Metabolism of 3-Chloro-, 4-Chloro-, and 3,5-Dichlorobenzoate by … · other chemicals employed were of analytical grade andwereobtainedfromMerck,Darmstadt,Germany. Biochemicals were

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1979, p. 421-4280099-2240/79/03-0421/08$02.00

Vol. 37, No. 3

Metabolism of 3-Chloro-, 4-Chloro-, and 3,5-Dichlorobenzoateby a Pseudomonad

J. HARTMANN, W. REINEKE, AND H.-J. KNACKMUSS*

Institut fur Mikrobiologie der Universitat und der Gesellschaft fur Strahlen- und Umweltforschung mbHMunchen in Gottingen, D-3400 Gottingen, Federal Republic of Germany

Received for publication 5 January 1979

Pseudomonas sp. WR912 was isolated by continuous enrichment in three stepswith 3-chloro-, 4-chloro-, and finally 3,5-dichlorobenzoate as sole source of carbonand energy. The doubling times of the pure culture with these growth substrateswere 2.6, 3.3, and 5.2 h, respectively. Stoichiometric amounts of chloride were

eliminated during growth. Oxygen uptake rates with chlorinated benzoates re-

vealed low stereospecificity of the initial benzoate 1,2-dioxygenation. Dihydrodi-hydroxybenzoate dehydrogenase, catechol 1,2-dioxygenase, and muconate cyclo-isomerase activities were found in cell-free extracts. The ortho cleavage activityfor catechols appeared to involve induction of isoenzymes with different stereo-specificity towards chlorocatechols. A catabolic pathway for chlorocatechols was

proposed on the basis of similarity to chlorophenoxyacetate catabolism, andcometabolism of 3,5-dimethylbenzoate by chlorobenzoate-induced cells yielded2,5-dihydro-2,4-dimethyl-5-oxo-furan-2-acetic acid.

Large amounts of man-made halogenated ar-omatic compounds are released into the environ-ment from chemical production plants as well asfrom intensive agriculture. Chlorobenzoic acidsare widely used as herbicides in the form of 2,5-dichloro-3-aminobenzoic acid, 2,3,6-trichloro-benzoic acid, or 2,6-dichlorobenzonitril. Further-more, chlorinated benzoates can originate fromcometabolism of polychlorinated biphenyls bysoil bacteria (1, 13, 21). 4-Chlorobenzoic acid isa metabolite of the herbicide Bidisin (18), and3,5-dichlorobenzoic acid possibly originatedfrom "Pronamid" (12). Halogenated aromaticcompounds without exception are markedlymore refractile to microbial attack than non-halogenated aromatics. When 3-chlorobenzoate(5), 4-chlorophenol (16), 2,4-dichlorophenol (33),4-chlorophenoxyacetate (10), 4-chloro-2-methyl-phenoxyacetate (14, 15), or 2,4-dichlorophenox-yacetate (9, 11, 30, 32, 33) were utilized as solesources of carbon and energy, halocatecholswere found to be central metabolites and subjectto ortho cleavage. The organically bound halo-gen is eventually eliminated as chloride from thenonaromatic metabolite generated from ringcleavage. Enzymes of unsubstituted benzoatemetabolism generally exhibit striking stereo-specificity, so that the affinities for chlorosubsti-tuted benzoic acids are low (24). Another criticalstep is the ring cleavage reaction (6, 7). WithPseudomonas sp. B13, which utilizes 3-chloro-benzoate, reduced turnover rates of halogenatedsubstrates are counterbalanced by overproduc-

tion of catabolic enzymes or induction of addi-tional isoenzymes with altered substrate speci-ficity (6, 7, 24, 25).

In the present paper we describe the metabolicactivities of a pseudomonad for the utilization of3-chloro-, 4-chloro-, and 3,5-dichlorobenzoicacid.

MATERIALS AND METHODSContinuous enrichment. The 200-ml chemostat

consisted of a cylindrical vessel (250 mm by 30 mm indiameter) and sampling device. Air was introducedthrough a G2 porous glass filter plate which coveredthe entire bottom of the vessel. The culture was main-tained at 28°C. The mineral medium (5) containingthe appropiate benzoates for enrichment was pumpedat a rate of 40 ml/day by a precision variable-speedperistaltic pump (model 12000, Varioperpex, LKB,Sweden).

Culture conditions. For growth in liquid culture,the mineral medium as described by Dorn et al. (5)contained the respective chlorobenzoates at 10 mM.Higher substrate concentrations inhibited exponentialgrowth. To avoid strong acidification during growthwith 3,5-dichlorobenzoate, the buffer concentrationcould be increased twofold without affecting thegrowth rate. Small quantities of cells were grown in500-ml Erlenmeyer flasks containing 50 ml of medium.The flasks were incubated at 28°C on a rotary shakerat 150 rpm. Large-scale growth of biomass was carriedout in 5- or 10-liter fermentors (Biostat from B. Braun,Melsungen, Germany) containing 4 or 8 liters of me-dium. Air was introduced at a rate of 5 liters per min,and the cultures were stirred at 600 rpm at 300C.

