedioate, twopathways have been described, both involving two double-bond reductions under consumption of NADH, eliminationof hydrogen chloride, and final production of succinate (15,23). The cleavage step takes place at the stage of 2-chloro-4-oxoadipate, yielding chlorosuccinate and acetate (23), or atthe stage of 3-oxoadipate, which means dehalogenationbefore cleavage (15). Since we identified 5-chloro-4-oxopen-tanoate as the dehalogenated decarboxylation product of2,5-dichloro-4-oxohex-2-enedioate, we suggest 2-chloro-3-oxoadipate as the consecutive metabolite formed by a dihy-drogenation-dehydrohalogenation sequence. This hypothe-sis is corroborated by our identification of 3-oxoadipate as ametabolite of 3- and 4-chlorocatechol degradation by strainsPS12 and PS14. A subsequent hydrolytical cleavage of2-chloro-3-oxoadipate would yield succinate and chloroace-tate.The data obtained for the oxidation of chlorobenzenes

(Table 1) in general are well correlated with those obtainedfor chloride-releasing rates shown in Table 3, even for theoxidation and dechlorination of 1,2,3,4-TeCB, which lostone of its four chlorine substituents. The oxygenolytic attackon this molecule has to take place at the two free hydrogen-substituted carbon atoms, yielding tetrachlorocatechol,which is slowly converted to 2,3,5-trichloro-4-oxohex-2-enedioate, representing the obvious dead-end metabolite ofthis crucial route (64a).

Interestingly, the oxidation of the structurally 1,2,4,5-TeCB-related 2,4,5-trichloronitrobenzene resulted in the un-

expected, obviously preferred elimination of the nitro group

as could be judged from the identification of temporarilyaccumulating nitrite and 3,4,6-trichlorocatechol. To our

knowledge, this is the first observation of a dioxygenase-mediated denitration of an aromatic compound. This reac-

tion and the converging pathways of 1,2,4-TCB and 1,2,4,5-TeCB degradation are shown in Fig. 6.

ACKNOWLEDGMENTS

This work was supported by The Federal Minister for Science andTechnology and a doctoral grant from DECHEMA to P.S.We thank V. Sinnwell for running the NMR spectra and C. Adami

for the preparation of photoprints.

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degradation of 1,2,4-trichloro- and 1,2,4,5-tetrachlorobenzene

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