Solid media were prepared by addition of 2% lona-ger (Oxoid) no. 2 to solutions of the basal medium.

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422 HARTMANN, REINEKE, AND KNACKMUSS

Stock cultures were maintained on nutrient agarslopes, subcultured monthly, and stored at 20°C.

Cells were harvested during exponential growthphase by centrifugation (10,000 rpm, 20 min at 10°C),suspended in 33 mM tris(hydroxymethyl)amino-methane-hydrochloride buffer (pH 8.0), and storedovernight at 8°C for preparation of cell-free extracts.Oxygen uptake experiments were carried out withfreshly harvested cells that were centrifuged andwashed once with 50 mM phosphate buffer (pH 7.4)and resuspended in the same buffer.

Preparation of cell-free extracts. Cell suspen-sions in 33 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 8.0) were disrupted by usinga French press (Aminco, Silver Spring, Md.) at 80 kg/cm2. Cell debris was removed by centrifugation at30,000 x g for 20 min. Protein content of cell-freeextracts was determined with the biuret method bythe method of Beisenherz et al. (3). Protein of wholecells was measured by a modified method ofLa Riviereaccording to Schmidt et al. (29).Enyzme assays. Specific activities are expressed

as micromoles of substrate utilized per minute pergram of protein at 25°C.

Dihydrodihydroxybenzoate dehydrogenase was de-termined by means of dihydrodihydroxybenzoate-de-pendent reduced nicotinamide adenine dinucleotideformation by the method of Reiner (27). For measur-ing catechol 1,2-dioxygenase activities, the modifiedprocedure of Ornston and Stanier (6, 7) was used.The activity of catechol 2,3-dioxygenase was as-

sayed by the method of Nozaki (20), by increasing theconcentration of substituted catechols fivefold toachieve substrate saturation. Catechol 1,2-dioxygenaseactivity was destroyed by heating cell-free extracts at55°C for 10 min (19, 28). Muconate cycloisomeraseactivity (muconate lactonizing enzyme) was assayedby the method of Ornston (22). Activity of this enzymecould only be measured at substrate concentrationsbelow saturation because assay concentrations of thebiologically produced muconates were only 50,iM.Oxygen uptake experiments. Rates of oxygen

uptake were measured polarographically by use of aClark oxygen electrode. Freshly harvested cells werewashed and suspended in 50 mM phosphate buffer(pH 7.4), containing 7 to 10 jig of protein per ml. Cellsuspensions were saturated with air in the cuvettes.After 5 min of constant endogenous oxygen uptake,the reaction was started by injecting 10,mol of assaysubstrate. Uptake rates were determined with begin-ning time course curves and corrected for endogenous02 uptake. Activities are expressed in micromoles of02 uptake per minute per gram of protein.Cometabolism of methylbenzoates. Chloroben-

zoate-grown cells were washed and resuspended in 20ml of 50 mM phosphate buffer (pH 7.4), to a finalprotein concentration of 4.4 mg/ml. Cells were incu-bated with the respective methylbenzoates in 250-mlErlenmeyer flasks at 30°C on a rotary shaker. Produc-tion of cometabolic products was followed by high-pressure liquid chromatography. Compounds wereidentified with authentic samples (17) by their reten-tion times and in situ scanning of their ultravioletspectra by use of a spectrophotometric detector(model 635, Varian-Techtron, Springvale, Australia).

For cometabolism of 3,5-dimethylbenzoate, a 10-literculture of Pseudomonas sp. WR912 was grown with10 mM 3,5-dichlorobenzoate as the growth substrateand 3,5-dimethylbenzoate (4 mM) as the cosubstrate.After the reaction had ceased (control with high-pres-sure liquid chromatography), the supernatant of theculture fluid was concentrated 30-fold by flash evapo-ration. After acidification (pH 2), the solution wasextracted repeatedly with diethyl ether. The pooledextracts were dried over MgSO4, and the solvent wasevaporated. The cometabolism product and unreactedbenzoates were separated by thin-layer chromatogra-phy (20- by 20-cm2 plates, 0.1-cm thickness, silica gel60 PF2s4nm, Merck, Darmstadt, Germany) with a sol-vent system of diisopropylether-formic acid-water(200:7:3, vol/vol/vol). The compounds were located byobservation under ultraviolet light at 254 nm. Afterbeing eluted with chloroform, the cometabolism prod-uct from 3,5-dimethylbenzoate was crystallized fromcooled diisopropylether/light petroleum, washed withlight petroleum, and dried over P205.

Analytical methods. Substituted benzoates andtheir metabolites in the culture fluid were analyzed byreverse-phase high-pressure liquid chromatography(chromatography model 4200 with solvent program-mer, Varian Associates, Palo Alto, Calif.; Micro PakCH-column, 250 mm by 2 mm, octadecylsilane chem-ically bonded to LiChrosorb 10-,lI particles). The mo-bile phase was 10 mM H3PO4 containing varying con-centrations of 2-propanol and methanol. A 254-nmdetector (Varian) and a computing integrator (AutolabSystem I, Spectra-Physics, Santa Clara, Calif.) wereused for quantification. Samples of culture fluids (5 to20 pI) were injected after cells had been removed bycentrifugation in 0.5-ml micro-test tubes for 3 min at20,000 rpm (Mikro-Hamatokrit, Heraeus Christ, Os-terode, Germany).

Chloride ion concentrations were measured with anion selective combination chloride electrode (model96/17, Orion Research Inc., Cambridge, Mass.), whichwas calibrated with NaCl (0.1 mM up to 50 mM) inmineral medium before each measurement. Ultravi-olet spectra were measured in water by use of a ZeissDMR 10 recording spectrophotometer. Infrared spec-tra were recorded on a Pye-Unicam SP 1000 spectro-photometer. Crystals were mulled in Nujol and placedbetween NaCl disks. Low- and high-resolution massspectra were measured with a DuPont mass spectro-photometer 21-492. The nuclear magnetic resonancespectrum was recorded with a Varian HA 100 instru-ment with tetramethylsilane as internal reference; thevalues are expressed in parts per million.

Chemicals. 3-Chloro-, 4-chloro-, 3-methyl-, 4-methyl-, and 3,5-dimethylbenzoic acid were obtainedfrom Fluka AG, Buchs, Switzerland, whereas 3,5-di-chlorobenzoic acid, 3-methyl-, and 4-methylcatecholwere purchased from EGA Chemie, Steinheim, Ger-many. The preparation of dihydrodihydroxybenzoatewas described by Reineke et al. (26). 3-Chloro- and 4-chlorocatechol were prepared by the method of Will-statter and Muller (37). 3,5-Dichlorocatechol was syn-thesized by chlorination of 2-hydroxybenzaldehyde bythe method of Biltz and Stepf (4) and by subsequentDakin reaction (2). The catechols were purified bysublimation before use. cis,cis-Muconic acid was syn-

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METABOLISM OF CHLOROBENZOIC ACIDS 423

thesized by the method of Wacek and Fiedler (34). Allother chemicals employed were of analytical gradeand were obtained from Merck, Darmstadt, Germany.Biochemicals were obtained from Boehringer, Mann-heim, Germany.

RESULTS

Isolation of the organism. Pseudomonassp. WR912, which utilizes chlorobenzoates, wasobtained by continuous enrichment from amixed population originating from soil samplesof the Gottingen area. During the initial opera-tion of the chemostat with 3-chlorobenzoate and4-methylbenzoate (20 mM each) as carbonsources, the mixed culture was supplementedwith two defined strains, the toluate-degradingPseudomonas putida mt-2 (19, 35) and the 3-chlorobenzoate-utilizing Pseudomonas sp. B13(5). Extensive cometabolism of 4-chloro- and 3,5-dichlorobenzoates was anticipated by the com-bined action of these organisms. After 4 weeksof operation, 4-chlorobenzoate was added to thefresh medium as an additional carbon source,the concentration being increased up to 20 mM.After being operated for another month thecontinuous culture was maintained with 4-chlo-robenzoate as sole carbon source. 3,5-Dichloro-benzoate was added in increasing concentrationincrements over 3 months to a maximum con-centration of 20 mM. Within 6 months of contin-uous enrichment, single colonies could be ob-tained from agar plates containing 3,5-dichloro-benzoate as the sole carbon source. The purityof the selected strains was verified by plating

both on nutrient agar and 3,5-dichlorobenzoate.Strain WR912, studied in detail, was able to use3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate inaddition to benzoate as the only source of carbonand energy.The methods of Stanier et al. (31) were used

for identification of the organism. The strainproved to be a gram-negative rod, motile by asingle polar flagellum. The following additionalcriteria were observed: no intracellular accumu-lation of poly-,f-hydroxybutyrate; no productionof fluorescent or phenazine pigments; no deni-trification, no growth on nutrient broth at 4 orat 410C; starch, gelatine, or extracellular poly-f8-hydroxybutyrate were not hydrolyzed, argi-nine dihydrolase and oxidase were positive; nogrowth factors were necessary. Among 120 sub-strates tested, only the following ones allowedgood growth: DL-valne, D-tryptophan, L-phen-ylalanine, pelargonate, adipate, sebacate, meso-tartrate, salicylate, nicotinate, mesaconate, cit-rate, lactate, and benzoate. Poor or no growthwas observed with carbohydrates and on meth-ylbenzoates. The strain resembles in some re-spects Pseudomonas ruhlandii (8).Growth with chlorobenzoates. As shown

in Fig. 1, the organism grew exponentially with3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate assole sources of carbon and energy, exhibitingdoubling times of 2.6, 3.3, or 5.2 h. Eliminationof organically bound chlorine as HCI was indi-cated by concomitant acidification of the me-dium. The amount of chloride eliminated cor-responded at any timne with the amount of sub-

16

14~ 7.2

102I8~

6*

0

4 8

time rhra]FIG. 1. Utilization of the following chlorobenzoic acids (0): (a) 3-chlorobenzoate; (b) 4-chlorobenzoate; (c)

3,5-dichlorobenzoate. Elimination of chloride (A) and pH (0) during growth (0) in a 5-liter fermentor.Concentrations ofsubstrates were followed by high-performance liquid chromatography; chloride ion concen-tration was determined with an ion-selective chloride electrode as described in the text. Growth was observedturbidimetrically at 546 nm.

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strates consumed. In the case of 3,5-dichloroben-zoate, liberation of 2 mol of HCI per mol ofsubstrate (Fig. lc) exceeded the buffer capacityof the medium so that phosphate concentrationhad to be doubled.During growth with 4-chlorobenzoate, the cul-

ture fluid turned greenish-yellow, exhibiting an

absorption maximum at 378 nm. On acidification(pH 2) the yellow color disappeared, but was

restored on addition of excess NaOH. This in-dicates excretion of chlorohydroxymuconic sem-

ialdehyde, originating from the action of cate-chol 2,3-dioxygenase on 4-chlorocatechol. Theamount of the meta cleavage product was cal-culated to be less than 1% of substrate utilizedbased on the molar extinction coefficient of 2-hydroxy-5-chloromuconic semialdehyde (28).Oxygen uptake experiments with chlo-

robenzoates. Since the initial enzyme, ben-zoate 1,2-dioxygenase, is unstable in cell-freeextracts, the relative rates of oxygen uptake bywhole cells with differently substituted ben-zoates were taken as a measure of enzyme spec-

ificity. The relative rates of respiration elicitedby benzoate and toluates have successfully beenused as a measure of enzyme specificity in P.putida mt-2 (35).As shown in Table 1, the relative rates of

oxygen uptake with chlorobenzoates were essen-tially the same regardless of whether the cellswere grown on benzoate or on one of the chlo-robenzoates. In contrast, the absolute oxygenuptake rates differed considerably with cellsfrom different growth substrates. 3,5-Dichloro-benzoate exhibited the lowest relative rates of02 uptake, but induced the highest specific ac-tivity of benzoate 1,2-dioxygenase. Enzymeoverproduction could not completely counter-balance the reduced turnover rate of 3,5-dichlo-robenzoate so that a twofold doubling time was

still observed during growth with this substratecompared to 3-chlorobenzoate.Catabolic enzyme activities in extracts.

Enzyme activities of dihydrodihydroxybenzoatedehydrogenase, catechol 1,2-dioxygenase, andmuconate cycloisomerase were induced in cells

TABLE 1. Relative rates of oxygen uptake with chlorosubstituted benzoates by freshly harvested cells ofPseudomonas sp. WR9120

Growth substrateAssay substrate

3-Chlorobenzoate 4-Chlorobenzoate 3,5-Dichlorobenzoate Benzoate

Benzoate 100 (109) 100 (121) 100 (202) 100 (90)3-Chlorobenzoate 99 97 92 994-Chlorobenzoate 83 88 88 933,5-Dichlorobenzoate 31 21 23 33

a The reaction rates are expressed as percentages of that for benzoate taken as 100%. Absolute activities asmicromoles of 02 per minute per gram of protein are given in parentheses for the relative rates reported as100%. For measurements of oxygen uptake rates see text.

TABLE 2. Specific activities of catabolic enzymes in cell-free extracts of succinate- and chlorobenzoate-grown cells ofPseudomonas sp. WR912'

Growth substrate

Activity Assay substrate 3-Chloro- 4-Chloro- 3,5-Di-benzoate benzoate chloro- Benzoate Succinatebenzoate benzoate benzoate

DHBb dehydrogenase DHB (290) (144) (387) (380) NDC

Catechol 1,2-dioxygenase Catechol 100 (182) 100 (72) 100 (247) 100 (208) 100 (9)3-Chlorocatechol 40 30 38 73 64-Chlorocatechol 40 30 34 69 143,5-Dichlorocat- 44 31 36 ND ND

echol

Catechol 2,3-dioxygenase Catechol 100 (14) 100 (13) l00 (11) 100 (17) 100 (12)4-Chlorocatechol 66 73 73 ND 52

a The cells were harvested during exponential growth. Enzyme activities were determined as described in thetext and are expressed as percentages of that substrate with the highest tumover rate. Absolute specificactivities (micromoles per minute per gram of protein) are given in parentheses for the relative rates reportedas 100%.

b DHB, Dihydrodihydroxybenzoate.'ND, Not determined.



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grown on unsubstituted and chlorosubstitutedbenzoates (Table 2). The levels of their specificactivities depended on respective growth sub-strates. Marked differences were found espe-cially for catechol 1,2-dioxygenase. The levels ofcatechol 1,2-dioxygenase increase in the se-quence 4-chlorobenzoate, 3-chlorobenzoate,benzoate, and 3,5-dichlorobenzoate as growthsubstrates. Only an uninduced level of this activ-ity was observed in succinate-grown cells. Therelative activity of substituted catechols com-pared to catechol increased in the following re-spective order: 4-chlorobenzoate, 3,5-dichloro-benzoate, 3-chlorobenzoate, and benzoate.Only an uninduced level of catechol 2,3-diox-

ygenase was detectable in benzoate- or chloro-benzoate-grown cells. The formation of insignif-icant amounts of chlorohydroxymuconic semi-aldehyde during growth on 4-chlorobenzoate canbe explained by the uninduced level of catechol2,3-dioxygenase, which exhibits relative high ac-tivity for 4-chlorocatechol.

Relatively high cycloisomerase activity wasfound only for chlorosubstituted cis,cis-mucon-ates irrespective of whether the cells were grownon benzoate or any of the substituted benzoates,i.e., 76% for 2-chloro-, 57% for 3-chloro-, and 18%for cis,cis-muconate referred to 2,4-dichloromu-conate as 100% (35 U/g of protein) in 3,5-dich-lorobenzoate-grown cells.Cometabolic activities with methylben-

zoates. 3,5-Dichlorobenzoate-grown cells read-ily cometabolized 3-methyl- and 4-methylben-zoate. During incubation, the "dead-end" me-tabolites 2,5-dihydro-4-methyl- and 2,5-dihydro-2-methyl-5-oxo-furan-2-acetic acid (compoundsI and II, X = CH3, Fig. 2) were quantitativelyaccumulated in the culture fluid. Initial dioxy-genation of unsymmetrically substituted ben-zoates generally fails to be highly selective (24),so that 3-methylbenzoate yields a mixture of Iand II (X = CH3). These metabolites wereformed at a ratio of 95:5 and identified withauthentic samples by comparison of their reten-tion times during high-pressure liquid chroma-tography and by in situ scanning of their ultra-violet spectra (17). Cells grown with 3,5-dichlo-robenzoate cooxidized the analogous 3,5-di-methylbenzoate to 2,5-dihydro-2,4-dimethyl-5-oxo-furan-2-acetic acid (III, X = CH3) accordingto the cometabolic sequence given in Fig. 2.Compound III was characterized by the follow-ing physical properties: melting point, 70 to720C; thin-layer chromatography in the pre-scribed solvent system on 0.25-mm layers ofSilica Gel GF254 (Merck, Darmstadt, Germany)with Rf value of 0.42; absorption spectrum,X.,.H20 = 211 to 212 nm (E = 10,200); infraredspectrum (CHC13) with a peak at 1765 cm-'

(a,/3-unsaturated y-lactone) and a shoulder at1735 cm-' (COOH); high resolution mass spec-trum, calculated for C8HI004, 170.0579, M+ foundat mle 170.0585, fragment peaks due to loss ofCH2COOH, CH3, COOH, CO, and H20; nuclearmagnetic resonance data as given in Fig. 3.

DISCUSSIONThe catabolic pathways of 3-chlorobenzoate,

4-chlorobenzoate, and 3,5-dichlorobenzoate (Fig.2) are proposed on the basis of the enzymeactivities found and the methylmuconolactonesI, II, and III (X = CH3), which were accumulatedduring cometabolism of the structurally analo-gous methylbenzoates. The substituted ben-zoates are hydroxylated yielding 1,2-dihydrodi-hydroxybenzoates, which are subsequently con-verted to catechols by dehydrogenation and de-carboxylation. The resulting 3-chlorocatechol, 4-chlorocatechol, or 3,5-dichlorocatechol are sub-ject to ortho cleavage with formation of therespective halogenated cis,cis-muconic acids.These are cycloisomerized with coincident orsubsequent elimination of chloride.

Cells of Pseudomonas sp. WR912 exhibit highbenzoate 1,2-dioxygenase activities not only for3-chlorobenzoate but also for 4-chloro- and 3,5-dichlorobenzoate. This resembles toluate-growncells of P. putida mt-2. Its plasmid-coded ben-zoate 1,2-dioxygenase was shown to function intoluate metabolism, consequently being stereo-unspecific and considerably more active towardshalogenated benzoates (24). In contrast, Pseu-domonas sp. B13 is restricted to the utilizationof 3-chlorobenzoate as growth substrate becauseits benzoate 1,2-dioxygenase shows narrow sub-strate specificity, which is generally observed inbenzoate-utilizing bacteria (24). The existence ofuninduced levels of meta-pyrocatechase exhibit-ing no function in chlorocatechol catabolismdemonstrated further similarity to P. putida mt-2. In Pseudomonas sp. WR912, chlorocatecholslead to expression of genes coding for the en-zymes of the ortho cleavage pathway. Murray etal. (19) also noted that chlorobenzoates failed toinduce meta pathway enzymes in P. putida mt-2. In cell-free extracts of chlorobenzoate as wellas benzoate-grown cells of Pseudomonas sp.WR912, high ortho cleavage activities for 3-chloro-, 4-chloro-, and 3,5-dichlorocatechol werefound. Differences in the relative ortho pyro-catechase activities for catechol and chlorocate-chols can freely be explained by the presence ofat least two enzymes distinctly different in theirspecificity towards substituted catechols. A sim-ilar situation was observed in Pseudomonas sp.B13, where two isoenzymes were identified (6).One of these, pyrocatechase II, showed highactivity for chlorocatechols and was exclusively

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COOH

x

I N

COOH

IOHOH

I NO

H

COO*HI 0X

COOH

Xb

* 66

Xe

COOH

x

COOH4 ON

OH

rOH

I

x

IeOOH

x

COOH1 1 0°ZX

I

COOH

0

COOH

xJ&x

COONlON

OH

ICON

XeX

,OH

x x

I

COON

COON

COOI( COO~Cl

COON

COON

0 1

Acetate H

CI- Succinate

Fumarate - clFIG. 2. Proposedpathway for the degradation of chlorobenzoates (X = Cl) and cometabolism ofanalogous

methylbenzoates (X = CH3) by Pseudomonas sp. WR912. Compound I, II and III (X = Cl) are hypothetical(unstable) intermediates. Late catabolic steps after halide elimination are proposed by the degradation of 4-chloro- and 2,4-dichlorophenoxyacetic acid by Pseudomonas sp. and Arthrobacter sp.

induced in 3-chlorobenzoate- or 4-chlorophenol- 3-chloro- and 3,5-dichlorocatechol. In the partic-grown cells. In contrast the highly specific iso- ular case of Pseudomonas sp. WR912, the chlo-enzyme, pyrocatechase I, has been demonstrated rocatechol-cleaving enzyme appears to be in-in benzoate- as well as chlorobenzoate-grown duced also when cells are grown on unsubsti-cells. This enzyme is inefficient in its cleavage of tuted benzoate. A similar catechol 1,2-dioxygen-



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1.89 7.33H30 H 1.56

2XCH30:2 0-CHH2COOH

2.73/2.93 11.06FIG. 3. 'H-nuclear magnetic resonance data of

2,5-dihydro-2,4-dimethyl-5-oxo-furan-2-acetic acid(CDCl3). 8 values are listed in the formula (in partsper million; STMS = 0). Coupling constants: JCH =

15.2 Hz; J3-H 4-CH3= 1.5 Hz.

ase with activity for chlorocatechols must alsobe induced in a 2,4-dichlorophenoxyacetate-grown Pseudomonas strain and an Arthrobacterstrain as reported by Evans et al. and Tiedje etal. (11, 32).Cometabolism of methyl-substituted sub-

strate analogs by chlorobenzoate-grown cells ofPseudomonas sp. WR912 supports a catabolicsequence for halocatechols as has been postu-lated for the 4-chloro- and 2,4-dichlorophenox-yacetate metabolism. The accumulation ofmethylmuconolactones I, II, and III (X = CH3)suggests a catabolic sequence for the chlorinatedsubstrates up to the level of cycloisomerizationand chloride elimination. At this level themethyl-substituted analogs cannot eliminateCH3 as a carbanion, so that "dead-end" metab-olites are formed (17). Chloride elimination as acatabolic step directly linked to cycloisomeriza-tion of 3-chloro-substituted muconates yieldingcompound IV has been demonstrated duringdegradation of 3-chlorobenzoate by Pseudo-monas sp. B13 (E. Schmidt and H.-J. Knack-muss, manuscript in preparation), 4-chlorophen-oxyacetate, and 2,4-dichlorophenoxyacetate. 5-OXo_1AI(5H)-afuranacetic acid or 4-chloro-5-oxo-AI(`H),-furanacetic acid (compounds IV and V,Fig. 2) have been identified as reaction products(10, 11, 30, 32). Elimination of chloride from thehypothetical intermediate I (X = Cl) cannot befreely rationalized. Although the structurallyanalogous methylmuconolactone I (X = CH3) isthe only cycloisomerization product of 2-meth-ylmuconic acid, an alternative mechanism forthe cycloisomerization of 2-chloromuconic acidwith 2,5-dihydro-5-oxo-furan-2-chloroacetic acidas an intermediate is currently being investi-gated. Further degradation to intermediates ofthe tricarboxylic acid cycle as well as eliminationof the second organically bound chlorine duringtotal breakdown of 3,5-dichlorocatechol is as-

sumed to be similar to the pathway proposed for2,4-dichlorophenoxyacetate catabolism (Fig. 2).The evolution of the unusually versatile chlo-

robenzoate-degrading capability of the present

strain seems to need supposition of prolongedadaptation under continuous culture conditions.In this case "the lag phase represents a periodduring which processes of plasmid exchange andrecombination result in the selection of one ormore organisms that finally have a battery ofenzymes capable of the complete degradation"(36). Transmissibility of the genes encoding en-zymes of the meta cleavage pathway for meth-ylarene metabolism and of the 2,4-dichlorophen-oxyacetate-degrading capability were reportedto be plasmid-borne (23, 38). Therefore it is notunreasonable to assume that the catabolic activ-ity for 4-chlorobenzoate and 3,5-dichloroben-zoate ofPseudomonas sp. WR912 might be com-posed of the initial benzoate 1,2-dioxygenasewith low stereospecificity, originally functioningin toluate catabolism, and of the halocatechol-metabolizing activity from the 3-chlorobenzoate-utilizing Pseudomonas sp. B13 or halophenoxy-acetate-utilizing bacteria. This concept is indeedrealized for the construction of halobenzoate-utilizing strains [W. Reineke and H.-J. Knack-muss, Nature (London), in press].

ACKNOWLEDGMENTWe thank E. Schmidt for supplying muconate preparations.

LITERATURE CITED1. Ahmed, A., and D. D. Focht. 1973. Degradation of

polychlorinated biphenyls by two species of Achromo-bacter. Can. J. Microbiol. 19:47-52.

2. Azouz, W. M., D. V. Parke, and R. T. Williams. 1955.Studies in detoxification 62. The metabolism of halo-benzene, ortho- and para-dichlorobenzenes. Biochem.J. 59:410-415.

3. Beisenherz, G., H. J. Boltze, T. Bucher, R. Czok,K. M. Garbade, E. Meyer-Arendt, and G. Pflei-derer. 1953. Diphosphofructose-Aldolase, Phosphogly-cerinaldehyd-Dehydrogenase, Milchsaure-Dehydro-genase, Glycerophosphat-Dehydrogenase und Pyruvat-Kinase aus Kaninchenmuskulatur in einem Arbeits-gang. Z. Naturforsch. 86:555-577.

4. Biltz, H., and K. Stepf. 1904. Uber die Chlorierung desSalicylaldehyds. Ber. Dtsch. Chem. Ges. 37:4022-4031.

5. Dorn, E., M. Hellwig, W. Reineke, and H.-J. Knack-muss. 1974. Isolation and characterization of a 3-chlo-robenzoate degrading pseudomonad. Arch. Microbiol.99:61-70.

6. Dorn, E., and H.-l. Knackmuss. 1978. Chemical struc-ture and biodegradability of halogenated aromatic com-pounds. Two catechol 1,2-dioxygenases from a 3-chlo-robenzoate-grown pseudomonad. Biochem. J. 174:73-84.

7. Dorn, E., and H.-J. Knackmuss. 1978. Chemical struc-ture and biodegradability of halogenated aromatic com-pounds. Substituent effects on 1,2-dioxygenation of cat-echol. Biochem. J. 174:85-94.

8. Doudoroff, M., and N. J. Palleroni. 1974. Genus I.Pseudomonas Migula 1894, 237 Nom. cons. Opin. 5,Jud. Comm. 1952, p. 217-243. In R. E. Buchanan andN. E. Gibbons (ed.), Bergey's manual of determinativebacteriology, 8th ed. The Williams & Wilkins Co., Bal-timore.

9. Duxbury, J. M., J. M. Tiedje, M. Alexander, and J. E.Dawson. 1970. 2,4-D metabolism: enzymatic conver-

VOL. 37, 1979


.asm.org/

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sion of chloromaleylacetic acid to succinic acid. J. Agric.Food Chem. 18:199-201.

10. Evans, W. C., B. S. W. Smith, P. Moss, and H. N.Fernley. 1971. Bacterial metabolism of 4-chlorophen-oxyacetate. Biochem. J. 122:509-517.

11. Evans, W. C., B. S. W. Smith, H. N. Fernley, and J. I.Davies. 1971. Bacterial metabolism of 2,4-dichloro-phenoxyacetate. Biochem. J. 122:543-551.

12. Fisher, J. D. 1974. Metabolism of the herbicide Pronamidin soil. J. Agric. Food Chem. 22:606-608.

13. Furukawa, K., K. Tonomura, and A. Kamibayashi.1978. Effect of chlorine substitution on the biodegrad-ability of polychlorinated biphenyls. Appl. Environ.Microbiol. 35:223-227.

14. Gaunt, J. K., and W. C. Evans. 1971. Metabolism of 4-chloro-2-methylphenoxyacetate by a soil pseudomonad.Preliminary evidence for the metabolic pathway. Bio-chem. J. 122:519-526.

15. Gaunt, J. K., and W. C. Evans. 1971. Metabolism of 4-chloro-2-methylphenoxyacetate by a soil Pseudomonad.Ring fission, lactonizing and delactonizing enzymes.Biochem. J. 122:533-542.

16. Knackmuss, H.-J., and M. Hellwig. 1978. Utilizationand cooxydation of chlorinated phenols by Pseudo-monas sp. B13. Arch. Microbiol. 117:1-7.

17. Knackmuss, H.-J., M. Hellwig, H. Lackner, and W.Otting. 1976. Cometabolism of 3-methylbenzoate andmethylcatechols by a 3-chlorobenzoate utilizing Pseu-domonas: accumulation of (+)-2,5-dihydro-4-methyl-and (+)-2,5-dihydro-2-methyl-5-oxo-furan-2-aceticacid. Eur. J. Appl. Microbiol. 2:267-276.

18. Kocher, M., F. Lingens, and W. Koch. 1976. Untersu-chungen zum Abbau des Herbizids Chlorphenprop-methyl im Boden und durch Mikroorganismen. WeedRes. 16:93-100.

19. Murray, K., C. J. Duggleby, J. M. Sala-Trepat, andP. A. Williams. 1972. The metabolism of benzoate andmethylbenzoates via the meta-cleavage pathway byPseudomonas arvilla mt-2. Eur. J. Biochem. 28:301-310.

20. Nozaki, M. 1970. Metapyrocatechase (Pseudomonas).Methods Enzymol. 17A:522-525.

21. Ohmori, T., T. Ikai, Y. Minoda, and K. Yamada. 1973.Utilization of polyphenyls and polyphenyl-related com-pounds by microorganisms. I. Agric. Biol. Chem. 37:1599-1605.

22. Ornston, L. N. 1966. The conversion of catechol andprotocatechuate to f?-ketoadipate by Pseudomonas pu-tida. III. Enzymes of the catechol pathway. J. Biol.Chem. 241:3795-3799.

23. Pemberton, J. M., and P. R. Fisher. 1977. 2,4-D plasmidand persistence. Nature (London) 268:732-733.

24. Reineke, W., and H.-J. Knackmuss. 1978. Chemical

structure and biodegradability of halogenated aromaticcompounds. Substituent effects on 1,2-dioxygenation ofbenzoic acid. Biochem. Biophys. Acta 542:412-423.

25. Reineke, W., and H.-J. Knackmuss. 1978. Chemicalstructure and biodegradability of halogenated aromaticcompounds. Substituent effects on dehydrogenation of3,5-cyclohexadiene-1,2-diol-1-carboxylic acid. Biochem.Biophys. Acta 542:424-429.

26. Reineke, W., W. Otting, and H.-J. Knackmuss. 1978.cis-Dihydrodiols microbially produced from halo- andmethylbenzoic acids. Tetrahedron 34:1707-1714.

27. Reiner, A. M. 1972. Metabolism of aromatic compoundsin bacteria. Purification and properties of the catechol-forming enzyme, 3,5-cyclohexadiene-1,2-diol-1-carbox-ylic acid (NAD+) oxidoreductase. J. Biol. Chem. 247:4960-4965.

28. Sala-Trepat, J. M., and W. C. Evans. 1971. The meta-cleavage of catechol by Arthrobacter species: 4-oxalo-crotonate pathway. Eur. J. Biochem. 20:400413.

29. Schmidt, K., S. Liaaen-Jensen, and H. G. Schlegel.1963. Die Carotinoide der Thiorhodaceae. I. Okenon alsHaupt-Carotinoid von Chromatium okenii Perty. Arch.Mikrobiol. 46:117-126.

30. Sharpee, K. W., J. M. Duxbury, and M. Alexander.1973. 2,4-Dichlorophenoxyacetate metabolism by Ar-throbacter sp.: accumulation of a chlorobutenolide.Appl. Microbiol. 28:181-184.

31. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff.1966. The aerobic pseudomonads: a taxonomic study. J.Gen. Microbiol. 43:159-271.

32. Tiedje, J. M., J. M. Duxbury, M. Alexander, and J. E.Dawson. 1969. 2,4-D metabolism: pathway of degra-dation of chlorocatechols by Arthrobacter sp. J. Agric.Food Chem. 17:1021-1026.

33. Tyler, J. E., and R. K. Finn. 1974. Growth rates of apseudomonad on 2,4-dichlorophenoxyacetic acid and2,4-dichlorophenol. Appl. Microbiol. 28:181-184.

34. Wacek, A., and R. Fiedler. 1949. Uber die Oxidation desBrenzcatechins zu Muconsaure. Monatsh. Chem. 80:170-185.

35. Williams, P. A., and K. Murray. 1974. Metabolism ofbenzoate and the methylbenzoates by Pseudomonasputida (arvilla) mt-2: evidence for the existence of aTOL plasmid. J. Bacteriol. 120:416-423.

36. Williams, P. A., and M. J. Worsey. 1976. Plasmids andcatabolism. Biochem. Soc. Trans. 4:466-468.

37. Willstatter, R., and H. E. Muller. 1911. Uber Chloro-derivate des Brenzcatechins und des ortho-Chinons.Ber. Dtsch. Chem. Ges. 44:2182-2191.

38. Worsey, M. J., and P. A. Williams. 1975. Metabolismof toluene and xylenes by Pseudomonas putida (ar-villa) mt-2: evidence for a new function of the TOLplasmid. J. Bacteriol. 124:7-13.



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Metabolism of 3-Chloro-, 4-Chloro-, and 3,5-Dichlorobenzoate by … · other chemicals employed were of analytical grade andwereobtainedfromMerck,Darmstadt,Germany. Biochemicals were